J Am Oil Chem Soc (2015) 92:423–430 DOI 10.1007/s11746-015-2605-8

ORIGINAL PAPER

FAME Production and Fatty Acid Profiles from Moist Chlorella sp. and Nannochloropsis oculata Biomass Rui C. M. Alves Sobrinho · Laércio Vauchinski · Renata Rodrigues de Moura · Ednei G. Primel · Paulo C. V. Abreu · Marcelo G. Montes D’Oca 

Received: 19 August 2014 / Revised: 17 October 2014 / Accepted: 28 January 2015 / Published online: 18 February 2015 © AOCS 2015

Abstract  In the present study, we investigated the production of fatty acid methyl esters (FAME) from moist Chlorella sp. and Nannochloropsis oculata biomass using a hydrolysis–esterification process. Additionally, we evaluated for the first time the fatty acid profile before and after this process. Hydrolysis of the lipid fraction was performed on a moist biomass in the presence of differing amounts of an acid catalyst in both 50 and 100 % w/w water relative to the biomass. The esterification of the crude extracts of the free fatty acids (FFA) was then investigated. The experiments show that in the presence of 50 % w/w water relative to the biomass, the hydrolysis–esterification process results in higher FFA and FAME yields. The analysis of the fatty ester profiles did not reveal any degradation of the FFA from the microalgae biomass under the hydrolysis–esterification conditions. The results were compared with both extraction–transesterification and direct transesterification processes using dry biomass. The extraction–transesterification and hydrolysis–esterification processes resulted in similar FAME yields and similar profiles of the fatty esters from dry and moist biomass materials, respectively. Keywords  Fatty acid profile · Microalgae · Moist biomass · Nannochloropsis oculata · Chlorella sp.

R. C. M. Alves Sobrinho · L. Vauchinski · R. R. de Moura · E. G. Primel · M. G. Montes D’Oca (*)  Laboratório Kolbe de Síntese Orgânica, Escola de Química e Alimentos, Universidade Federal do Rio Grande, Av. Itália km 08, Rio Grande, RS, Brazil e-mail: [email protected] P. C. V. Abreu  Laboratório de Ecologia do Fitoplâncton e de Microorganismos Marinhos, Instituto de Oceanografia, Universidade Federal do Rio Grande, Av. Itália km 08, Rio Grande, RS, Brazil

Introduction Biodiesel (or fatty acid methyl esters, FAME) can be produced from a wide range of biomass materials, including inedible vegetable oils and residual oils [1, 2]. The combustion properties of biodiesel are similar to those of petroleum-based diesel; thus, biodiesel may be used either as a substitute for diesel fuel or, more commonly, in fuel blends [3]. An alternative biomass used for biodiesel production is microalgae [4]. Microalgae have a high lipid content and grow rapidly, and their oils can be converted into biodiesel fuel using existing technology [5, 6]. Of the methods used to produce biodiesel from microalgae, the most widely employed is the extraction–transesterification process [7, 8]. This two-stage process involves the extraction of lipids from the microalgae followed by transesterification. Various methods can be used to extract the lipids, such as ultrasound baths, magnetic stirring, microwave irradiation, and supercritical carbon dioxide [9–12]. The particular method is chosen based on the type of microalga and the chemical composition of the cell wall (e.g., silica, chitin, cellulose, or CaCO3) [10, 13]. According to Chisti, certain microalgae are rich in oils, and others can be grown under conditions that favor the accumulation of large quantities of oil [14]. Algal oils may be similar to other vegetable oils, or they may be composed primarily of hydrocarbons, depending on the algal species used to produce them. Because of the wide range of polarity of the lipids found in microalgae, different types of polar solvents have been used in the extraction process [15–17]. For example, chloroform:methanol mixtures, methanol, ethanol, chloroform, ethanol:hexane:water combinations, and methylene chloride:methanol mixtures have all been used to extract lipids [11, 18, 19].

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According to the literature, the extraction–transesterification process has been replaced by direct transesterification, a one-stage process in which extraction and transesterification occur simultaneously [20, 21]. In a previous report, we compared the yields of FAME obtained by direct transesterification and extraction–transesterification from a dry Chlorella pyrenoidosa biomass [11]. However, both the extraction–transesterification and direct transesterification methods have disadvantages. One such disadvantage is the requirement of a moisturefree biomass because the presence of water decreases the biodiesel yield due to hydrolysis reactions of the FAME. Microalgae are cultivated in an aqueous environment, and removal of their water content beyond that of a paste consistency is labor intensive. In a recent life cycle assessment (LCA) study, a negative energy balance was attained during the process of producing biodiesel from a dry microalgae biomass when Chlorella vulgaris was cultured in a nitrogen-limited medium [22]. A previous study has shown that using wet-paste microalgae biomass can have an adverse effect on the in situ transesterification process [20]. That study has shown that microalgae biomass with a water content of more than 31.7 % completely inhibited direct transesterification, leading to a negligible biodiesel conversion. A possible explanation for this phenomenon is the occurrence of undesirable hydrolysis reactions during transesterification. In the presence of water, a triglyceride can be easily hydrolyzed to a diglyceride and an FFA. Hence, wet microalgae biomass must be initially dried to ensure an efficient and optimal performance during direct transesterification. Note that alcohols are miscible in water, and transesterification cannot proceed optimally when there is previously solubilized water in the methanol/ethanol solvent. Therefore, it is suggested that extensive drying of the biomass be undertaken prior to direct transesterification to avoid the occurrence of any side reactions and to simplify the subsequent separation processes. According to the literature, adding distilled water to dry Chaetoceros gracilis cells (in a range increasing from 10 to 100 % w/w of the biomass) caused progressively decreasing yields of FAME, with a 100 % w/w water-to-biomass ratio yielding only 50 % of the expected FAME in the direct transesterification [23]. Recently, the hydrolysis of lipids from microalgae with a high water content has been described in the literature as a pretreatment for direct esterification [24]. The results indicated that the amount of FAME obtained by esterification of hydrolysates was increased by 181.7 % compared with the amount of FAME obtained by direct transesterification at the same water content (80 %). In the present study, however, palmitic acid and tripalmitin were used as

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the feeding materials in order to observe the effect of water on the transesterification and esterification processes. The corresponding FAME were produced from commercial microalgae. Reddy et al. [25] have described a single-step process for the direct conversion of wet algae to crude biodiesel under supercritical ethanol conditions. Ethanol was used for the simultaneous extraction and transesterification of lipids in algae to produce fatty acid ethyl esters under supercritical conditions. The results reveal that the maximum yield of fatty acid ethyl esters (FAEE) was 67 % relative to the dry biomass. To avoid the requirement of drying the biomass, we investigated the production of fatty acid methyl esters by a hydrolysis–esterification process, using the microalgae Chlorella sp. and Nannochloropsis oculata. Additionally, due to the presence of both unsaturated and polyunsaturated fatty acids in the microalgae, the variation in the fatty acid profile was examined before and after the hydrolysis– esterification processes.

Materials and Methods General To investigate the hydrolysis–esterification process, commercially available Chlorella sp. biomass (Purifarma, São Paulo, Brazil) was used. This biomass had a 120-mesh particle size, containing 50.0 % protein, 21.0 % carbohydrate and 2.3 % chlorophyll. The microalga species Nannochloropsis oculata (Eustigmatophyceae) used in this study was obtained from the collection at the Ecology of Phytoplankton and Aquatic Microorganisms Laboratory of the Federal University of Rio Grande (catalog names NANN OCUL-1 and THAL WEIS-1, respectively) [26]. The cells were grown in batch cultures (3 replicates) in 1,600-L culture tanks. The culture medium employed in these studies consisted of inexpensive commercial fertilizers containing ammonium sulfate, urea, calcium superphosphate, ferric chloride and vitamins B1, B6 and B12 [27]. The following culture conditions were used: salinity of 28, mean temperature of 20 °C, and natural light; the photoperiod used for these experiments was 12 h of light and 12 h of darkness. The cells were mixed by bubbling atmospheric air through the tanks, and the cells were concentrated by flocculation using a commercial flocculant (Flopam® 4880, SNF Floerger, France) after 15 days of growth. The algal biomasses was dried in an oven at 60 °C until a constant weight was obtained. Prior to the extractions, the samples were maintained in a freezer at −5 °C in a container protected from the light.

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The solvents and reagents used in these experiments were obtained from commercial sources (Synth, Brazil and Merck, Germany). The fatty acid profile was determined using a GCMS-QP2010Plus chromatographic system (Shimadzu, Japan) equipped with a split/splitless injector coupled with a mass detector. The acid index was determined using a Titrino Plus 848 potentiometer titrator (Metrohm, Switzerland). Extraction and Quantification of the Lipids A total of 1 g of biomass and 6 mL of a chloroform:methanol mixture (2:1 v/v) was added to a test tube at room temperature (20 °C) and subjected to magnetic stirring. The samples were then centrifuged for 5 min. The organic phase was carefully collected, and the solvent was evaporated under reduced pressure. The lipid fraction was dried to a constant weight in an oven at 60 °C. The total lipid fraction was calculated by determining the difference between the weights of the original and final flasks. All of the procedures were performed in triplicate [11, 28]. Determination of the Acid Value A total of 200 mg of the lipid fraction and 50 mL of a diluent solution (toluene:isopropanol:water, 1:0.95:0.5 v/v) was added to a beaker and titrated with a solution of 0.1 mol L−1 KOH in isopropanol, standardized with 200 mg potassium biphthalate. The acid values (in mg KOH/g) of the lipid fraction were measured by potentiometric titration according to ASTM D 664-11a (Standard Test Method for Acid Number of Petroleum Products by Potentiometric Titration, Philadelphia, 2009). Fatty Acid Profile The derivatization of the lipid fraction of the microalgae was performed according to a previous study [11, 29]. Briefly, the sample containing the lipid fraction (300 mg) was placed in a test tube to which 3 mL of a boron trifluoride/methanol solution was added. The mixture was heated in a water bath at 70 °C for 20 min. The derivatized mixture was washed in a separatory funnel with 15 mL of hexane and 20 mL of distilled water. The organic and aqueous phases were then separated. The organic phase containing the fatty esters was dried, and the solvent was evaporated at 50 °C. Afterward, the fatty acid profile was determined using a GCMS-QP2010Plus chromatographic system (Shimadzu) equipped with a split/splitless injector coupled with a mass detector. The detector operation temperatures were as follows: interface, 280 °C; source, 230 °C. The detection was set to the full-scan mode to scan

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from 30 to 500 m/z in 0.2 s. Electron impact at 70 eV was used as the ionization mode. The operating conditions of the chromatograph were as follows: injector, 250 °C; column, 80 °C (initial temperature, 0 min), followed by a gradient of 10 °C/min up to 180 °C and then increasing by 7 °C/min to reach a final temperature of 330 °C; He gas flow, 1.3 mL/min; pressure, 88.5 kPa; average linear velocity, 42 cm/s; injection volume, 1 mL with a split ratio of 1:100. A 5 % Crossbond diphenyl/95 % dimethyl polysiloxane (30 m × 0.25 mm × 0.25 µm; Restek, Bellefonte, PA, USA) column was used. The compounds were identified by their retention times and their identities were confirmed by mass spectrometry. Hydrolysis–Esterification Process Step 1: Hydrolysis of the Lipids A total of 20 g (±1 g) of oven-dried biomass in distilled water (50 or 100 % w/w relative to the biomass) was mixed in a round-bottom flask equipped with a condenser. H2SO4 was used as a catalyst (at 20, 40, or 60 % w/w relative to the biomass), and 100 mL of hexane was added. The reaction was performed at 100 °C under constant stirring for 240 min. After the reaction was complete, the mixture was vacuumfiltered in a Büchner funnel to separate the moist biomass. Then, 100 mL of hexane was added to the biomass, and the mixture was stirred and filtered again. The organic fractions containing the crude fatty acid were evaporated under reduced pressure to remove the hexane and dried to a constant weight in an oven at 60 °C for subsequent esterification. Step 2: Esterification of the Crude Fatty Acids To the flask containing the crude fatty acids was added half the volume of methanol suitable for a molar ratio of 30:1 (alcohol:fatty acid). The H2SO4 acid catalyst (10 % w/w relative to the fatty acid mass), diluted in the other half of the methanol volume, was then added. The reaction remained under constant stirring at 100 °C for 240 min. When the reaction was complete, the reaction mixture was neutralized with a solution of NaOH/methanol and evaporated under reduced pressure. The crude product was treated with hexane (100 mL) for 120 min, and the soluble fraction in the hexane layer (containing the fatty esters) was separated from the insoluble fraction (residue) by vacuum filtration through a Büchner funnel, and the solvent was removed under reduced pressure. The reaction was monitored by thin-layer chromatography performed on glass plates coated with silica gel. A mixture of hexane:diethyl ether (80:20 v/v) was used as the eluent, and the products were visualized using iodine

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water after the flocculation step in order to monitor the quality of biomass while avoiding contamination by commercial fertilizers. The Chlorella sp. and Nannochloropsis oculata biomasses were dried and then re-mixed with distilled water to reproduce the harvested microalgae. Hydrolysis was performed on the moist algal biomass in the presence of sulfuric acid and hexane. The esterification process was then investigated for the conversion of crude fatty acids to FAME (Fig. 2). The FAME were also produced using both the conventional process (extraction–transesterification) and by direct transesterification from dried biomass in the presence of sulfuric acid in methanol. The results were compared with those of the hydrolysis–esterification process.

Microalgae

dry biomass direct transesterification (in situ process) conventional process

solvent extraction residual biomass

total lipids transesterification

FAME

FAME Production from Chlorella sp and Evaluation of the Fatty Acid Profiles

Fig. 1  Methods of FAME production from microalgae

vapor. The fatty esters were purified on a silica gel/Al2O3 column to yield the pure FAME. All of the procedures utilizing these methods were performed in triplicate.

Results and Discussion In the production of biodiesel from microalgae using acid catalysts (typically H2SO4), FAME are produced by a process involving extraction followed by transesterification of the algal oil, or alternatively by direct transesterification (Fig. 1) [11, 12]. In the present study, we investigated the production of FAME by commercially available Chlorella sp. and the cultivated Nannochloropsis oculata using a hydrolysis– esterification process and evaluated the fatty acid profile after the two-step procedure. To investigate the hydrolysis–esterification process using cultivated Nannochloropsis oculata, the wet biomass had to be washed with distilled

Hydrolysis was performed on the algal biomass in the presence of 50 and 100 % w/w water relative to the biomass. In this experiment, the addition of hexane was required to promote the simultaneous extraction of the free fatty acids produced from the hydrolysis of the lipids. Next, esterification of the FFA was performed in a homogeneous system using methanol and H2SO4 as a catalyst (10 % relative to the crude fatty acid mass) for the conversion of fatty acids into fatty acid alkyl esters [30]. Figures 3 and 4 show the yields of the crude free fatty acids (FFA) and fatty acid methyl esters (FAME) based on the moist algal biomass from Chlorella sp. In the hydrolysis–esterification process the yields of FFA obtained from experiments using 50 % water and 20 % H2SO4 in the hydrolysis step at 100 °C for 240 min and the yields of the FAME obtained following esterification at both 60 and 100 °C for 60 min were similar relative to the initial biomass (Fig. 3, entries 1 and 2, respectively). At a fixed temperature of 100 °C in the esterification step (for 240 min), the yield of the FAME was higher, at 6.8  ± 0.3 % relative to the biomass (Fig. 3, entry 3, gray bar).

Fig. 2  Production of FAME from moist algal biomass using the hydrolysis–esterification process

O OH O Moist algal biomass

H2SO4

H2SO4

OH OH

Hexane 100 oC

O OH O Algal Free Fatty Acids

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MeOH reflux

O R

OMe FAME

R= fatty chain

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Fig. 3  Yields of the crude free fatty acids (FFA) and fatty acid methyl esters (FAME) from Chlorella sp. by the hydrolysis–esterification process using 50 % water and H2SO4 as a catalyst (20, 40, and 60 % w/w relative to the biomass)

Fig. 4  Yields of the crude free fatty acids (FFA) and fatty acid methyl esters (FAME) from Chlorella sp. by the hydrolysis– esterification process (entries 1–3) using 100 % water and H2SO4 as a catalyst (20, 40, and 60 % w/w relative to the biomass) and the conventional and in situ processes

We subsequently evaluated the use of different amounts of water and H2SO4 catalyst (20, 40, and 60 % based on the dry biomass) in the hydrolysis step at 100 °C for 240 min. Figures 3 (entries 3–5, dark bars) and 4 (entries 6–8, dark bars) show the crude FFA yields obtained from the hydrolysis process in different amounts of water, 50 and 100 % w/w, respectively. In these cases, larger amounts of catalyst resulted in higher FFA yields from the hydrolysis process. The results show that the crude FFA yields obtained using 100 % w/w water (Fig. 4, entries 6–8, dark bars) were less than those obtained using 50 % w/w water (Fig. 3, entries 3–5, dark bars) for all of the amounts of catalyst tested. This decrease could be caused by the greater amounts of water present and the consequent dilution of the catalyst (H2SO4). Additionally, because the hydrolysis step occurs in two phases (an apolar phase containing hexane and a polar phase containing water, biomass, and catalyst), an increase in the volume of the polar phase causes greater fluidity in the reaction medium, reducing the amounts and complicating the extractions of fatty acids from the medium.

After the hydrolysis step for experiments in which 50 % water was used, the FAME yields obtained from the esterification process for 20, 40, and 60 % catalyst were 6.8 ± 0.3, 6.9 ± 0.2, and 7.3 ± 0.8 % relative to the initial biomass, respectively (Fig. 3, entries 3–5, gray bars). The respective yields obtained under the same experimental conditions, but instead using 100 % water, were 5.9 ± 0.6, 6.3 ± 0.2, and 6.8 ± 0.5 % (Fig. 4, entries 6–8, gray bars). These results reveal that maximum yields of FAME generated by Chlorella sp. in the hydrolysis–esterification process were obtained using 50 % w/w water and 60 % H2SO4 at 100 °C for 240 min in the hydrolysis step followed by esterification at 100 °C for 60 min. The extraction–transesterification process (the conventional process) using Chlorella sp. was subsequently compared to the hydrolysis–esterification process, and the FAME yields are expressed as their weight calculated relative to the dry biomass. The production of the FAME was performed according to a previous study [11]. First, the lipid content, acid index of the lipid fraction, and fatty acid profile of the Chlorella sp. microalga were determined. When we used a chloroform:methanol (2:1

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v/v) mixture [27] as the extractor solvent, the lipid yield was 14.0 ± 0.7 % based on the dry biomass weight (Fig. 4, conventional process, black bar). The lipid fractions of the microalga Chlorella sp. contained fatty chains that varied from 16 to 18 carbon atoms, with 23.8 wt% palmitic (16:0), 11.9 wt% palmitolenic (16:2), 15.3 wt% hiragonic (16:3), 3.3 wt% estearic (18:0), 22.9 wt% linoleic (18:2) and 20.8 wt% linolenic (18:3) acids. Next, the acid values (calculated as mg KOH/g) of the lipid fractions obtained using the chloroform:methanol mixture were measured by potentiometric titration (we chose this method over conventional titration because the lipid fraction extracted from the microalga possessed a dark color). The acid value of the lipid fractions of Chlorella sp. was 24.8 ± 0.4 mg KOH/g. The FAME yield obtained by acid transesterification of the lipid fraction extracted with chloroform:methanol (2:1 v/v) was 7.1 ± 1.8 % relative to the initial biomass (Fig. 4, conventional process, gray bar). This result confirms that the conversion of lipids to esters was 50.7 % relative to the lipid fraction using the chloroform:methanol extraction. This observation is in accordance with our previous results obtained for the extraction–transesterification process [11]. The experiments showed that the FAME yield obtained from the extraction–transesterification process resulted in a similar yield compared with the hydrolysis–esterification process in 50 % water, 7.3 ± 0.8 % relative to the initial biomass (Fig. 3, entry 5, gray bar). In addition, the analysis shows that the extraction– transesterification and hydrolysis–esterification processes resulted in a similar profile of the fatty acid methyl esters (Table  1) from the microalga Chlorella sp. Uncommon fatty acids were not observed in any of the cases. These results indicate that the double bonds present in the unsaturated and polyunsaturated fatty acids from the microalgal lipids are not suffering from the addition of water under the acidic conditions of the hydrolysis–esterification process. Otherwise, this process would result in the lowest FAME yields in addition to different fatty acid profiles than were obtained using the extraction–transesterification process. The FAME production from Chlorella sp. by direct transesterification (an in situ process) was then compared with the FAME produced by the hydrolysis–esterification process, and the FAME yield is expressed as FAME weight relative to the dry biomass. The direct transesterification process involves the simultaneous extraction and transesterification of the lipids by mixing a microalgal cell suspension with solvents in the presence of the catalyst. In contrast to the extraction–transesterification process, the presence of sulfuric acid in the direct transesterification is important for disrupting the cell

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J Am Oil Chem Soc (2015) 92:423–430 Table 1  Profile of the fatty acid methyl esters (%) obtained from the microalga Chlorella sp. by both hydrolysis–esterification and conventional processes Entry

Fatty estera

Hydrolysis–esterification (60 % catalyst, 50 % water)

Conventional process

1 2 3 4 5 6

C16:0 C16:1 C16:2 C16:3 C18:0 C18:2

21.8 1.7 7.2 14.1 3.1 20.3

26.8 1.7 6.5 10.9 3.6 22.1

7

C18:3

31.9

29.2

a

  Hexadecanoic (palmitic, C16:0), 9-hexadecenoic (C16:1), 9,12-hexadecadienoic (C16:2), 7,10,13-hexadecatrienoic (hiragonic, C16:3), octadecanoic (stearic, C18:0), 9,12-octadecadienoic (linoleic, C18:2), 9,12,15-octadecatrienoic (linolenic, C18:3)

walls in situ in order to release the lipids into the solvent mixture, resulting in high FAME yields. To evaluate the direct transesterification process applied to the dry Chlorella sp. biomass, we performed reactions at 100 °C in methanol for 240 min with 20 % H2SO4 as the acid catalyst, in accordance with a previous study [11]. The use of methanol for the in situ transesterification process provided yields of 11.6 ± 0.4 % FAME based on the dry biomass (82.8 % relative to the lipid fraction). These results show that direct transesterification from the Chlorella sp. biomass results in higher FAME yields (Fig. 4, in situ process, gray bar). In addition, the fatty esters identified in the FAME obtained by direct esterification from the microalga Chlorella sp. were similar to those generated in both the hydrolysis–esterification and extraction–transesterification processes. FAME Production from Nannochloropsis oculata and Evaluation of the Fatty Acid Profiles We also investigated the production of FAME obtained through the hydrolysis–esterification process from Nannochloropsis oculata grown in batch cultures. First, the lipid content of Nannochloropsis oculata was determined using a mixture of chloroform:methanol (2:1 v/v) as the extractor solvent, giving a yield of 15.0 ± 0.3 % based on the dry biomass weight. The lipid fractions of the microalga Nannochloropsis oculata contained fatty chains that varied from 14 to 20 carbon atoms, showing 4.60 wt% myristic (14:0), 25.2 wt% palmitic (16:0), 18.1 wt% palmitoleic (16:1), 3.02 wt% estearic (18:0), 8.01 wt% oleic (cis-18:1), 6.85 wt% elaidic (trans-18:1), 4.80 wt% linoleic (18:2), 0.85 wt% gadoleic (20:1), 5.26 wt% eicosatetraenoic (20:4) and 20.8 wt% eicosapentaenoic (20:5) acids.

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Hydrolysis was performed on the algal biomass at 100 °C for 240 min in the presence of 50 % w/w water and sulfuric acid concentrations of 40 and 60 % w/w relative to the biomass. After the hydrolysis was complete, esterification was investigated in the same experimental conditions using H2SO4 as the catalyst (10 % catalyst relative to the crude fatty acid mass) for the conversion of fatty acids into fatty acid alkyl esters. The FFA yields from hydrolysis using 40 and 60 % H2SO4 catalyst in 50 % w/w water, based on the dry biomass, were 8.6 ± 0.3 and 8.5 ± 0.2 %, respectively (Fig. 5, entries 1 and 2, black bars). The FAME yields obtained from these experiments after the esterification process were 4.8 ± 0.3 and 5.1 ± 0.2 % relative to the initial biomass, respectively (Fig. 5, entries 1 and 2, gray bars). The FAME profiles from Nannochloropsis oculata were also established using the direct transesterification process from dried biomass in the presence of sulfuric acid in methanol. These results were compared with the profiles obtained using the hydrolysis–esterification process. The direct transesterification was performed at 100 °C in methanol for 240 min using H2SO4 as the catalyst (20 % acid relative to the dry biomass). The in situ transesterification yielded 5.7 ± 0.5 % FAME from the dry biomass (Fig. 5, in situ process, gray bar). These findings show that the direct transesterification from the Nannochloropsis oculata biomass results in higher FAME yields than the hydrolysis–esterification process. In this case, however, the difference in the FAME yields was lower between the two processes compared with those results observed for the hydrolysis–esterification (Fig. 3, entry 5, gray bar) and

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direct transesterification processes (Fig. 4, in situ process, gray bar) using the Chlorella sp. biomass under the same experimental conditions. The significant differences in the observed FAME yields most likely resulted from the unique interactions between the cell walls of different microalgae and the conditions for each method employed. The fatty ester profiles from Nannochloropsis oculata obtained from both the hydrolysis–esterification procedure, using 40 and 60 % H2SO4 catalyst in 50 % w/w water, and the direct transesterification process (in situ) are shown in Table  2. These results show that the hydrolysis–esterification and in situ processes produced similar profiles of the fatty acid methyl esters. However, the results revealed a significant difference between the fatty acid profile obtained from the lipid extracts using a chloroform:methanol mixture (2:1 v/v) as the extractor solvent and the profile of the methyl esters obtained by the hydrolysis–esterification and direct transesterification processes for the microalga Nannochloropsis oculata. The percentages of certain fatty acids changed after the hydrolysis–esterification and in situ processes compared with those in the lipid extracts when using chloroform:methanol; for example, there was a lower concentration of C16:0 and C20:5 fatty acids following both processes. This result is not thought to be due to the degradation of FFA by the hydrolysis–esterification and direct transesterification conditions. It is more likely that the significant differences in the observed fatty acid profiles resulted from interactions between the Table 2  Profile of the fatty acid methyl esters (%) obtained from the microalga Nannochloropsis oculata by the hydrolysis–esterification and in situ processes Entry

Fatty estera

Hydrolysis– esterification (40 % catalyst, 50 % water)

Hydrolysis– esterification (60 % catalyst, 50 % water)

In situ process

1 2 3 4 5

C14:0 C16:0 C16:1 C18:0

7.29 19.04 14.57 4.17 8.44

6.88 18.10 15.82 4.28 8.41

7.28 17.35 14.99 4.33 7.85

6

Fig. 5  Yields of the crude free fatty acids (FFA) and fatty acid methyl esters (FAME) from Nannochloropsis oculata by the hydrolysis–esterification process (entries 1 and 2) using 50 % water and H2SO4 as a catalyst (40 and 60 % w/w relative to the biomass) and in situ processes

cis-C18:1

7 8 9

trans-C18:1 C18:2 C20:1 C20:4

10

C20:5

7.5

6.64

6.72

5.91 3.57 7.09

5.3 3.79 7.49

5.38 4.09 7.76

15.51

15.06

14.65

a  Tetradecanoic acid (myristic, C14:0), hexadecanoic (palmitic, C16:0), 9-hexadecenoic (C16:1), octadecanoic (stearic, C18:0), cis9-octadecenoic (oleic, C18:1), trans-9-octadecenoic (elaidic, C18:1), 9,12-octadecadienoic (linoleic, C18:2), n-11-eicosenoic (gadoleic, C20:1), 5,8,11,14-eicosatetraenoic (arachidonic, C20:4) and 5,8,11,14,17-eicosapentaenoic (EPA, C20:5)

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different solvents used in the lipid extraction, hydrolysis– esterification and in situ processes.

Conclusions This study has examined the hydrolysis–esterification process for moist biomass from the commercially available Chlorella sp. and Nannochloropsis oculata grown in batch culture tanks. The experimental results reveal that maximum yields of FAME from Chlorella sp. and Nannochloropsis oculata were obtained using 50 % w/w water relative to the initial biomass. Despite the presence of both unsaturated and polyunsaturated fatty acids from the microalgal lipids in the reaction medium, no further degradation of the FFA under the hydrolysis–esterification conditions was observed compared with the lipid extracts or the extraction–transesterification and direct transesterification processes. This was reasoned in accordance with the fatty acid methyl ester profiles observed. Acknowledgments  The authors would like to thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Petrobras/Centro de Pesquisas e Desenvolvimento Leopoldo Américo Miguez de Mello (CENPES) for their financial support. Fellowships from CNPq are also acknowledged.

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