• International Journal of Technology (IJTech)
  • Vol 15, No 4 (2024)

Performance of In-Situ Stirring Batch Reactor Transesterification of Nannochloropsis sp Microalgae into Biodiesel

Performance of In-Situ Stirring Batch Reactor Transesterification of Nannochloropsis sp Microalgae into Biodiesel

Title: Performance of In-Situ Stirring Batch Reactor Transesterification of Nannochloropsis sp Microalgae into Biodiesel
Yustia Wulandari Mirzayanti, Lenny Marlinda, Hery Irawan, Muhammad Al Muttaqii, Zuhdi Ma'sum, Nyoman Puspa Asri, Jia-Ming Chern

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Cite this article as:
Mirzayanti, Y.W., Marlinda, L., Irawan, H., Al Muttaqii, M., Ma'sum, Z., Asri, N.P., Chern, J.-M., 2024. Performance of In-Situ Stirring Batch Reactor Transesterification of Nannochloropsis sp Microalgae into Biodiesel. International Journal of Technology. Volume 15(4), pp. 859-869

Yustia Wulandari Mirzayanti Department of Chemical Engineering, Faculty of Technology Industrial, Institut Teknologi Adhi Tama Surabaya, Surabaya 60117, Indonesia
Lenny Marlinda Department of Industrial Chemistry, University of Jambi, Jambi 36361, Indonesia
Hery Irawan Department of Mechanical Engineering, Faculty of Technology Industrial, Institut Teknologi Adhi Tama Surabaya, Surabaya 60117, Indonesia
Muhammad Al Muttaqii Research Center for Chemistry, Badan Riset dan Inovasi Nasional, South Tangerang 15314, Indonesia
Zuhdi Ma'sum Department of Chemical Engineering, University of Tribhuwana Tungga Dewi, Malang 65144, Indonesia
Nyoman Puspa Asri Department of Food Technology, University of Ciputra, Surabaya, 60219, Indonesia
Jia-Ming Chern Department of Chemical Engineering and Biotechnology, Tatung University, Taipei, 10491, Taiwan
Email to Corresponding Author

Performance of In-Situ Stirring Batch Reactor Transesterification of Nannochloropsis sp Microalgae into Biodiesel

In-situ stirring batch reactor transesterification is a widely used method for the conversion of various biomass into biodiesel, particularly Nannochloropsis sp microalgae. Therefore, this study aims to investigate the effect of CaO/Hydrotalcite catalyst loading ratio and stirring rate on in situ stirring batch reactor transesterification of Nannochloropsis sp microalgae to produce biodiesel. In-situ transesterification was carried out using stirring with a four-blade paddle (50 w), while CaO/Hydrotalcite catalyst preparation was performed using incipient wetness impregnation. Nannochloropsis sp microalgae were then converted into biodiesel with varying loading ratio of catalyst (1:1, 3:1, and 5:1 %wt/wt) and stirring speed of agitation (50, 100,150, 200, and 250 rpm) for 4 hours (60oC). Subsequently, biodiesel obtained from the process was analyzed using gas chromatography (GC). The results showed that the surface area of CaO/Hydrotalcite was 45.756 m2/g. The conversion yields of Nannochloropsis sp microalgae to biodiesel with CaO/Hydrotalcite reached 38.36% with an agitation rate of 250 rpm. Based on the fatty acids analyzed using GC methods, the products predominantly contained olefine (61.49%) and gasoline (38.51%). 

Biodiesel; CaO/Hydrotalcite; In-Situ transesterification; Nannochloropsis sp.


Renewable energy, such as biofuels, is a valuable alternative to fossil fuels due to its beneficial properties. In addition, biofuels are classified into generations based on the raw materials used during their synthesis, including edible and non-edible oils, biomass, lignocellulose, and residues (Lomeu et al., 2023; Asri et al., 2021; Budianto et al., 2018). Among the raw materials, microalgae are biomass with the potential to serve as a feedstock, primarily due to their high lipid content, accelerated growth, and higher photosynthetic biomass yield compared to land plants (Larida and Bañaga, 2021; Shirazi et al., 2020). In line with previous studies, microalgae primarily comprise starch, proteins, lipids, triglycerides, and fatty acids, particularly for biofuels production. These algae also offer various potential benefits over lignocellulosic source crops due to the absence of competition for agricultural land with other food crops. However, a notable challenge lies in their rigid cell wall structure, which typically requires solvent for extraction compared to vegetable oils (Sardi et al., 2022; El-Sheekh, 2021; Purkan et al., 2021; Culaba et al., 2020; Cercado, Ballesteros, and Capareda, 2018). In response to this challenge, several technologies have been developed for the conversion of microalgae biomass and are classified into different types, including biochemical conversion, thermochemical conversion, chemical reaction, and direct combustion.

According to previous studies, biodiesel is predominantly produced using transesterification method, which comprises mixing lipid (triglyceride) and alcohol, with catalysts being used to facilitate the synthesis of ester and glycerol. High free fatty acids (FFA) cannot be directly tranesterified using an alkaline catalyst, which gives low yield and low quality of biodiesel (Wahyono et al., 2022). The best biodiesel production method typically comprises the use of lipid with carbon chain length ranging from saturated C14 to C22, reduced unsaturated free fatty acids groups, and lower saturation states. This is primarily because the use of high unsaturation levels can negatively affect the crucial aspect of the product obtained (Koech, Kumar, and Siagi, 2020). Consequently, several reports have explored the use of Nannochloropsis sp., which has high oil content and can serve as a suitable green and low-impact environmental alternative fuel. This organism has also been reported to possess various advantages, such as biodegradability, nontoxicity, renewability, and safety.      

Several studies have explored the conventional conversion of Nannochloropsis sp. microalgae to biodiesel using extraction with solvents, followed by transesterification process (Ghedini et al., 2022; Makareviciene and Sendzikiene, 2022; Purkan et al., 2021; Wood, 2021;Kazemifard et al., 2019; Salam et al., 2016). For example, a previous investigation used NiO nanocatalyst for in-situ transesterification synthesis of biodiesel from Nannochloropsis oculata, consisting of 2 stages, including in-situ extraction and transesterification (Purkan et al., 2021; Salam et al., 2016). However, this conventional method typically requires a high conversion cost. To overcome the limitation, various reports have proposed the use of in-situ transesterification, where extraction and esterification processes are combined to save time and enhance efficiency. In this method, the alcohol compound serves as both a solvent to extract lipids and a reactant for esterification reaction. In addition, co-solvents and catalysts can also be added to facilitate rate of transesterification reaction. Despite the potential, the effect of stirring rate parameters (Purkan et al., 2021) on biodiesel yield has not been explored (Salam, Velasquez-Orta, and Harvey, 2016). An in-depth understanding of the impact of mixing on transesterification process has been reported to be an essential parameter in the scaling-up and design of reactors (Tabatabaei et al., 2019). Industrial agitators and mixers for biodiesel production process often include anchor agitators, helix mixers, high-shear mixers, magnetic stirrers, mechanical stirrers, turbines agitators, micro-mixers, motionless or static mixers, two flat-blade paddle agitators, and ultrasonic mixers.   

       Heterogeneous catalyst are gaining more interest because its have advantages over homogeneous catalysts (Aisyah et al., 2023; Rahma and Hidayat, 2023; Cercado, Ballesteros, and Capareda, 2018). Based on previous studies, calcium oxide (CaO) is a base heterogeneous catalyst with good potential for chemical reaction, particularly transesterification reaction (Zul et al., 2021). Catalyst has also been shown to have high alkalinity, low solubility, and can easily be separated from the product. However, CaO has a weak catalytic activity, leading to reduced product yield. This is because the oxygen ions (O2-) on its surface can quickly form hydrogen bonds with methanol or glycerol, neccesitating the support of an active metal, such as hydrotalcite. According to previous studies, hydrotalcite is a hydroxycarbonate form of magnesium and aluminum with the molecular formula Mg6Al2(OH)16CO3.4H2O and is also a derivative of Mg(OH)2 (Mirzayanti, Alisa, and Sari, 2020a). The use of this compound as catalyst in transesterification reaction has the potential to increase biodiesel yield (Zhang et al., 2019). Therefore, it is necessary to combine CaO with hydrotalcite to improve its catalytic performance.   

        The novelty in this current study lies in the use of a metal catalyst (CaO/Hydrotalcite) to convert Nannochloropsis sp microalgae into biodiesel using in-situ transesterification method as well as exploring the effectiveness of the agitation rate parameters on reactor batch in-situ transesterification. CaO is an alkali metal oxide with various advantages, while hydrotalcite is a heterogeneous catalyst that can accelerate transesterification reaction. The investigation primarily focuses on the performance of the combination of CaO metal and hydrotalcite as catalyst. Reactor's design was in-situ stirred batch reactor with a unique design comprising a four-blade paddle. Catalyst used was an alkali metal, which comprised the combination of CaO/Hydrotalcite heterogeneous catalyst and methanol as a solvent and a reactant in in-situ transesterification process, with n-hexane serving as a co-solvent. Therefore, this study aims to determine the effect of CaO/Hydrotalcite loading ratio and stirring rate in converting Nannochloropsis sp. microalgae. 

Experimental Methods

This section described the principal materials and the methodologies used in the experimental procedures. 

2.1.    Material, Equipment, and Apparatus

Nannnochloropsis sp microalgae were obtained from the Brackish Water Aquaculture Fisheries at Jepara, Central Java, Indonesia. Meanwhile, calcium carbonate, methanol, n-hexane (Merck & Co, 99%), and hydrotalcite synthetic (Sigma Aldrich, 99%) were purchased from an Indonesian supplier. The major equipment was in-situ stirred batch reactor (material: stainless steel type 304), vertical cylindrical shape equipped with a stirrer. The top and bottom covers were flange-shaped, with a total tank volume of 20 liters and a stirrer of 50 w (four-blade paddle) (Figure 1a). Figure 1b illustrated a stirred tank batch reactor with dimensions of outside diameter (do) = 7.48 in; cylinder height (Ls) = 7.48 in; cylinder thickness (ts) = 3/16 in; and leg support (l) = 20 in (Scale 1:1). The heating circulation had a maximum speed of 500 rpm, a maximum temperature of 250oC, and H of 20.5 in (scale = 1:1).  

2.2.    Synthesis and Characterization of CaO/Hydrotalcite

The incipient wetness impregnation method was used for the synthesis of the CaO/Hydrotalcite catalyst, which was placed in an oven for 12 hours at 100oC. The calcium carbonate (CaCO3) was calcined into CaO for 3 hours at 900oC and then weighed with loading ratio of CaO to hydrotalcite (1:1, 3:1, and 5:1 wt/wt). Subsequently, CaO was dissolved in distilled water, and the solution was sprayed onto hydrotalcite slowly and evenly until it seeped into hydrotalcite pores at room temperature. The sample was dried at 100oC for 12 h and treated in the furnace at 900oC for 3 hours. The final product was CaO/Hydrotalcite catalyst powder, which was characterized using X-ray diffraction (XRD), X-ray Fluorescence (XRF), and Brunaur Emmett and Teller (BET) to investigate the surface area. The characterization techniques of XRD investigations were performed using instruments possessing Cu K-Alpha source, 40 kV and 30 mA generator settings, and no beam monochromator. 

Figure 1 a) In Situ stirred batch reactor transesterification, b) stirred tank batch reactor, and c) heat circulation

2.3.   In-Situ Transesterification Reaction

The experimental setup consisted of in-situ stirred batch reactor, as shown in Figure 1. The weighed amount of Nannochloropsis sp microalgae biomass was mixed with CaO/Hydrotalcite catalyst (10%wt of microalgae) and methanol. The mixture obtained was then blended for 4 hours at 60oC with varying agitation rates of 50, 100, 150, 200, and 250 rpm. After the reaction had occurred, it was cooled to separate biodiesel from glycerol and suspended solids. Subsequently, the filtered residue was washed using 30 mL of a mixture of methanol-n-hexane (1:1 v/v) to obtain fatty acids methyl ester (FAME). The filtrate was separated by adding n-hexane with a 1:1 (v/v) ratio, shaken, and allowed to stand until 2 layers formed for 30 minutes. The bottom layer was then removed from the separating, and n-hexane was added with a 1:1 (v/v) ratio. The solution was shaken and allowed to stand until 2 layers formed for 8 hours. The top layer was washed to remove any catalyst and glycerol in the mixture. The product obtained was distilled (70oC) to separate FAME and solvent. Gas Chromatography-Mass Spectrometry (GC-MS) was used to analyze biodiesel for detected fatty acids in higher yields in all variables. The peak area from GC-MS data could be read by the normalization method based on the peak area per percentage of identified components used to measure the GC-MS results. All hydrocarbon components were identified using Wiley275 and NIST02 mass spectral data libraries. Hydrocarbon components with a probability equal to or above 80% were considered in line with previous studies (Marlinda et al., 2022; Mirzayanti et al., 2020b). This current study optimized the process variables for the highest yield, namely the effectivity of the speed of stirring conversion to crude biodiesel and loading of CaO/Hydrotalcite catalyst by microalgae, density, acid number, and %FFA. The limitation of this report was that it solely focused on the effect of the CaO and hydrotalcite loading ratio and stirring speed on yield and FFA. The best yield obtained was subjected to GC-MS testing to determine the content of hydrocarbon compounds produced.     

2.4.   Analysis of Crude Biodiesel

Analysis and determination of the %yield of crude biodiesel was calculated with the formulations presented below (Equation 1)

where MB is the mass of crude biodiesel (g); and MA is microalgae (g) mass. Compound analysis biodiesel components (ASTM D4007) were analyzed using GC-MS.

Results and Discussion

3.1. Characteristics of Nannochloropsis sp Microalgae

        Characteristics of lipid of liquid Nannochloropsis sp microalgae biomass are presented in Table 1. The results showed that the highest fatty acids content in Nannochloropsis sp microalgae was oxygenated compounds (51.80%), followed by carboxylic acid (46.61%). In addition, oxygenated compounds were hydrocarbons containing at least 1 oxygen atom as a part of their chemical structure, and are commonly referred to as fuels. The 4 general classes of hydrocarbons included alkanes, alkenes, alkynes, and arenes. Carboxylic acid, in organic chemistry, is an organic acid containing a carboxyl group (C(=O)OH) attached to an R-group. This compound was characterized by its carbon (C) atom being bonded to an oxygen (O) atom by a double bond as well as to a hydroxyl group (?OH) by a single bond. Based on Table 1, Nannochloropsis sp microalgae could be used as raw materials for biodiesel.

Table 1 Fatty acid content in Nannochloropsis sp microalgae








Oxygenated compounds



Carboxylic acid


        The total lipid content of microalgae biomass largely depended on the specific cultivation technology. Microalgae culture condition, nutrients, and light intensity could be optimized to increase the oil content, leading to enhanced biodiesel production. In addition, the growth rate of this organism and maximum biomass production depended on their culture systems (light, temperature, pH, salinity, consumption of O2, CO2, nutrients, and toxic chemicals) (Medipally et al., 2015). According to previous studies, nutrients for microalgae growth included nitrogen (N) and/or phosphorus (P) (Kazemifard et al., 2019). Nannochloropsis sp microalgae could serve as biodiesel due to the abundance of its oleic acid content (73.40%) among the remainder of the MUFA composition (Kanagesan et al., 2020). The specific growth rate of Nannochloropsis sp microalgae strains generally ranged from 0.11 to 0.21 per day, with total lipid content of 37-60% of dry weight (DW) (Ma et al., 2016).    

3.2. Catalyst Characterization

        Figure 2 showed a series of XRD diffractions of CaO/Hydrotalcite. The initial incipient wetness impregnation at 900oC in 3 hours predominantly contained CaO and MgO with few amounts of Al2O3. In addition, the XRD pattern of CaO/Hydrotalcite catalyst had related curves in terms of uniformity. The peaks obtained were compared with the Joint Committee on Powder Diffraction Standards (JCPDS) file. From Figure 3, the principal components of CaO/Hydrotalcite catalyst included CaO, Ca2Al, and MgO. Diffraction 2? from JCPDS for using CaO was 32.2o, 37.3o, and 53.8o. Therefore, the pattern in Figure 2 showed the intensified and narrow peaks at 2 = 17.88o, 28.47o, 33.92o, 42.88o, 47.07o, and 50.60o, as reported in a previous study (Mohamad et al., 2018). Catalyst characterization also used XRF analysis to determine the chemical compositions. The elemental chemical compositions of catalyst in this study are presented in Table 2. According to the XRF, the CaO content of the various ratio of CaO with hydrotalcite (1:1, 3:1, and 5:1 %wt/wt) increased gradually. The highest amount of CaO was obtained at ratio of 5:1 %wt/wt (84.13 %m/m), as shown in Table 2. The results suggested that the decomposition of carbonates was achieved at 900oC for approximately 3 hr. In a previous study, the majority of variation ratio catalyst above 50% were CaO species (Amusan et al., 2019). The total content of MgO and Al2O3 was below 50% because hydrotalcite was decomposed into metal oxides when calcined at 900oC for 3 hours (Amusan et al., 2019; Mohamad et al., 2018).

        The surface area of CaO/Hydrotalcite particles synthesized to catalyst (obtained by calcination at 900oC for 3 h) from BET and BJH was 45.756 m2/g. Furthermore, the surface area of the obtained catalyst showed good activity for converting Nannochloropsis sp oil into biodiesel. This result was consistent with a previous study, which distributed CaO or K2CO3 in various support materials (Manurung et al., 2023; Zhang et al., 2019). Navajas et al. (2018) also reported the best results for hydrotalcite activity as a heterogeneous methanolysis catalyst. These outcomes were attributed to the high basicity properties of the solid that was subjected to the rehydration process after calcination with a surface area of 12 and 66 m2/g. Apart from the strong basic sites, the presence of interlayer hydroxide anions at the accessible crystal edges enhanced catalyst's activity. However, hydrotalcite synthesized using a variety of processes had a surface area of 131-153 m2/g at pH 10 and 150oC (Navajas et al., 2018). This result was better than previous studies, which obtained an average value of 34.6 m2/g (Anr et al., 2017). The CaO/Hydrotalcite area was more significant compared to a previous study at 4.34 m2/g (Win and Khine, 2017).

Figure 2 XRD diffractogram of CaO/Hydrotalcite calcined at 900oC for 3 h (5:1 %wt/wt)

Table 2 XRF analysis of CaO/Hydrotalcite at 900oC for 3 hours



%Massa (m/m)







































3.3. Catalytic Performance for Biodiesel Production

        In-situ stirring batch reactor transesterification reaction was performed using CaO/Hydrotalcite, and the reaction conditions were investigated by varying the different parameters. The effect of stirring speed and CaO loading on transesterification was also studied in the reaction.  

3.3.1.  Effect of stirring rate and CaO loading on %yield crude biodiesel

        Figure 3a presents stirring speed of the different CaO/Hydrotalcite ratio of CaO/Hydrotalcite catalyst. In addition, the highest yield of crude biodiesel at 250rpm was obtained with a 1:1 wt/wt ratio of catalyst (38.36%). A high mixing must be used during in situ transesterification to obtain high FAME and yield (Salam, Velasquez-Orta, and Harvey, 2016). The results showed that the yield increased with an increase in mixing speed. The process showed that the mixing speed at 250 rpm was 38.36%. Figure 3 also showed the presence of an anomaly in the decrease of %yield crude biodiesel of CaO/Hydrotalcite catalyst (1:1 %wt/wt; 150 rpm and 3:1 %wt/wt; 100 and 200 rpm). Loading ratio of CaO/Hydrotalcite catalyst at 1:1 produced the highest yield. This was due to the best metal composition in the 1:1 %wt/wt composition. When catalyst loading ratio was increased (5:1 %wt/wt), the results showed a decrease in %yields, as reported in previous studies (Koech, Kumar, and Siagi, 2020). The outcome observed was caused by interaction with other molecules/compounds, leading to increased by-products. The high catalyst concentration led to a corrosive nature, which hindered transesterification reaction. 

Figure 3 Effect of stirring speed (a) on %yield crude biodiesel based on the effect of CaO/Hydrotalcite loading ratio (b) on FFA crude biodiesel based on the effect of CaO/Hydrotalcite loading ratio    

3.3.2.  Effect of stirring rate and CaO loading on %FFA crude biodiesel

       Figure 3b showed that the lowest FFA in loading ratio CaO/Hydrotalcite of 1:1 %wt/wt (250 rpm) was 1.39%. In addition, the higher stirring speed, the higher the level of decrease in %FFA. The results also showed that stirring rate had a significant effect on the FFA of crude biodiesel, where the higher stirring rate, the smaller the FFA observed. The acid number obtained affected the quality of biodiesel, and the higher the acid number in biodiesel, the lower its quality. This parameter could also affect the shelf life and the level of corrosiveness of the engine. The results showed that high acid numbers created deposits in the fuel system and reduced the quality of fuel system components.

3.4. Properties of Nannochloropsis sp Microalgae Biodiesel

        Table 3 presented the physicochemical characteristics of Nannochloropsis sp microalgae biodiesel. The properties showed that Nannochloropsis sp microalgae could be used as biodiesel raw material. According to a previous study, biodiesel obtained from this species had a density quality of 0.839 g/m3 (Koech, Kumar, and Siagi, 2020; Salam, Velasquez-Orta, and Harvey, 2016). The results in Table 3 confirmed the good characteristics of Nannochloropsis sp microalgae for conversion into biodiesel.    

Table 3 Physicochemical properties Characteristics of Nannochloropsis sp Microalgae Biodiesel  



Test method


Values of algae Biodiesel


Density (g/cm3)

SNI 7182:2015

0.850 – 0.890



Acid value (KOH/g)

ASTM D 664

max. 0.5



Free Fatty Acid value


max. 2


        Based on the results of data processing of GC-MS, Nannochloropsis sp microalgae biodiesel using CaO/Hydrotalcite consisted of toluene (61.49%area) and Octane (38.51%), as shown in Table 4. Toluene was a hydrocarbon compound belonging to the olefin/alkenes group with a closed chain/aromatic type. Several studies had shown that it had the molecular formula of C6H5CH3 and was a derivative of benzene compounds, namely methylbenzene. Meanwhile, octane was a hydrocarbon compound with an open/aliphatic chain type in the Alkane group. According to previous reports, it had the molecular formula of C8H18 was a compound belonging to the alkane group. Petroleum fractions were classified based on the amount of carbon in the hydrocarbon bond chain where C4-C12 was included in gasoline, C10-C16 was kerosene, and C12-C22 was categorized as biodiesel. Olefins contained in biofuels were C7 hydrocarbons, and constituents were hydrocarbons with the number of carbon chains C8 included in one of the gasoline, which had a range of carbon numbers C4-C12 (Salam, Velasquez-Orta, and Harvey, 2016).

        Transesterification process in the presence of CaO occurred through various steps. Protons (H+) were extracted from CH3OH at the CaO base site to form CH3O-. In triglyceride compounds, the carbon atom of the carbonyl functional group was attacked by CH3O- to produce the alkoxy carbonyl compound, which was rearranged into FAME (the desired product) and the diglyceride anion. Subsequently, the diglyceride anion attacked the calcium hydroxide cation to form diglyceride and CaO, leading to the recovery of catalyst. Based on the results and previous studies, CaO/Hydrotalcite catalyst was a solution to obtain catalyst system that combined the characteristics of a bimodal pore distribution and micropores. In addition, it provided basic sites and high surface density along with the presence of a suitable macro or mesopore network responsible for triglyceride transport. This allowed rapid access to the active site from the bulk reaction medium and removal of products (glycerol and FAME) from catalyst (Nisar et al., 2021). The reaction mechanism comprising the use of CaO is presented in Figure 4.

Figure 4 A chemical reaction mechanism involving CaO

Table 4 Nannochloropsis sp microalgae biodiesel compounds using GC-MS analysis  



Retention Time











In conclusion, Nannochloropsis sp microalgae was subjected to in situ extraction-transesterification with methanol (solvent) and n-hexane (co-solvent) using CaO/Hydrotalcite catalyst. The highest yield obtained was 38.36%, with an FFA content of 1.39% (250 rpm; acid number 7.63 mg NaOH/g sample, and density 0.908 g/cm3). The GC-MS analysis showed the presence of toluene-like olefin (61.49%) and octane-like gasoline (38.51%)s group, showing the effectiveness of CaO/Hydrotalcite catalyst in converting Nannochloropsis sp microalgae to biodiesel using in situ stirring batch reactor transesterification.  


    The authors are grateful to the Ministry of Education, Culture, Research, and Technology for supporting this study in the Regular Fundamental Research Project. The number of contract was 183/E5/PG.02.00.PL/2023; 074/SP2H/PT/LL7/2023.


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