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 (60^oC). 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.

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 (d_o) = 7.48 in; cylinder height (L_s) = 7.48 in; cylinder thickness (t_s) = 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 250^oC, 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 100^oC. The calcium carbonate (CaCO₃) was calcined into CaO for 3 hours at 900^oC 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 100^oC for 12 h and treated in the furnace at 900^oC 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 60^oC 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 (70^oC) 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 M_B is the mass of crude biodiesel (g); and M_A is microalgae (g) mass. Compound analysis biodiesel components (ASTM D4007) were analyzed using GC-MS.

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

        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 900^oC 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 32.2^o, 37.3^o, and 53.8^o. Therefore, the pattern in Figure 2 showed the intensified and narrow peaks at 2 = 17.88^o, 28.47^o, 33.92^o, 42.88^o, 47.07^o, and 50.60^o, 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 900^oC 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 900^oC 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 900^oC 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 150^oC (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 900^oC for 3 h (5:1 %wt/wt)

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

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  

        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  

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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