Cell Disruption of <i>Chaetoceros calcitrans</i> by Microwave and Ultrasound in Lipid Extraction

Nogueira, Daniela Almeida; da Silveira, Juliane Machado; Vidal, Évelin Mendes; Ribeiro, Natália Torres; Veiga Burkert, Carlos André

doi:https://doi.org/10.1155/2018/9508723

International Journal of Chemical Engineering

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Abstract Introduction Materials and Methods Results and Discussion Conclusion Data Availability Conflicts of Interest Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2018 | Article ID 9508723 | https://doi.org/10.1155/2018/9508723

Cell Disruption of Chaetoceros calcitrans by Microwave and Ultrasound in Lipid Extraction

Daniela Almeida Nogueira,¹Juliane Machado da Silveira,¹Évelin Mendes Vidal,¹Natália Torres Ribeiro,¹and Carlos André Veiga Burkert¹

Academic Editor: Xunli Zhang

Received22 Feb 2018

Revised03 Jul 2018

Accepted25 Jul 2018

Published02 Sept 2018

Abstract

Downstream processing, such as cell disruption and extraction, constitutes a key step in microalgal-based industrial bioprocesses, mainly due to high costs and environmental impact. In this context, extraction technologies need to be improved, including the use of nonconventional cell disruption techniques suitable for scale-up, such as microwave and ultrasound. Therefore, this study aimed at investigating the effects of different methods of cell disruption (microwave and ultrasound) on lipid extraction from biomass of the diatom Chaetoceros calcitrans cultured in mixotrophic conditions in a medium with natural sea water and residual glycerol, with different treatment times. Both techniques applied to the biomass were efficient; that is, the results were 24.6 ± 1.3% lipids (ultrasound for 5 min) and 24.2 ± 0.9% lipids (microwave for 40 s), with no significant differences between them (). Likewise, there was no significant difference regarding the chemical disruption with hydrochloric acid 2 M as control (24.2 ± 1.0%). The ultrasound method consumed less energy than the microwave method. Both cell disruption methods applied to the biomass resulted in changes in the fatty acid profiles, that is, percentages of saturated fatty acids increased from 7.7% (control) to 16.6% (microwave) and 15.5% (ultrasound), whereas polyunsaturated ones increased from 12.8% (control) to 22.8% (microwave) and 21.8% (ultrasound). Concerning monounsaturated fatty acids, percentages decreased from 79.5% (control) to 60.6% (microwave) and 62.7% (ultrasound).

1. Introduction

Population growth and the constant search for quality of life have led to greater demand for products obtained in sustainable ways. Microalgae stand out in the industrial sector due to their potential for yielding several metabolites, such as carbohydrates, pharmaceuticals, pigments, and enzymes [1, 2]. Furthermore, microalga biomass can easily be yielded in any season, since it neither depends on harvest periods nor competes with agriculture for arable land [3].

Several studies have focused on lipid production by microalgae, as some species have high contents of lipids, such as Chlorella sp. [4, 5], Phaeodactylum tricornutum [5–7], Tetraselmis suecica [7], Nannochloropsis oculata [5, 7], Isochrysis galbana [5, 7], and Chaetoceros calcitrans [5, 7]. Moreover, they may become a sustainable source to be used by both food and biofuel industries, depending on their contents of saturated and unsaturated fatty acids [7, 8]. The marine diatom Chaetoceros calcitrans, which belongs to the class Bacillariophyceae, stands out among the species of microalgae as a potential producer of intracellular lipids [9]. Diatoms are peculiar microalgae not only because they do not have any flagella but also because their cell wall is composed of overlapping halves called frustules, formed by polymerized opaline silica [10].

In the extraction of lipids from microalgae, cell disruption techniques must be carefully chosen, since lipids are yielded intracellularly. The method of cell disruption should be effective in order to liberate the intracellular lipids from microalgal cells and take into account the amount of energy needed for the process [11]. However, in general, conventional techniques (i.e., mechanical, chemical, and thermal/thermochemical methods) used for cell disruption and extraction are expensive and are often hindered by low efficiency levels [12]. Regarding emerging cell disruption techniques, treatments that use ultrasound and microwave have been viewed as promising ones because they are simple, easy, and efficient methods of lipid extraction that require little time and maintain the quality of the extracts [13–16]. For example, Menéndez et al. [17] verified that lipid extraction from Nannochloropsis gaditana biomass with microwave and ultrasound has many advantages, requiring a lower amount of solvent, lower energy consumption, and a shorter extraction time, leading to a lower environmental impact of the process.

In cell disruption by ultrasound, the phenomenon called cavitation forms gas microbubbles that grow and collapse violently [18]. As a result, the pressure and temperature of the adjacent tissue increase and cause the disruption of the cell wall, release soluble compounds, improve mass transference, and enable the solvent to access the cell content [16, 19]. In cell disruption by microwave, nonionizing electromagnetic waves with a frequency of 300 MHz to 300 GHz are used, and electromagnetic energy is converted into calorific energy by two mechanisms: ionic conduction and dipole rotation [18]. Efficiency of disruption by ultrasound and microwave usually depends on the cell structure, the lipid content, and the microwave and ultrasound conditions [14, 16, 19]. However, despite these advantages, there is a lack of information on these techniques applied to Chaetoceros calcitrans biomass.

Therefore, this study aimed to evaluate techniques of cell disruption by microwave and ultrasound in the recovery and profile of fatty acids of lipids from Chaetoceros calcitrans cultured in mixotrophic conditions.

2. Materials and Methods

The marine microalga Chaetoceros calcitrans, which was used in the experiments, was donated by the Laboratório de Biologia Marinha e Biomonitoramento (LABIOMAR), a laboratory that belongs to the Universidade Federal da Bahia (UFBA), located in Salvador, Bahia, Brazil. Residual glycerol (82.09% purity) was supplied by BSBIOS Indústria e Comércio de Biodiesel Sul Brasil S/A, a company located in Passo Fundo, Rio Grande do Sul, Brazil. It is the byproduct of the alkaline-catalyzed transesterification of soybean oil and methanol.

The Conway medium [20] was used for the inoculum (10% of final volume), and it was prepared with sterile sea water with the addition of saline solution (2 mL·L⁻¹), vitamin solution (0.1 mL·L⁻¹), and silicate solution (2 mL·L⁻¹). The saline solution contained 45 g·L⁻¹ C₁₀H₁₄O₈Na₂·2H₂O (Na₂EDTA), 33.6 g·L⁻¹ H₃BO₃, 100 g·L⁻¹ NaNO₃, 0.36 g·L⁻¹ MnCl₂·4H₂O, 1.3 g·L⁻¹ FeCl₃·6H₂O, 20 g·L⁻¹ NaH₂PO₄·2H₂O, and 1 mL·L⁻¹ of trace metals solution containing 21 g·L⁻¹ ZnCl₂, 20 g·L⁻¹ CoCl₂·6H₂O, 9 g·L⁻¹ (NH₄)₆Mo₇O₂₄·4H₂O, and 20 g·L⁻¹ CuSO₄·5H₂O. The vitamin solution contained 0.05 g·L⁻¹ vitamin B12 and 1 g·L⁻¹ vitamin B1, and the silicate solution contained 40 g·L⁻¹ sodium silicate. The sea water was collected at the Marine Aquaculture Station (FURG), located at the Cassino Beach (Rio Grande, Brazil). It was previously filtered on a qualitative filter paper, and the salinity was adjusted to 28 PSU (practical salinity units) using a manual salinometer (Biobrix, Model 211, Brazil). The sea water, the saline solution, and the silicate solution were sterilized by autoclaving at 121°C for 15 min. The vitamin solution was sterilized separately by filtration through a 0.22 μm filter.

In cultures aimed at biomass to be used in cell disruption assays, the microalga was cultured in 2 L Erlenmeyer flasks with 1800 mL Conway medium, in accordance with Walne [20], with modifications. The medium was prepared with the addition of 5.61 g·L⁻¹ residual glycerol, and concentrations of silicates and sodium nitrate in the solutions were modified to 70 g·L⁻¹ and 50 g·L⁻¹, respectively, as recommended by previous studies (unpublished data).

The inoculum (10% of final volume) containing 0.55 g·L⁻¹ of biomass was added to the flasks, and these were placed in an incubator with a photoperiod (Eletrolab EL-202, Brazil), on a 12 h light/dark cycle, at 30°C. Light was provided by fluorescent light bulbs simulating natural daylight, with irradiance of 3000 lx. Atmospheric air was directly and constantly injected at a flow rate of 0.2 L·min⁻¹ by a pump and glass wool filter system to ensure air sterility. After 10 days, the biomass was recovered by centrifugation (18,800 ×g; 15 min), washed with distilled water, centrifuged again (18,800 ×g; 15 min), and lyophilized.

In cell disruption assays, portions of 0.3 g lyophilized biomass were used for testing: (1) chemical disruption (control), with the addition of 5 mL HCl 2 M, followed by immersion in a water bath for 1 h at 80°C; (2) disruption by microwave, with a frequency of 2.45 MHz and power of 1.4 kW (Brastemp, model BMS35BBHNA, Brazil), with the addition of 20 mL distilled water to the sample, in accordance with Lee et al. [14], and exposure times of 40, 105, and 300 s; (3) disruption by ultrasound, with a frequency of 20 kHz and power of 130 W (Cole Parmer, model CPX 130, USA), with the addition of 20 mL distilled water, in accordance with Lee et al. [14], and exposure times of 5, 10, and 20 min. At the end of the treatments, lipids were extracted and quantified, as proposed by Bligh and Dyer [21], with amounts of solvent recommended by Manirakiza et al. [22].

The consumption of energy (EC) (expressed as GJ·m⁻³) was estimated by using the following equation, based on Alagöz et al. [23]:where is the power, is the time treatment, and is the volume of the sample.

The specific energy (SE) was defined as the energy supplied per unit of mass of microalgae [23]:where is the concentration of cell suspension. In both equations, the corresponding units were converted properly.

Esterification of the lipid fraction extracted from the biomass was conducted according to the method adapted from Metcalfe et al. [24]. The fatty acid profile was determined by gas chromatography. In order to separate and quantify the fatty acid mixture, a gas chromatograph (Shimadzu, model 2010 Plus, Japan), equipped with a split/splitless injector, a capillary column RTX®-1 (30 m × 0.25 mm internal diameter × 0.25 μm particle diameter), and a flame ionization detector (FID), was used. Helium, at a flow rate of 1.25 mL·min⁻¹, was the carrier gas. Temperatures of the injector and the detector were adjusted to 260°C; the injected volume was 1 µL. The chromatographic conditions of separation were as follows: initial temperature of the column was 50°C, which was raised to 200°C at 6°C·min⁻¹, and maintained for 4 min. In the second temperature ramp, the temperature was raised to 240°C at 2°C·min⁻¹ and maintained for 10 min. Comparison between retention times and methyl ester standards was used to identify fatty acids, which were quantified by area normalization.

The assays were performed in triplicate, and the results were submitted to analysis of variance (ANOVA) and Tukey’s test at a 95% confidence level () or the t-test at a 95% confidence level (). Statistica 5.0 (Stat Soft Inc., USA) software was used.

3. Results and Discussion

3.1. Disruption by Microwave and Ultrasound

The content of lipids extracted after the biomass was submitted to ultrasound waves decreased significantly () when exposure time increased from 5 min to 10 min, with no significant differences () between 10 min and 20 min (Figure 1). Consequently, the best condition corresponded to the 5 min treatment of biomass by ultrasound, since total lipids reached 24.6 ± 1.3% and did not differ significantly () from the values of the chemical disruption (24.2 ± 1.0%). On the other hand, the longest exposure times led to significantly different () percentages of total lipids, in relation to the chemical disruption. Values decreased to 20.7 ± 1.7% (10 min) and 20.4 ± 0.3% (20 min).

The pretreatment using microwave for 300 s (Figure 1) resulted in the lipid content (16.3 ± 1.0%) differing significantly () from 105 s (22.3 ± 1.6%) and 40 s (24.2 ± 0.9%), which differed () neither from each other nor from the chemical disruption (24.2 ± 1%).

A comparison of both methods (Figure 1) led to the conclusion that they achieved the best results at the lowest exposure times, differing neither from each other (microwave for 40 s with 24.2 ± 0.9% lipids and ultrasound for 5 min with 24.6 ± 1.3% lipids) nor from the control (24.2 ± 1.0%). Decreases in the recovery of lipids from Chaetoceros calcitrans due to the increase in exposure times in both methods under evaluation may be associated with lipid degradation in drastic conditions of biomass treatment. According to Naghdi et al. [25], prolonged exposure to sonication can produce free radicals that may deteriorate the quality of lipids through oxidation, while Prommuak et al. [26] found that increased exposure time led to a decrease in lipid yield from Chlorella vulgaris and Haematococcus pluvialis caused by lipid oxidation. Furthermore, Kalil et al. [18] observed that treatments such as microwave and ultrasound could lead to the thermal degradation of some microbial compounds by temperature increase.

According to Lee et al. [14], efficiency in lipid extraction from microalgae depends on the species and the method of extraction. Viswanathan et al. [13] studied the effects of three methods of cell disruption (autoclave, ultrasound, and high-pressure homogenization) to recover lipids from a consortium of microalgae (Chlorella minutissima, Chlamydomonas globosa, and Scenedesmus bijuga) and found an increase in the lipid content, from 10.78% to 12.22%, when ultrasound was applied, in comparison with the sample with no treatment.

Lee at al. [14] evaluated five methods of cell disruption (autoclave, bead milling, microwave, ultrasound, and osmotic shock with 10% NaCl) when they studied the microalgae Botryococcus spp., Chlorella vulgaris, and Scenedesmus spp. They found that the highest lipid contents were achieved by microwave. However, in the case of the microalga Botryococcus spp., ultrasound yielded low values of lipid content (8.8%), evidence that different microalgae have distinct behaviors when disruption techniques are applied because of differences in the constitution of their cell walls. Kurokawa et al. [27] established the most effective ultrasound frequency for the cell disruption of Chaetoceros gracilis (2.2 MHz), Chaetoceros calcitrans (3.3 MHz), and Nannochloropsis spp. (4.3 MHz) based on the rate of algal cell disruption measured by hemocytometry.

Ma et al. [16] compared cell disruption of the microalga Chlorella spp. by microwave and ultrasound and showed that the former was faster and more efficient in cell disruption aiming at lipid extraction than the latter. The authors attributed the results not only to the high pressure that was generated but also to fast heating and humidity inside the cell in the microwave process, whereas in the ultrasound process, cells exploded due to the shock of bubbles formed by cavitation. In the work of Moura et al. [28], the microwave method was successfully applied to 4 different microalgae species in a shorter treatment time in comparison with the ultrasound method. This behaviour was similar to that in our results, since the same lipid extraction performance was observed for both methods. However, while the treatment time using the microwave method was 40 s, for ultrasound, it was 5 min.

Considering the above results, in relation to the best values for lipid recovery, the treatments with microwave for 40 s and ultrasound for 5 min were chosen to evaluate fatty acid profile and energy consumption.

3.2. Fatty Acid Profiles

Observation of fatty acids (Figure 2) shows that both treatments resulted in an increase in saturated fatty acids (SFAs) and polyunsaturated fatty acids (PUFAs). Concerning SFAs, palmitic acid had a 3.3-fold increase after disruption by microwave and a 3-fold increase after disruption by ultrasound, compared with the chemical disruption. Regarding PUFAs, linoleic acid (C18 : 2n9t) had approximately a 2.3-fold increase, whereas γ-linolenic acid (C18 : 3n6) had an increase of approximately 2.8-fold. In the case of α-linolenic acid (C18 : 3n3), the highest percentage was achieved by the chemical disruption. In terms of monounsaturated fatty acids (MUFAs), tetradecenoic acid (C14 : 1) was reduced by 28% when the biomass was submitted to microwave and by 21.6% when it underwent ultrasound. Likewise, palmitoleic acid (C16 : 1) was reduced by 25.8% and 26.2%, respectively.

Li et al. [29], in their study of soybean oil extraction by ultrasound, found that a decrease in contents of unsaturated fatty acids and increase in contents of saturated fatty acids is used for evaluating the extent of oxidation because the former are more susceptible to oxidation, whereas the latter are more stable. This may explain the significant increase in contents of palmitic acid and the decrease in contents of palmitoleic acid that were found by this study when ultrasound and microwave were applied.

Ma et al. studied cell disruption in the microalga Chlorella spp. and found 1.3% and 6.6% increases in palmitic and linoleic acids, respectively, when treated by microwave. The authors also observed decreases of 10% and 3% in palmitoleic and α-linolenic acids, respectively, in disruption by microwave, whereas the use of ultrasound led to decreases of 7.7% and 8.4%, respectively. According to Pingret et al. [30], this behaviour may be attributed to lipid oxidation due to hydroxyl free radicals (OH˙) which are generated during exposure to ultrasound and microwave, since they cleave double bonds of unsaturated fatty acids as the result of their strong activity and oxidation capacity.

3.3. Electric Energy Consumption

Table 1 shows the estimated energy consumed by microwave (40 s) and ultrasound (5 min) in cell disruption. There is an expressive difference in relation to energy consumption, which corresponds to an increase of approximately 50% when microwave is used instead of ultrasound (from 1.95 GJ·m³ to 2.80 GJ·m³, resp.). Halim et al. [11] studied cell disruption of the microalga Chlorococcum spp. by ultrasound. 200 mL cell suspension containing the biomass was sonicated at a maximum power of 130 W during 25 min, which represents an energy consumption of 0.975 GJ·m⁻³. Lee et al. [14] studied several methods of cell disruption in three species of microalgae. The microwave technique resulted in the highest lipid extraction, that is, 30% for Botryococcus spp., 10% for Chlorella vulgaris, and 10% for Scenedesmus spp. The energy consumed by microwave was 2.1 GJ·m⁻³(100 mL cell suspension, 5 kg·m⁻³ cell concentration, 700 W power, and 5 min).

Both techniques are suitable for scale-up, require a single unit, and are environmentally friendly [12]. Despite the higher treatment time, the ultrasound technique was chosen because it required less energy than that of the microwave. However, further optimization of its process parameters is required to make the overall process economically sustainable, that is, concentration of cell suspension, in order to reduce the specific energy consumption.

4. Conclusion

Techniques of ultrasound (5 min) and microwave (40 s) applied to cell disruption of the microalga Chaetoceros calcitrans were efficient, since lipid content values of 24.6% and 24.2% w/w (dry basis) were obtained, respectively, with no significant differences () in relation to the chemical disruption (24.2%). However, they used fewer chemicals and were less time-consuming. Regarding fatty acid profiles, both disruption techniques led to higher percentages of SFAs and PUFAs. Considering that the treatment of ultrasound consumed less energy than microwave, this technique is recommended for extracting lipids from Chaetoceros calcitrans.

Data Availability

Data are available upon request from the corresponding author.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

The authors wish to thank the CAPES (Coordination of Superior Level Staff Improvement) and CNPq (National Council for Scientific and Technological Development) for the awarding of scholarships.

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Copyright © 2018 Daniela Almeida Nogueira et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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