Abstract
Microalgae were isolated, identified, and cultivated for enhanced biomass production and lipid accumulation. A marine microalgae was isolated from coastal rock pools of Tuticorin, Tamil Nadu, and identified as Nitzschia sp. RRSE2 upon microscopic examination and molecular sequence analysis. The experimental results showed that the maximum growth, biomass, and lipid content were obtained at pH 8 using the F/2 medium. These parameters revealed a notable difference when NaCl was added at 3% concentration. Meanwhile, the nutrients NaNO3 (18.75 mgL-1) and NaH2PO4 (3.48 mgL-1) were shown to be suitable nitrogen and phosphorus sources, respectively, for the production of lipids. On day 14, the maximum lipid concentration of 77.5 mgL-1 was produced using optimized culture conditions. Additionally, the maximum number of 17×105 cells mL-1 and the biomass concentration of 0.69 gL-1 were achieved on this same day. Finally, the fatty acid composition of the algal lipid was analyzed by gas chromatography/mass spectrometry (GC/MS) analysis.
Download PDF
Full Article
Enhanced Production of Lipid as Biofuel Feedstock from the Marine Diatom Nitzschia sp. by Optimizing Cultural Conditions
Anandapadmanaban Baala Harini,a Renganathan Rajkumar,a,* and Mohd Sobri Takriff b,*
Microalgae were isolated, identified, and cultivated for enhanced biomass production and lipid accumulation. A marine microalgae was isolated from coastal rock pools of Tuticorin, Tamil Nadu, and identified as Nitzschia sp. RRSE2 upon microscopic examination and molecular sequence analysis. The experimental results showed that the maximum growth, biomass, and lipid content were obtained at pH 8 using the F/2 medium. These parameters revealed a notable difference when NaCl was added at 3% concentration. Meanwhile, the nutrients NaNO3 (18.75 mgL-1) and NaH2PO4 (3.48 mgL-1) were shown to be suitable nitrogen and phosphorus sources, respectively, for the production of lipids. On day 14, the maximum lipid concentration of 77.5 mgL-1 was produced using optimized culture conditions. Additionally, the maximum number of 17×105 cells mL-1 and the biomass concentration of 0.69 gL-1 were achieved on this same day. Finally, the fatty acid composition of the algal lipid was analyzed by gas chromatography/mass spectrometry (GC/MS) analysis.
Keywords: Isolation; Identification; Nitzschia; Biomass; Lipid
Contact information: a: Department of Environmental Sciences, Bharathiar University, Coimbatore – 641 046, Tamil Nadu, India; b: Research Centre for Sustainable Process Technology, Faculty of Engineering & Built Environment, University Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia;
* Corresponding authors: micro_rajkumar@yahoo.co.in; sobritakriff@ukm.edu.my
INTRODUCTION
Microalgae have received much attention as a potential renewable energy source as part of the efforts to mitigate global warming. Microalgae can easily convert solar energy into chemical energy via atmospheric carbon dioxide fixation, which is currently under consideration as a promising raw material for biodiesel production (Spolaore et al. 2006; Chisti 2007; Mata et al. 2010).
In spite of the various advantages microalgae could bring to biodiesel production, their biomass and lipid production cost is relatively very high due to many factors associated with the microalgal cultivation process (Chisti 2013; Singh et al. 2015). To improve the economic feasibility of microalgal cultivation, it will be necessary to identify a suitable microalgal strain and explore its response to different cultivation conditions (Chen et al. 2015; Negi et al. 2015; Singh et al. 2016). Recently, many researchers have worked to optimize various factors, such as nitrogen starvation or limitation (Zhila et al. 2005; Jiang et al. 2012; Pancha et al. 2014), silicon deficiency (Lynn et al. 2000), phosphate limitation (Mandal and Mallick 2009; Chu et al. 2013), salinity (Rao et al. 2007; Zhila et al. 2011), pH of the medium (Breuer et al. 2013), iron content (Liu et al. 2008), light intensity (Ruangsomboon 2012), and harvesting time (Zhila et al. 2011), to improve the lipid accumulation by microalgae. When evaluating microalgae under such conditions, Hsieh and Wu (2009) stated that various microalgal strains responded appreciably when the lipid content was increased 30% to 60% of the dry cell weight.
The biomass content of the microalgae can be enhanced when the microalgae are cultivated in photobioreactors. A previous study reported that a high concentration of microalgae cultures was cultivated in photobioreactors that facilitate high light intensity surfaces and high mass transfer rates to improve the biomass productivity (Ugwu et al. 2008). It has also been reported that the biomass productivity of Chlorella vulgaris was increased 50% in the membrane-sparged helical tubular photobioreactor (MSTR) (Fan et al. 2008).
Among these microalgae, diatoms (class Bacillariophyceae) include a large diversity and produce the greatest biomass in the marine environment, though they are not well recognized for biofuel purposes. Diatoms have been identified as promising candidates to produce biodiesel because they give up to 60% of their cellular biomass as triacylglycerols (TAG) that can be converted into biodiesel through the transesterification process (Blanchemain and Grizeau 1996; Yu et al. 2009). In general, diatoms have been studied widely for their biochemical constituents that can be used for the larval diet in the aquaculture field. However, only a few reports demonstrate the use of diatoms for biofuel production. For example, Nitzschia palea was found to exhibit a high amount of lipid production according to the results given by the Aquatic Species Program (Sheehan et al. 1998).
The effect of nutrients and pH on marine diatoms, especially Nitzschia sp., for enhancing lipid and biomass production remains an infrequently studied research area. Thus, the present study focused on the isolation of indigenous diatoms from Tuticorin along the Tamil Nadu coast, and studied the effects of cultural conditions on the production of biomass and lipid by the isolate.
EXPERIMENTAL
Materials
Isolation and growth conditions
Seawater samples were collected in sterile plastic vials from coastal rock pools in Tuticorin, Tamil Nadu, India. Pure cultures were isolated and purified by streaking 1.5% agar plates with F/2 medium (Guillard and Ryther 1962). This media was composed of: NaNO3, 75 mgL-1; NaH2PO4.2H2O, 5 mgL-1; Na2SiO3.9H2O, 30 mgL-1; trace metal stock solution, 1 mL (per 1 L: FeCl3.6H2O, 3.15 g; Na2EDTA.2H2O, 4.36 g; 1 mL CuSO4.5H2O, 9.8 gL-1; 1 mL Na2MoO4.2H2O, 6.3 gL-1; 1 mL ZnSO4.7H2O, 22.0 gL-1; 1 mL CoCl2.6H2O, 10.0 gL-1 and 1 mL MnCl2.4H2O, 180.0 gL-1), and 0.5 mL of vitamin stock solution (per 1 L: vitamin B1, 200 mg; 1 mL vitamin H, 1.0 gL-1 and 1 mL vitamin B12, 1 gL-1). Media chemicals were bought from Himedia, Mumbai, India. The individual colonies were inoculated into the above liquid medium and incubated at 25 ± 1°C in a thermostatically controlled room, with a light intensity of 30 μEm-2 s-1, and a photoperiod of 16:8 h (light: dark). The same cultivation conditions were maintained for all the subsequent experiments. Preliminary identification was made for morphological characteristics under a microscope (John et al. 2003; Bellinger and Sigee 2010). The purity of the individual culture was confirmed by repeated streaking techniques on the nutrient agar plate. Based on the highest lipid productivity and growth rate, the strain was chosen for further study.
Nile Red Staining
Nile Red (9-(Diethylamino)-5H-benzo[a]phenoxazin5-one) (Himedia, Mumbai, India) staining was performed to observe intracellular lipid droplets according to the method of Greenspan et al. (1985). Isolated algal cells were cultured for two weeks and tested with Nile Red staining. The growing culture (0.5 mL) was taken by centrifugation at 1500×g for 10 min and cleaned with a physiological saline solution (0.5 mL). Once the obtained cells were re-suspended in the same saline solution (0.5 mL), the Nile Red solution (0.1 mg mL-1 dissolved in acetone) was further added to cell suspensions (1:100 v/v) and kept for 10 min. Subsequently, stained algal cells were observed under fluorescent microscopy.
Molecular Identification
The selected algal strain of approximately 50 mL in the log to stationary phase was used for molecular identification. The isolation of the genomic DNA of the microalgal strain was performed using a plant genomic DNA isolation kit (Sigma-Aldrich, Bangalore, India) following the manufacturer’s protocol. The primers SSU-1+ (AAC CTG GTT GAT CCT GCC AGT) and SSU-B (CCT TCT GCA GGT TCA CCT AC) (Sigma-Aldrich, Bangalore, India) were subjected to the polymerase chain reaction (PCR) (Medlin et al. 1988; Li et al. 2015). The polymerase chain reaction (PCR) amplification was performed in a thermal cycler (Agilent Technologies, Santa Clara, CA, USA) using the following program: initial denaturation for 5 min at 95 °C; 20 cycles for 1 min at 94 °C, 45s at 55 °C, 4 min at 72 °C; and a final extension for 10 min at 72 °C. Electrophoresis was performed with 1% agarose gel (Himedia, Mumbai, India) to separate the PCR products. The amplified product was sequenced at Acme Progen Biotech Pvt. Ltd., Salem, Tamil Nadu, India, and the obtained sequences were analyzed on the National Center for Biotechnology Information (NCBI, Bethesda, MA, USA) server using the Basic Local Alignment Search Tool (BLAST). Further, the nucleotide sequence was submitted to the NCBI to obtain the accession number.
Effect of pH, Salinity, Nutrients, and Cultivation Time on Biomass and Lipid Production
For all experiments, the selected microalgae were grown in an F/2 medium adjusted with different pH ranges (6 to 9). For the analysis of salinity tolerance, the isolated culture was transferred into the same medium added with 0%, 3%, 6%, and 9% of the NaCl concentration. The effect was 0, 18.75, 28.125, 37.5, and 46.875 mgL-1 (NaNO3 as nitrogen source). Quantities of 0, 2.32, 3.48, 5, and 7 mgL-1 (NaH2PO4 as the phosphorus source) were also tested separately. Other than the above cultural parameters, the composition of other elements was the same as the F/2 medium. Experiments were carried out in 500 mL Erlenmeyer flasks containing 200 mL medium. All the flasks were inoculated with the 10% v/v cells of two-week-old culture and incubated in a thermostatically controlled room for 16 days to analyze the growth and lipid productivity every 2 days. The optimum cultural parameter was selected at each experiment based on lipid productivity, and further cultivation was completed in a photobioreactor under the optimized conditions.
Cultivation in Photobioreactor
The chosen microalga was cultivated in a photobioreactor (Lark Innovative Fine Teknowledge, Chennai, India) (2 L capacity) within an optimized F/2 medium to determine the effect of selective factors (Fig. 1).
Fig. 1. Photograph of the photobioreactor for the cultivation of Nitzschia sp. RRSE2
The photobioreactor provided a shaft that prevented the culture from settling. Sparger supplied CO2 with air. Turbines helped to mix the media and culture. A glass vessel was autoclaved and used to grow the Nitzschia sp., which was helpful in the overall increase in both biomass and lipid content. For this experiment, 10% of the inoculum was added to the F/2 media (pH 8) with optimized components, such as a salinity of 3%, a nitrate concentration of 18.75 mgL-1 (NaNO3), and a concentration of phosphorous of 3.48 mgL-1 (NaH2PO4). The reactor ran until the decline of the growth phase of the algae, which was up to 14 days. The pH was controlled to remain between 7.9 and 8.1. To maintain a level of 5% CO2 and to supplement nutrients, the sample was mixed at 150 rpm. The conditions surrounding the reactor were a light: dark maintained as 16:8 h, with 2500 to 3000 lux of light, and a temperature of approximately 25 to 27 °C. A sample of approximately 30 mL was collected every 2 days starting from day 0. The cell concentration and the lipid content were recorded during the cultivation process. On day 14, the grown biomass was collected by filtering with Whatman No.1 filter paper. The collected biomass was oven-dried dried at 65 °C for 1 h, and the crude lipid was extracted. Finally, the extracted lipid was subjected to gas chromatography/mass spectrometry (GC/MS) analysis.
Analytical Methods
Growth and biomass estimation
The cultures were harvested by centrifugation at 10000 rpm for 5 min. Then, the cells were washed twice with distilled water, and the pellet was dried at 70 °C for 1 h. The dry weight of the biomass was determined gravimetrically. The algal growth was expressed in terms of dry cell weight (DCW) per liter (gL-1). Meanwhile, microalgal growth was also monitored by the cell-counting method using a Neubauer hemocytometer (Rohem, India).
Extraction and estimation of lipid
Lipids were extracted from the biomass using the method of Folch et al. (1957). Ten mL of grown culture was centrifuged. Then, the pellet was collected and ground with chloroform:methanol (1:2, v/v). Physiological saline was added for phase separation between chloroform and methanol. The above experiment was left overnight. The lower chloroform phase was collected and air-dried. The presence of lipid was treated with the concentrated sulphuric acid and kept at a high temperature for 10 min. Then, vanillin reagent was added to the solution and read at 520 nm. Cholesterol (Himedia, Mumbai, India) with different concentrations was used to prepare for the standard graph to find the unknown weight of the isolated lipid. Simultaneously, transesterification of fatty acids was done by the method of Ichihara et al. (1996). The extracted lipid was air dried at room temperature and the obtained lipid was measured gravimetrically. About 10 mg of lipid was dissolved using 2 mL of hexane and 200 µL of 2 M methanolic KOH and vortexed for 2 to 5 min. The FAME containing upper layer (hexane phase) was recovered for further analysis.
Analysis of fatty acid methyl esters
Fatty acid methyl esters were analyzed from the extracted lipid by GC/MS according to the method of Tadashi et al. (2009). Gas chromatography/mass spectrometry was performed for the isolated crude lipid and was run in SQ8C GC/MS (Perkin Elmer, Waltham, MA, USA). The crude lipid underwent the basic principle of separation of compounds present in the sample in DB-5ms capillary standard non-polar column (30 m length; 0.32 mm; 0.25 mm; with a flow rate of 0.25 µm). A sample of 1 mL was injected. The oven’s initial temperature was 150 °C and progressively raised to 240 °C. A total of 2 µL of sample was injected with flame ionisation detector (FID) temperature of 260 °C. The nitrogen carrier gas was used as a stationary phase that serves as a column with the solvent delay of 3 min. Retention time, a time at which each particular component eluted from the sample, helped in differentiating the elements. Lastly, the GC/MS analysis of this fatty acid composition was performed at Tamil Nadu Agricultural University, Coimbatore, India.
Statistical analysis
Statistical analyses were performed using SPSS version 20.0 (IBM Corporation, USA) for one-way ANOVA test. All the experiments were carried out in triplicate. Experimental data were calculated as mean ± SE, and the mean values were determined by using DMRT (Duncan’s Multiple Range Test). Significant differences were considered to be at P < 0.05.
RESULTS AND DISCUSSION
Isolation and Identification of the Isolate
Nitzschia sp. RRSE2 strain, which was the dominant microalga at the collection site, was isolated from coastal rock pools in Tuticorin by serial dilution, then subjected to plating techniques. The strain revealed the following morphological characteristics under a light microscope: that it was comprised of single cells with bilateral symmetry and isopolar frustules, and in each cell there were two chloroplasts, elongated valves, and a rounded polar end. The cell length was 18.5 μm, and the width was 4.5 μm (Fig. 2a). Nile Red staining of algal cells was also performed to detect intracellular lipid droplets stained in red using fluorescence microscopy (Fig. 2b). The strain was identified as genus Nitzschia, confirmed by 18S rDNA gene sequencing followed by BLAST analysis, demonstrating over 95% similarity with other Nitzschia sp. (KT072977.1). Hence, the strain was identified as Nitzschia sp. RRSE2 and the sequences were submitted to the NCBI, and an accession number (MK785417) was obtained.