Bioconversion of Glycerol to Docosahexaenoic Acid by Thraustochytrium WB-02 an Indigenous Indonesian Microalga Strain

Glycerol is a by-product of the biodiesel industry, and it can be processed to produce many useful derivatives. This study is aimed at examining the bioconversion of glycerol to docosahexaenoic acid (DHA) using local microalgae. Glycerol to docosahexaenoic acid converting microalgae were obtained from the mangrove area in the coastal sea of Lampung Province. The single colony was then generated by the scratching technique in its isolation and purification process. By using 18S rDNA, a potential strain namely WB-02, was identified as Thraustochytrium sp. Gas chromatography analysis was performed to identify its product conversion. As a result, Thraustochytrium WB-02 was identified to utilize glycerol as a single carbon source and convert to DHA. A maximum DHA yield of more than 3.4 g/L was obtained when the glycerol concentration in the medium was 8%. Thraustochytrium WB-02 was regarded as a potential microalgae resource in producing DHA due to its high level of production.


Introduction
Glycerol is the primary by-product in the biodiesel industry. In general, in the production of 100 kg of biodiesel, 10 kg of glycerol is usually produced [1]. The Ministry of Energy and Mineral Resources reported that in 2016 Indonesia's biodiesel production reached 3.656 billion liters [2], so that the potential of glycerol produced was around 0.366 billion liters.
Several avenues for utilizing glycerol have been investigated. For example, glycerol can be converted to propylene glycol [3] or acetol [4]. It can also be used in the fermentation process to produce 1,3-propanediol [5], lipids [6], pigments [7], and a mixture of succinic acid, butanol, ethanol and hydrogen [8]. Other studies have also shown that glycerol can be used to produce DHA [9].
Studies have shown that docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are important for proper fetal development -including the development neuronal, retinal, and immune function. DHA and EPA may also affect many aspects of cardiovascular function-including inflammation, peripheral artery disease, major coronary events, and anticoagulation. In addition, EPA and DHA have been linked to promising results in prevention, weight management, and cognitive function in those with very mild Alzheimer's disease [10].
The major commercial source of DHA is fish oil [11]. However, global catches have been in decline since the late 1980s and the number of overfished stocks has been increasing exponentially since the 1950s [12]. Furthermore, the presence of chemical contaminants (such as, mercury) in fish oil can be harmful to consumers. In addition, fish oil is not suitable for vegetarians and the odor makes it unattractive. There are several alternative sources of EPA and DHA -such as bacteria, fungi, plants and microalgae -that are currently being explored for commercial production [13]. The development of microalgae containing DHA from glycerol is an opportunity to provide alternative omega-3 sources. The omega-3 fatty acids can be extracted from algae and used to enrich the nutrition of other foods. Microalgae cell biomass can also be used directly as a feed additive in various animal industries such as aquaculture [14] or poultry [15].
The microalgae belonging to the Labyrinthulomycetes class and family Thraustochytriaceae [16] are unicellular protists that are present in the marine ecosystem. They play a key role in the initial stage of microalgae chain food, as organic matter degraders. The geographic distribution of these microorganisms includes Antarctica, the North Sea, India, Micronesia, Japan, and Australia [17]. Recently, the Thraustochytrium strain was isolated from the coast of the King George Islands Antarctic [18], Vancouver Island, British Columbia [19], Yundang Lake, Xiamen City, China [20], the coastal waters of Southern China [21], Tasmania and Queensland, Australia, [22], Subic Bay, Philippines [23] and the mangrove areas in Malaysia [24].
However, to the author's knowledge, there is limited research regarding the isolation of Thrautochytrium sp. from Indonesia. This study is aimed at examining the Thraustochytrium spp isolated from Indonesia that have the ability to convert glycerol to DHA. These microalgae are expected to be a source for commercial DHA production from glycerol.
Sampling. Sample of fallen leaves were obtained from the mangrove area in Pahawang Islands, Lampung Province, Indonesia. This location was chosen because Thraustochytrium spp are monocentric protists and are easily found in marine habitats and mangrove areas. The fallen leaf samples were collected and washed with sterile sea water to remove any particulates, which can be a source of contaminants [25]. The samples were then placed in a container filled with ice and taken to the laboratory in less than four hours to isolate microalgae.
Isolation of DHA-producing microalgae. The isolation of DHA-producing microalgae was carried out by the direct plating technique according to the method described by Perveen et al. [26] with a slight modification. The small cut of leaf samples were directly placed on agar plates containing 0.5% (w/v) glucose, 0.1% (w/v) yeast extract, 0.1% (w/v) peptone, 1% (w/v) agar, streptomycin and penicillin G (0.3 g/L each) in 50% (v/v) sea water (referred to as the By+ media) and incubated at 28 o C for several days [25]. The colonies were taken from the plate and re-grown in a plate containing the By+ media. The isolates grown on the By+ media were then purified by the scratching technique until a single colony was obtained. Pure isolates were then inoculated on an agar slant containing the By+ media and incubated at 30 o C for 72 hours and stored at 4 o C as a stock culture. The microorganisms were maintained in slant cultures with a glucose-yeast extract -pepton agar media, containing 2% glucose, 1% polypeptone, 0.5% yeast extract, and 1.5% agar in 50% sea water [27]. The natural seawater in this study was used after filtration.
Bioconversion of glycerol to DHA. Thraustochytrium WB-02 was inoculated to 10 mL culture media in a 50 mL conical flask. The culture media consisted of 3.0% glycerol and 1.0% yeast extract in 50% (v/v) sea water. The pH of the medium, which was originally around 6, was not adjusted unless otherwise stated. The Thraustochytrium WB-02 strain was previously precultured at room temperature (28-30 o C) on a shaker at a speed of 180 rpm for one day. Then about 0.1 mL of the pre-culture broth (5,4 x 10 6 CFU/ml) was transfer to 10 mL of culture media in 50 mL conical flask to produce an optical density with wavelength of 600 nm (OD 600 ) value of 0.1 or an inoculum size of about 1% (v/v). Fermentation was then carried out on a reciprocal shaker at room temperature (28-30 o C) for 2-3 days under the same conditions. Total lipid content was determined using Bligh and Dyer's method of extraction [28].

Microalgae cell growth and lipid analysis.
The microalgae cell growth was determined by dry cell weight. Cells (usually from 2 mL of culture broth) were harvested and washed with water by centrifugation, dried at 105°C for 3 h and weighed. The fatty acids were directly transmethylated from the dried cells with 10% methanolic HCl and methylene chloride [29]. As an internal standard, 1.0 mg icosanoic acid was usually added to the reaction mixture. The esterified fatty acids were extracted with n-hexane, and the resultant extracts were applied to a gas chromatography analysis.
Gas chromatography analysis. Gas chromatography analysis was carried out using the Shimadzu GC-MS QP 2010 equipped with a flame ionization detector with capillary column Rtx-5ms (30 m  0.25 mm i.d., film thickness 0.25 mm). Helium was used as the carrier gas and the speed was maintained at 0.90 mL/minute and split ratio 100.0. As mentioned above, 1 mL of fatty acid methyl ester was injected with the injection port temperature at 250 ºC, oven column temperature at 80 ºC, and pressure at 56.9 kPa. The column temperature was maintained at 80 ºC for 2 minutes, 210 ºC for 1 minutes and increased to 280 ºC after 10 minutes. The identification of fatty acid methyl ester components was carried out using the Wiley 7 and NIST 147 library software found in the Shimadzu GC-MS QP 2010. Total fatty acids were calculated from the number of chromatogram peak areas relative to the internal standard peak area. The result of DHA were determined by the composition of DHA multiplied by the total fatty acid content [27].
Microalgae identification. Genomic DNA from a pure culture of the best potential, WB-02, was extracted and purified. The DNA was then amplified by a polymerase chain reaction with a 27F primer (5'-GAG TTT GAT CCT GGC TCA G-3') and 1525R primer (5'-AGA AAG GAG GTG ATC CAG CC-3'). The polymerase chain reaction program consisted of pre-denaturation at 96°C for 3 minutes, and then 30 cycles of denaturation at 96°C for 45 seconds, annealing at 56°C for 30 seconds, and elongation at 72°C for 2 minutes. Post elongation was done at 72°C for seven minutes and finally held at 4°C until the process was completed. The 18S rDNA sequences (500 nt) of WB-02 were then analyzed for similarities using the Basic Local Alignment Search Tool (http: //www.ncbi.nlm.nih.Gov/blast/) [30].
The Effect of glycerol concentration on the conversion of glycerol to DHA. The isolate with the best potential Thraustochytrium WB-02 was inoculated to a 10 mL media in a 50 mL flask. The media contained glycerol at various concentrations (0%, 1%, 2%, 3%, 4%, 5%, 8%, 10%, 12%, 15%) and 1.0% yeast extract in 50% (v/v) sea water. The Thraustochytrium WB-02 strains were previously pre-cultured at room temperature (28-30 ºC) on a shaker at a speed of 180 rpm for one day, and then about 0.1 mL of pre-culture broth (5.4 x 10 6 CFU/mL) was transferred to 10 mL of culture media in a 50 mL conical flask with an OD 600 value of 0.1 nm or the inoculum size around 1% (v/v). Fermentation was then carried out using the reciprocal shaker at room temperature (28-30 ºC) for 2-3 days. At the end of the fermentation cell growth measurements were carried out on OD 600 , dry cell weight and the amount of DHA produced by GC-MS chromatographic analysis.

Results and Discussion
Isolation of microalgae. From purified isolates regenerated on the By+ media, 23 isolates showed the ability to produce DHA. After observing the GC-MS chromatography data, WB-02 was chosen as the isolate with the strongest ability to produce DHA from glycerol.

Characterization and identification of isolates.
The micrograph of the WB-02 isolate (Figure 1) showed that the WB-02 strain is a spherical, single cell organism. In addition, the 18S rDNA sequence showed that the WB-02 strain has 93% similarity to Thraustochytrium sp. Therefore, the strain WB-02 was named Thraustochytrium WB-02.
Analysis of DHA conversion. Fatty acid chromatograms as a result of the conversion of glycerol to fatty acids are shown in Figure 2, while fatty acid components are shown in Table 1. From Table 1 it can be observed that by using glycerol as a carbon source, Thraustochytrium WB-02 can produce several polyunsaturated fatty acids, namely: 5,8,11,14-eicosatetraenoic acid (0.42%), 5,8,11,14,17-eicosapentaenoic acid (0.84%) and 4,7,10,13,16,19-docosahexaenoic acid (44.88%). It appears that DHA is the main component with a portion reaching almost 45%. Thus, it can be said that Thraustochytrium WB-02 is a potential microbial converting glycerol to DHA.   Thraustochytrium WB-02 -which is an indigenous Indonesian microalga strain isolated from Lampung Province, Indonesia-has the potential to convert glycerol to DHA. Figure 3 shows the effect of glycerol concentration on Thraustochytrium WB-02 growth and the DHA produced. The experiment showed the highest Thraustochytrium WB-2 growth when the glycerol con-centration was 8%, with the highest concentration of DHA produced as 3.4 g/L. In Chi et al.'s study [31] optimizing the conditions of the culture resulted in the production of 4.91 g/L of DHA. Several attempts have been made to increase the production of DHA produced by Thraustochytrium. By changing the carbon and nitrogen sources in the culture media of the Schizochytrium sp, Sahin et al. [32] could affect the production of biomass, fatty acids and DHA. The highest biomass and yield were obtained by using June 2019  Vol. 23  No. 2 peptone proteose as the only nitrogen source. The combination of peptone proteose as a source of nitrogen and glycerol as a carbon source, as well as the addition of ethanol with the selection of the right time proved to be beneficial for obtaining higher DHA yields [32]. Furthermore, by optimizing the use of response surface methodology, Manikan et al. [33], could increase the amount of DHA produced by Thraustochytrium from Malaysia by 4.5 g/L or 25% higher than the initial production before optimization. The addition of sodium sulfate to the medium can affect the growth and production of DHA compared to sodium chloride. The results of the experiment show that sodium sulfate (30 g/L), peptone (15 g/L) and sucrose (20 g/L) are the most effective media for higher DHA production [34].
Comparing the result of this study with the other studies above, this study still needs to be continued to achieve higher productivity so that it is feasible for commercial production, by optimizing the conditions of cultivation and media composition during glycerol fermentation to DHA.

Conclusion
It can be concluded that Thraustochytrium WB-02which is an indigenous Indonesian microalgae strain isolated from Lampung Province, Indonesia -has the potential to convert glycerol to DHA. Its productivity in producing DHA can, however, still be improved by optimizing the conditions of cultivation and media composition during fermentation.