Analysis of Swamp Microalgal Isolates from South Sumatra as Biofuel Candidates

2.1 Sampling method

Sampling was carried out at several water locations in South Sumatra, namely, Palembang, Banyuasin, Ogan Ilir and Ogan Komering Ilir. The four of sampling location are the representative swamps in South Sumatera has not been explored. Each sampling location was determined by its coordinates by using GPS (Figure 1).

At each sampling location, physical and chemical factors of the aquatic environment were evaluated by determining pH levels, temperature, dissolved oxygen levels, turbidity, total dissolved solids and total suspended solids in situ [26]. Samples were collected in the morning between 08:00 and 11:00 from the top and bottom of the waters by using a plankton net compositely to obtain the dominant microalgae in every level. Furthermore, researchers conducted water sampling by using a 10 liter bucket. The collected water was then filtered 10 times with a plankton net. Afterwards, each 100 ml sample was collected in a bottle and subjected to low-temperature conditions in the laboratory. The label of each sample bottle contained the following information: name of the observation location, day, date and time of sampling.

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Figure 1. Map of South Sumatra swamps

2.2 Microalgal abundance

The abundance of microalgae was calculated by counting the number of microalgal cells by using the Sedgwick Rafter Counting Cell through the hand counting method. The Sedgwick Rafter Counting Cell was cleaned first. Afterwards, the water sample to be calculated for density was dripped as much as 1 ml. The sample was added with one drop of gelatine to slow down the movement of microalgae and closed with a cover glass. Furthermore, the Sedgewick Rafter Counting Cell was observed under a microscope, and the number in each box was calculated using the hand counting method [27].

2.3 Microalgal biomass isolation and cultivation

The isolation and cultivation of microalgae aimed to obtain potential biomass as a source of biofuel and were carried out in a laboratory scale (batch culture). The isolation and propagation of isolates were conducted with agar and liquid culture media. The isolates were then propagated using culture flasks in the laboratory by using microalgal culture media (BBM and BG11). The isolation and cultivation process applied in this study followed the procedure used by Lee et al. [26, 28]. Isolation was carried out to separate the algal population and obtain a single type of algae from water samples by using the standard plating method. Samples from the field were serially diluted to facilitate the isolation process. The agar medium (40 ml) was poured into a sterile Petri dish and used as growth medium for the thawed sample.

The sample liquid (1 ml) was flattened onto the surface of the agar medium. Researchers transferred the microalgal colonies into sterile media in Petri dishes, which were incubated at room temperature (20℃-25℃) for 14 days until the algae grew. This process was repeated to obtain microalgal unicells. Diluted microalgal cells resulted in low likelihood of bacterial contamination. Grown algal cultures were then separated in various growth media under sterile conditions and placed at room temperature in the laboratory. This step was repeated until a single culture of algae was obtained. Stock cultures were maintained once a month by regrowing on agar media. The result of this stage was pure isolates of local microalgae, which were obtained from fresh waters in South Sumatra.

Cells were transferred into dilute media in culture tubes, placed in the culture bottle under low light at an appropriate constant temperature and checked microscopically for growth or waited until macroscopic growth could be detected (3–4 weeks after transfer). The isolates that grew were identified on the basis of the colony morphology found on the agar culture medium. A general classification method was carried out to differentiate the formation of colonies morphologically. Liquid culture results were identified through microscopic observations for cell morphology down to the genus level. Each isolate was labelled and photographed at different magnifications. Species identification was performed in accordance with the methods of the references [29-31].

2.4 Morphological identification

Phenotypic identification was carried out on the basis of morphological characteristics to determine the diversity of microalgal species. Algal morphology was observed using a specific microscope for microalgae. Identification was carried out by referring to the algal identification key as applied in several studies [29-31] and the online identification key from the Center for Freshwater Biology website [32]. Species diversity data were analysed descriptively and presented using the table of species composition based on the taxonomic rank.

2.5 Lipid content analysis

The analysis of total lipid content was carried out through the Bethien–Diemar (1963) method [33] by utilising nonpolar petroleum ether as solvent. The percentage of the total dry weight of lipids was calculated using Eq. (1):

$\%$ total lipid $=\frac{(A-B)}{C} \times 100$                    (1)

where, A is weight of flask + weight of lipid after extraction (mg), B is weight of flask before extraction (mg) and C is the dry weight of the sample.

4.1 Swamp microalgae survey and isolation

Based on the results of the isolation in Table 1, most of the isolates come from Palembang, showing that the swamp area in Palembang is rich in minerals needed by microalgae naturally. The growth of microalgae in nature is strongly influenced by environmental factors, such as light intensity, temperature, pH and nutrients [34]. A high diversity of microalgal genera at swamps in Palembang compared with those in other locations is presumed to be because the swamp areas of sampling locations in Palembang contain a variety of different minerals needed by microalgae to grow, such as N, P, K, Mg, S and H [35]. A previous study also found that the waters of Palembang have six types of microalgae, namely, Microcystis aeruginosa, Nannochloropsis oculata, Oscillatoria sp., G. radiata, Dictyospherium pulchellum and C. vulgaris [36]. In the fresh waters of Banyuasin, a previous study only found four species, namely, C. vulgaris, Scenedesmus obliqus, Nannochloropsis oculata and Ankistrodesmus falcatus [37]. These results indicate that the diversity of microalgae in South Sumatra especially in Palembang is high.

4.2 Identification of swamp microalgal morphology

The dominance of Chlorophyceae is presumed because this group has high adaptability especially in freshwater in solitary and in colonies [38]. The results of the morphological identification of the microalgal isolates are described in Figure 1. Morphologically, C. vulgaris Beyerinck isolates have the following characteristics: (i) round or ovoid cells, (ii) unicellular, (iii) size of 10 µm, (iv) dominance of green colour, (v) chloroplasts and pyrenoids [39] and (vi) hard cell walls because of cellulose and pectin [40]. C. vulgaris Beyerinck can live at an optimum salinity of 10–20 ppt and can still live at 40℃ [40]. In addition, C. vulgaris Beyerinck can live in solitary or in colonies [40]. D. granulatum Hindák isolates have the following characteristics: (i) round colonies, (ii) many cells (4–64 cells), (iii) mucus-like mass covering, (iv) cell colony size of about 33.75 µm, (v) green colour, (vi) round cell with a size of 3–5 µm and (vii) chloroplasts with pyrenoids [39]. Nannochloropsis sp. isolates have the following characteristics: (i) unicellular; (ii) round cell; (iii) size of about 3.5 µm; (iv) dominance of the colour of the chloroplast pigment, i.e. yellowish green [39]; (v) cell walls, mitochondria, chloroplasts and membrane-enclosed nucleus and (vi) bell-shaped chloroplasts located at the edge of the cell, a light-sensitive stigma (eyespot) and cell walls made of cellulose components, which are distinct characteristics of this microalga [41, 42]. Furthermore, Nannochloropsis sp. is cosmopolitan in nature and can be found in almost all types of water, e.g. marine and freshwaters. Nannochloropsis sp. can grow at high salinity. In addition, the optimum condition for its growth is 25‰–35‰ and optimal temperature range of 25℃-30℃ [43, 44]. S. bernardii G. M. Smith isolates have the following characteristics: (i) cells with cylindrical forms (sometimes elliptical or crescent-shaped forms), (ii) colonial nature with 4–16 cells attached to the centre side by side to form flattened colonies, (iii) long spinelike structures at both ends of each cell and (iv) cell length of 30 µm and width of about 5 µm [39]. G. radiata Chodat isolates have the following characteristics: (i) round shape, (ii) cilia on the cell surface, (iii) size of 15.25 µm and (iv) green colour [39]. Although identification results show that the isolates obtained from the swamps of South Sumatra are members of the Chlorophyceae group, each isolate has its respective morphological characteristics.

4.3 Analysis of lipid content of microalgae from swamp areas in south Sumatra

The analysis of lipid content is one indicator to determine the potential of microalgae as a candidate for biofuel. This analysis was carried out using the Bethien–Diemar (1963) method (in Endrawati, 2012 [33]). The results of the analysis indicate that the highest percentage is found in the C. vulgaris Beyerinck isolate (Figure 2). Pratono et al. indicated that C. vulgaris Beyerinck possesses 15.30% crude fat, which is exactly as that obtained in this study. Rebolloso-Fuentes et al. [15, 45, 46] showed that Nannochloropsis sp. contains high lipids, i.e. around 31%–68%. However, in the present study, only 6.94% lipid is found in Nannochloropsis sp. The results of the analysis also show that the G. radiata Chodat isolate has the lowest lipid content (3.21%).