ABSTRACT

This study discusses the perspectives regarding the green alga Dunaliella salina Toed for biodiesel manufacturing purposes. The alga was cultivated under controlled lab conditions. Biomass concentration at early stationary grown microalga was 2.6 mg/L dry weight, while the algal oil was about 27.1% of the biomass. Algal oil was esterified and analyzed using GLC technique. Fourteen fatty acid methyl esters were identified. The amount of saturated and unsaturated fatty ester fractions was 35% and 65% respectively. The physicochemical properties of fatty acids comprising biodiesel were discussed. However, culture optimization coupled with genetic improvement will definitely represent contributions to bring about innovation in oil hyper-producing D. salina that will ultimately meet with success.

1. Introduction

Owing to the rising price of fuel and the worry about global warming that is associated with burning fossil fuels as well as the depleting nature of fossil fuel resources [1], biofuel is gaining a significant value as an attractive fuel. Microalgae can offer numerous different types of renewable biofuels. These comprise methane produced by anaerobic digestion of the algal biomass [2]; photobiologically produced biohydrogen [3,4] and biodiesel resultant from microalgal oil [1,5]. In this situation, the employing of microalgae as a sustainable source of energy seemed extremely attractive to the energetic market [6,7]. Microalgae exhibit several important aspects for innovative research on renewable energy. Easy and inexpensive nutrient system, faster growth rate, high biomass productivity and smart biochemical profile recommend strong application as a bioenergy foundation [8].

Biodiesel is a mixture of monoalkyl esters of long chain fatty acids derived from a renewable lipid feedstock [9]. It is composed of 90% - 98% triglycerides, and smaller amounts of monoand diglycerides and free fatty acids, besides residual amounts of phospholipids, phosphatides, carotenes, tocopherols, sulphur compounds and water [10]. Algal oil can be converted into biodiesel through a process called transesterification. Transesterification is a chemical reaction between triglycerides and alcohol in the presence of a catalyst to produce monoesters that are termed as biodiesel [11-13]. The success of algal biodiesel depends on the structure of the economy and energy replacement. From an economic perspective, developed countries are more likely to adopt clean energies because they have more flexible economies [14].

Depending on species, many microalgae have been described as lipid-rich strains for their hydrocarbons and other complex oils. However, not all algal oils are satisfactory for the production of biodiesel [15,16]. Many microalgal species can be promoted to accumulate high lipid contents [17], although average lipid contents vary between 1% and 70%, some species may reach 90% (w/w DW) under certain conditions [18-20]. Studies are considered necessary to achieve a fundamental understanding of the relationship between the combination of environmental parameters and algal mass production with high lipid content. Maximum biomass and lipid content of Botryococcus braunii are attained by photoperiod of 12 h light: 12 h dark, temperature of 23˚C, and 0.8755% NaCl, and 30 - 60 W/m² irradiance of light intensity [21].

Dunaliella salina Toed is a motile unicellular wall-less green alga that accumulates β-carotene and has a cell size of about 6 - 14 μm. It grows in a wide range habitat. It has a high tolerance to salt, temperature and light. This microorganism is quite easy to cultivate and has a relatively high growth rate and lipid content [22].

The objective of this investigation is to determine the significance of Dunaliella salina as a prospective source for potential future biodiesel. Fatty acids profile of the alga as a tool for testing the resulting biodiesel was studied. The following biodiesel properties were discussed: kinematic viscosity, cetane number, oil stability and oil density.

2. Materials and Methods

2.1. Organism and Cultivation Conditions

Dunaliella salina is a green halophilic alga that sharing among the most basic and essential components of aquatic environment. It was obtained from the algal culture collection of the phycology laboratory, Botany and Microbiology Department, Faculty of Science, University of Alexandria. This organism was originally isolated from the brine water of salty lagoon at El Max district, Alexandria, Egypt.

Axenic samples of known volume were inoculated into sterilized 1 L Erlenmeyer flasks containing 400 ml MH medium [23]. These cultures were maintained in a photoincubator set at 25˚C ± 1˚C, a light intensity of 60 μmol photon s⁻¹∙m⁻² (as supplied by cool-white fluorescence tubes) and a 12:12 h dark: light photoperiod with manual shaking twice per day for 16 days.

2.2. Biomass Estimation

Algal growth was characterized based on cell counts. 1 ml of algal culture was removed from the replicate flasks at regular intervals throughout the experiment. It preserved with iodine solution prior to counting using a haemocytometer slide under bright-field contrast microscopy.

The cultures were harvested by centrifugation at 5000 rpm for 15 min. Pellets were then washed twice with distilled water and dehydrated at 30˚C until a constant weight was achieved. The dry weight of the algal biomass was estimated from the average of at least three representative samples.

2.3. Lipid Extraction

At early stationary phase after 12 days of experimentation, lipids were extracted by using chloroform: methanol (2:1) solvent mixture according to the methods of Bligh and Dyer [24]. The extracted lipid was separated into two layers, the upper layer methanol together with water was removed and the chloroform layer including lipid was collected. The residues were subjected to repeated extraction twice. The entire extracts were mixed together forming crude oil extract and the chloroform was evaporated.

2.4. Fatty Acid Methyl Ester Analysis

Via direct methylation, FAMEs were prepared as described by Christie [25]. The sample was esterified in 1% sulfuric acid in absolute methanol. The mixture left overnight at 50˚C then water containing sodium chloride is added and the required esters are extracted with hexane to separate the layers. The hexane layer is washed with water containing potassium bicarbonate and dried over anhydrous sodium sulfate. The solvent was evaporated using rotary evaporator. The composition of FAME was quantified and identified on a Shimadzu gas-liquid chromatography equipped with a flame ionization detector with packing column material Hp-5. The carrier gas was nitrogen and the short speed was 5 mm/min. Fatty acid methyl esters of the micro-alga were deduced by comparing their retention times with those of standards. Quantification was based on the internal standard method.

2.5. Statistical Analysis

All analyses were carried out in triplicate, and the standard deviations (SD) were determined.

3. Results and Discussion

3.1. Algal Biomass and Oil Content

Figure 1 shows that the number of cells after 12 days of experimentation achieved a maximum value of 7.1 × 10⁶ cells/ml. This value corresponding to 2.6 mg/L dry weight and a relatively high oil content of 27.1% (Table 1). For the purpose of biodiesel production, high oil content and rapid growth rates are major factors in selecting an algal strain [18]. Rodolfi et al. [26] reported that alga with high biomass productivity may manifest in a relative low lipid productivity and vice versa.