Fatty acid composition of the thermophilic Gloeocapsa gelatinosa under different combinations of temperature, light intensity, and NaNO3 concentration Fatma Zili, Nahla Mezhoud, Lamia Trabelsi, Imed Chreif & Hatem Ben Ouada Journal of Applied Phycology ISSN 0921-8971 J Appl Phycol DOI 10.1007/s10811-014-0296-4
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Author's personal copy J Appl Phycol DOI 10.1007/s10811-014-0296-4
Fatty acid composition of the thermophilic Gloeocapsa gelatinosa under different combinations of temperature, light intensity, and NaNO3 concentration Fatma Zili & Nahla Mezhoud & Lamia Trabelsi & Imed Chreif & Hatem Ben Ouada
Received: 21 September 2013 / Revised and accepted: 17 March 2014 # Springer Science+Business Media Dordrecht 2014
Abstract The combined effect of temperature, light intensity, and NaNO3 concentration on lipid biosynthesis and fatty acid composition was investigated for the thermophilic cyanobacterium Gloeocapsa gelatinosa (Kützing 1843) isolated from a thermal spring in Tunisia. Under optimal growth conditions, the lipid content was 7.3 % DW. Fatty acid analysis revealed the predominance of 16:0 and 18:0 (23.7 and 18.2 %, respectively) as main straight carbon chains of saturated fatty acids. Unsaturated fatty acids were also identified with 18:1n9c (18.8 %) and 16:1n7 (5 %) being the predominant components. The effect of environmental factors on fatty acid composition was monitored by using principal component analysis and central composite design. Variation of light intensity (20 to 150 μmol photons m−2 s−1), temperature (20 to 60 °C), and nitrogen concentration (0 to 3 g L−1) induced a significant variation in the amount of fatty acid proportions, whereas lipid content was only slightly modified. Results showed that light intensity had the strongest effect on the composition of fatty acids. Temperature had a synergic effect with light intensity while nitrogen concentration had a trivial effect. The combined effect of high light intensity (150 μmol photons m−2 s−1) and high temperature (60 °C) increased the proportion of saturated 16:0 and 18:0 fatty acids along with long-chain fatty F. Zili (*) : N. Mezhoud : L. Trabelsi : H. Ben Ouada Laboratory of Marine Biodiversity and Biotechnology, National Institute of Marine Sciences and Technology, BP 59, 5000 Monastir, Tunisia e-mail:
[email protected] I. Chreif Laboratory of Biochemistry, UR: “Human Nutrition and Metabolic Disorder” Faculty of Medicine of Monastir, 5019 Monastir, Tunisia N. Mezhoud Earth Observation and Hydroclimatology Laboratory, Masdar Institute of Science and Technology, PO Box 54224, Masdar City, Abu Dhabi, United Arab Emirates
acids to 82 % which was twofold higher than that in optimal growth conditions. This induced fatty acid profile makes G. gelatinosa-based biofuels adaptable for higher energetic efficiency and higher oxidative stability. Keywords Thermophilic . Gloeocapsa gelatinosa . Lipids . Fatty acids . Induction . Environmental factors
Introduction When put into stressful environments (e.g., temperature, light intensity, and nutrient starvation), algae may switch carbon allocation from reproduction to oil production (Illman et al. 2000; Pruvost et al. 2009; Lv et al. 2010). This oil from algae can be extracted and turned into biodiesel through a chemical process called trans-esterification (Vasudevan and Briggs 2008). The screening of microalgae strains suitable for biodiesel production has been the subject of several investigations since 1978 (Sheehan et al. 1998). Recently, some studies on the screening of oleaginous microalgae have been reported, focusing on optimizing culture conditions to increase lipid productivity and evaluation of the potential for biodiesel production (Rodolfi et al. 2009; Gouveia et al. 2009; Li et al. 2010). The main indexes determining the potential of microalgal strains as biodiesel feedstock were considered as growth rate, lipid content, and lipid productivity. It was found that many eucaryotic microalgae, such as Chlorophyta and diatoms, can accumulate oils at a large extent (of 15–77 % total dry weight) (Liang et al. 2006; Chisti 2007). The properties of biodiesel are largely determined by the structure of its component fatty acid esters (Knothe 2005). Biodiesel produced from feedstocks that are high in PUFAs has good cold-flow properties. However, these fatty acids are particularly susceptible to oxidation. On the other hand, saturated fats produce a biodiesel with superior oxidative stability
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and a higher cetane number, but rather poor low-temperature properties (Stansell et al. 2011). It has been pointed out that microalgal fatty acid compositions react considerably to stress conditions imposed by chemical and physical stimuli, either acting individually or in combination (Illman et al. 2000; Liu et al. 2008). Cyanobacteria have also been subjected to screening for lipid production (Cobelas and Lechado 1989; Basova 2005). Unfortunately, considerable amounts of total lipids have not been found in cyanobacteria species examined in the laboratory (Hu et al. 2008). Nevertheless, cyanobacteria have many significant advantages when compared to other microalgae. These advantages are rapid growth rate (Parmar et al. 2011), high metabolic plasticity, and rapid adaptation to environmental conditions through lipid class synthesis (Karatay and Dönmez 2011). Cyanobacteria could therefore constitute an excellent model in a production process to favorably manipulate fatty acid composition in a dynamic manner and to predict the effect of stressful environmental changes that may influence the biodiesel quality. Few publications address the issue of enhancing the fatty acid profile of cyanobacteria (Knothe 2013). Nowadays, increased attention is being paid to the isolation and characterization of new algal strains from thermal springs. According to Vladimirova and Semenenko (1962) and Richmond (1986), organisms with high optimal growth temperature possess wider possibilities for adaptation. As reported in hot springs worldwide (Ward and Castenholz 2002; Debnath et al. 2009; Ionescu et al. 2010), cyanobacteria are the most common microbial group constituting the mats which occur in these ecosystems. Furthermore, the majority of cyanobacteria species isolated from thermal springs are facultative thermophiles (also called moderate thermophiles) which can thrive at a wide range of temperatures ranging from 0 to 75 °C (Castenholz 1969). This exceptional metabolic flexibility offers the potential to modify cyanobacterial fatty acid compositions using environmental variables and highlights the necessity of exploring thermophilic species as potentially excellent models for a lipid composition pathway to reveal the most prospective strains for further investigation. Such investigation should not then be applied separately and over a limited interval because different species respond to different variables in different ways (Guschina and Harwood 2006). We have selected the hot spring cyanobacterium Gloeocapsa gelatinosa (Kützing 1843) as a model for the investigation of thermophilic microorganism lipids. The aim of this study was to investigate the lipid content and fatty acid variation of G. gelatinosa by exposing concentrated cultures to different combinations of stress conditions of temperature, light intensity, and NaNO3 concentration (nitrogen source), using an experimental design.
However, robust algal growth and high lipid production are usually mutually exclusive (Sheehan et al. 1998). High lipid contents produced under stress conditions are usually associated with relatively low biomass productivity and, consequently, low overall lipid productivity (Li et al. 2011). Based on these considerations, we suggest a two-phase cultivation strategy, with a first optimal-growth biomass production phase followed by a lipid induction phase under stress conditions. Instead of changing temperature, light intensity, and nitrogen concentration, this study describes an attempt to modulate the fatty acid composition of G. gelatinosa in the culture in phase II of the proposed two-phase process which is much more feasible and easier to operate.
Materials and methods Samples were taken from “Aïn Atrous,” a hot spring (60 °C) located in the northern part of Tunisia (36° 49′ N, 10° 34′ E). Sampling materials were composed of microbial mats anchored to submerged stones. Mats collected were treated by filtration, centrifugation, and dilution techniques according to standard microbiological protocols (Stanier et al. 1971; Rippka et al. 1979). A purified strain was identified morphologically according to the keys and description established by Naz et al. (2004) as G. gelatinosa (Kützing, 1843). This cyanobacterium was initially subjected to different media and to different temperature and light intensity conditions to define the optimal growth conditions. These preliminary inlab experiments indicated that the species showed the highest growth in modified BG11 liquid medium containing (in g L−1) 1.5 NaNO3, 0.08 K2HPO4, 0.15 MgSO4 7H2O, 0.072 CaCl2 2H2O, 0.012 C6H8O7 H2O, 0.012 FeSO4, 0.002 Na2EDTA 2H2O, and 0.04 Na2CO3 and 1 mL trace metal solution (g L−1): 2.86 H3BO3, 1.81 MnCl2 4H2O, 0.39 Na2MoO4 2H2O, 0.079 CuSO4 5H2O, 0.049 Co(NO3)2 6H2O, and 0.222 ZnSO4 7H2O (Stanier et al. 1971; Rippka et al. 1979) at 40 °C and 85 μmol photons m−2 s−1. Growth condition culture Pre-cultures were achieved under conditions that optimize the algal culture growth (temperature 40 °C, light intensity 85 μmol photons m −2 s −1 , and NaNO 3 concentration 1.5 g L−1). Cultivation was carried out, in triplicate, in 10-L sterilized flasks containing 5 L of modified BG11 medium and equipped with a device for aseptic removal of samples. Each culture was inoculated with an initial biomass concentration of 0.17 mg mL−1. Cultures were stirred continuously with air at a constant flow rate (0.1 v/v/min). Biomass concentration was determined daily and expressed as g L−1 dry weight. The dry weight was obtained by placing the sample in a dry (105 °C for 24 h) tared crucible
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with a 1.5-μm pore size, 24-mm-diameter glass microfiber filter (Whatman, UK), rinsing with distilled water, and drying at 105 °C for 24 h. The change in the weight of the crucibles with the addition of the rinsed algae after drying off all of the water was considered the dry weight. Lipid synthesis induction Cultures were conducted in multiple batch reactors and exposed separately to different temperatures (20–40–60 °C), light intensities ranging from 20 to 120 μmol photons m−2 s−1, and sodium nitrate concentrations (N) (as nitrogen source) from 0 to 3 g L−1, as shown in Table 1. Cells were harvested from pre-cultures at the end of the exponential growth phase (day 5), concentrated by centrifugation, and resuspended equally (0.71 mg mL−1) in 250-mL Erlenmeyer flasks containing 110 mL of the appropriate medium. Aliquots of each culture (50 mL) were harvested after 2 days of treatment, by centrifugation (at 4 °C, 4,000 rpm for 5 min). The pellet was washed three times with deionized water to eliminate medium salts. The obtained cells were freeze-dried and subsequently weighed and stored at −20 °C until lipid analysis. All experimental cultures were conducted under controlled light in a temperature-programmable chamber with continuous illumination. Phyto-Claude halogen lamps (400 W) were used to illuminate chambers. The intensity of incident light was measured using the silicon sensor HD 8366. Lipid extraction For all samples, lipid analysis was conducted in triplicate. Pellets of each sample were added to 5 mL of a methanol/ water/HCl (30:3:1, by vol) mixture into clean Teflon-lined screw-capped glass test tubes and held at 55 °C for 6 h (Rezanka et al. 2003). A total of 15 mL of cold water–hexane (2:1, by vol) mixture was added to the sample and vortex
Table 1 Quantitative values of the encoded factors: temperature, light intensity, and nitrogen source Range of factors for designing the experiment −1a Temperature (°C) 20 Light intensity (μmol photons m−2 s−1) 20 Sodium nitrated (g L−1) 0 a
Low level
b
Central value
c
High level
d
Nitrogen source
0b
+1c
40 85 1.5
60 150 3
mixed for 20 s. The hexane layer was filtered and concentrated to dryness under a stream of nitrogen at 10 °C. The residue was extracted three times with 5 mL chloroform, then filtered and concentrated to dryness under a stream of nitrogen at 10 °C. The dried hexane and chloroform extracts were combined and dissolved in 1 mL of hexane–chloroform (1:1, by vol) mixture. The solvent was removed by being flushed with nitrogen, and the total lipid content was estimated gravimetrically (Rezanka et al. 2003). The comparison of lipid content values was performed by using one-way analysis of variance (ANOVA), and the significance of the differences (P
