Elsevier

Algal Research

Volume 5, July 2014, Pages 1-6
Algal Research

Semicontinuous nitrogen limitation as convenient operation strategy to maximize fatty acid production in Neochloris oleoabundans

https://doi.org/10.1016/j.algal.2014.03.007Get rights and content

Abstract

Neochloris oleoabundans is a green microalga known for the ability to increase its fatty acid (FA) content when grown under nitrogen (N) depletion. Accumulation of FAs and their composition were compared by cultivating N. oleoabundans in N limitation and N depletion conditions. We adopted an innovative approach that consists in semicontinuous cultivation mode as a strategy to optimize lipid accumulation. To identify the optimal conditions for the production of different FAs, a temporal profile of how different conditions may influence the microalgae culture in terms of biomass, FA concentration and composition was evaluated. N limited culture attained higher values of algal productivity and total FA concentration (0.42 mg L 1 d 1 and 91.2 mg L 1) than N deplete culture (0.15 mg L 1 d 1 and 53.2 mg L 1). Considering lipid accumulation, concentration and percentage of triacylglycerols (TAG) increased in all culture methods, with respect to the N replete control. In particular, N limitation led to a significant increase of polyunsaturated TAG (PUFA), while N depletion led to the highest level of monounsaturated TAG. These results suggest that N. oleoabundans biomass and FAs were strongly affected by the N supply and that the quality of the TAG fraction could be modulated in accordance to the utilization purpose in a wide range of applications. Semicontinuous culture resulted as a promising operational strategy for FAs production. More specifically, semicontinuous cultivation coupled to N limitation was found to be a convenient method to increment PUFA fraction and therefore to yield a high value product.

Introduction

The use of microalgae has several advantages when compared to other available feedstock (e.g., soybean, rapeseed, sunflowers and palm oil) [1]. Microalgae have higher growth rates and areal productivities of biomass and lipids than conventional crops, which eventually results in a lower demand of land area. Furthermore, since microalgae can be cultivated in non-arable land, they do not compete with agriculture. Microalgae cultivation is not seasonally limited and allows daily harvests [2]. Moreover, other compounds can be extracted from microalgae residual biomass such as polyunsatured fatty acids (PUFAs), sugars, pigments and antioxidants, which can be used in many commercial applications such as cosmetics, pharmaceutical and nutraceutical industries [3].

Recent studies highlighted that oleaginous algal strains cultivated under nitrogen (N) deficiency increase their lipid content without a substantial decrease in biomass productivity [4], [5]. Most studies report data in terms of maximum lipid content, without a correlation with the biomass productivity and without considering that the maximum lipid content is often attained when the biomass productivity is at a minimum. Instead, a key-parameter to evaluate lipid production should be lipid productivity [6], meant as the product of biomass productivity and the lipid content. In addition, lipid composition is an important aspect to be evaluated, because different fatty acids (FAs) affect the quality of the final product. Depending on the use of the oil (energy production, food or feed), the required characteristics may vary. For biodiesel production, a high presence of monounsaturated FAs (MUFA as oleic and palmitoleic acids), a reduced presence of PUFA, and a controlled saturated FA content are recommended [7], [8]. On the other hand, PUFA, especially Ω-3 PUFA, have been shown to be effective in preventing or treating several diseases [9], [10], [11], [12]; therefore, this is the most interesting FA fraction for human as well as for animal nutrition [13], [14].

Different cultivation conditions may affect the lipid content and the FA composition; under N deficiency, the protein content and the chlorophyll level decrease, while carbohydrate and lipid content increases [15], [16]. A higher lipid content is mainly due to an accumulation of triacylglycerols (TAG), which are the preferred substrate for biodiesel production. In green algae, the variation in the FA composition usually results in increased oleic acid contents, with a consequent decrease in the average degree of polyunsaturation [17], [5]. The cultivation of microalgae under N stress is carried out either by N depletion or N limitation. Under N depletion, microalgae grow in a medium lacking a N source, while under N limitation there is a constant but insufficient supply of N. Using N depletion to increase the lipid content in algae, especially TAG fraction, is a well- known process [17], [2], [18], [19], [4], [5]; whereas N limitation is less studied or it is meant as progressive depletion [4], [20], [21]. Neochloris oleoabundans is a microalga also known for its potential in biodiesel production [17], [22], [23], [18], [24]. This species when grown in batch N depletion may reach a total lipid content up to 40% [22], [18], and has been tested in a wide range of cultural conditions [25], [26].

The aim of the present study is to investigate the effect on biomass growth, lipid production and FA composition of N. oleoabundans under N depletion and N limitation. While other studies have investigated the effect of the culture conditions on the final lipid content, lipid and biomass concentration and the temporal trend of these parameters were seldom reported. Here, a temporal profile of how different conditions (sufficient/limited/deplete) may influence the microalgal culture in term of biomass, lipid concentration and FA composition is reported, with the aim to define the optimal conditions for the production of different lipids.

Section snippets

Pre-cultivation conditions

N. oleoabundans UTEX 1185 was obtained from the University of Texas Culture Collection of Algae (UTEX). N. oleoabundans was inoculated in 250 mL Erlenmeyer flasks containing 100 mL of liquid medium. Flasks were maintained in sterile condition in a CO2 incubator (Sanyo CO2 Incubator Mco-19Aic) flushed with air/CO2 (97/3, v/v) to support growth and maintain pH within a desired range. In the incubator, the temperature was 25 ± 2 °C and a continuous artificial illumination of 200 μmol m 2 s 1 was provided

Biomass concentration

Biomass concentrations of microalgae growth in N replete/limited/deplete conditions are shown in Fig. 1.

As expected, the highest biomass concentration was achieved in N replete condition. After the first 3 days of adaptation phase, in N replete and limited cultures the daily productivity compensates the daily harvesting, thus keeping constant the algal concentration at the sampling time. This condition was maintained throughout the experiment by a constant N supply (Fig. 1a). The deplete culture

Conclusions

This study shows that biomass and TFA are strongly affected by the cultivation method. TFA concentration was positively affected by N limitation, which maximized biomass production and TFA accumulation. Semicontinuous is confirmed as an effective cultivation method as it allows a greater availability of light per cell when compared to batch mode [18], [19]. In semicontinuous culture, the biomass concentration can be controlled by varying the dilution rate and it can attain a constant biomass

Acknowledgments

This work was partly supported by Finpiemonte, project AlgaeNRG Grant n. 147-405 and by two grants MIUR XXVI Cycle from the Doctoral School of Sciences and Innovative Technologies, University of Turin. The authors are grateful to Rocco Mussat Sartor, Elena Ghiglione, Angelica Lucrezia Gibiino and Ambra Giuganino for their help in lipid extraction.

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