Semicontinuous nitrogen limitation as convenient operation strategy to maximize fatty acid production in Neochloris oleoabundans
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.
References (35)
Biodiesel from microalgae
Biotechnol. Adv.
(2007)- et al.
Microalgae for biodiesel production and other applications: a review
Renew. Sustain. Energy Rev.
(2010) - et al.
The impact of N starvation on the dynamics of triacylglycerol accumulation in nine microalgae strains
Bioresour. Technol.
(2012) Essential fatty acids in health and chronic disease
Am. J. Clin. Nutr.
(1999)- et al.
Photosynthetic performance, lipid production and biomass composition in response to nitrogen limitation in marine microalgae
Plant Physiol. Biochem.
(2012) - et al.
Simultaneous growth and neutral lipid accumulation in microalgae
Bioresour. Technol.
(2013) - et al.
Lipid composition of the N starved green alga Neochloris oleoabundans
Enzym. Microb. Technol.
(1983) - et al.
Investigation of biomass and lipids production with Neochloris oleoabundans in photobioreactor
Bioresour. Technol.
(2009) - et al.
Systematic investigation of biomass and lipid productivity by microalgae in photobioreactors for biodiesel application
Bioresour. Technol.
(2011) - et al.
Lipid accumulation and growth of Chlorella zofingiensis in flat plate photobioreactors outdoors
Bioresour. Technol.
(2011)
Characterization of the lipid accumulation in a tropical freshwater microalgae Chlorococcum sp
Bioresour. Technol.
Growth of three microalgae strains and nutrient removal from an agro-zootechnical digestate
Chemosphere
Biomass and lipid productivity of Neochloris oleoabundans under alkaline–saline conditions
Algal Res.
Neochloris oleoabundans grown in enriched natural seawater for biodiesel feedstock: evaluation of its growth and biochemical composition
Bioresour. Technol.
Effect of oxygen at low and high light intensities on the growth of Neochloris oleoabundans
Algal Res.
Cultivation of microalgae for oil production with a cultivation strategy of urea limitation
Bioresour. Technol.
pH-upshock yields more lipids in nitrogen-starved Neochloris oleoabundans
Bioresour. Technol.
Cited by (31)
Semi-continuous cultivation strategy for improving the growth of Synechocystis sp. PCC 6803 based on the growth model of volume average light intensity
2022, Algal ResearchCitation Excerpt :The results demonstrated that renewal rate of 30 % and nutrient concentration of 8 Mm NaNO3 could optimize the cell, protein, carbohydrate, chlorophyll a, and exopolysaccharide productivity of Chroomonas under semi-continuous culture conditions. Bona et al. [35] studied the influence of nitrogen depletion and nitrogen limitation on biomass growth, lipid production, and fatty acid composition of Neochloris oleoabundans under semi-continuous cultivation. The research found that semi-continuous culture resulted in a promising operational strategy for fatty acid production.
Biochemical engineering approaches to enhance the production of microalgae-based fuels
2022, 3rd Generation Biofuels: Disruptive Technologies to Enable Commercial ProductionAquaculture wastewater treatment through microalgal. Biomass potential applications on animal feed, agriculture, and energy
2021, Journal of Environmental ManagementCitation Excerpt :In the 3rd trial, it was decided to add a nitrogen supplementation of 20 mg N L−1 every other day in the 2nd and 3rd reactors so that the culture could lower COD levels. In these reactors, the nitrogen levels were too low to allow the culture to grow, a situation that has already been verified by Bona et al. (2014) and Markou et al. (2016). The remediation rate in the aquaculture effluent for total nitrogen and total phosphorus was also 100% for the two trials and two microalgae (Table 4).
Seaweeds and microalgal biomass: The future of food and nutraceuticals
2021, Future Foods: Global Trends, Opportunities, and Sustainability ChallengesBioprospection of green microalgae native to Paraná, Brazil using a multi-criteria analysis: Potential for the production of lipids, proteins, and carotenoids
2020, Bioresource Technology ReportsCitation Excerpt :After cultivation, each microalgal culture was distributed into nine 125-mL Erlenmeyer flasks containing 70-mL of BG11 culture medium. Four conditions were evaluated: high light intensity (87.5 μmol m−2 s−1), osmotic stress (addition of NaCl 3% w/v), nitrogen limitation (reduction to 10% of the original nitrogen source) and normal conditions (laboratory conditions/control) (Bona et al., 2014). The other growth conditions, inoculum, temperature, and photoperiod were maintained as in the previous cultures (Section 2.2).