Combined metals and EDTA control: An integrated and scalable lipid enhancement strategy to alleviate biomass constraints in microalgae under nitrogen limited conditions

Highlights

• A. obliquus showed highest lipid productivity amongst all seven microalgal strains.

• Combined metals stress eased the constraint of low biomass under limited nitrogen.

• Combined metals stress enhanced the overall lipid productivity (1.99 fold).

• EDTA addition further improved the lipid productivity (2.18 fold).

• This strategy showed 2.08 fold increase in lipid productivity at 3000 L cultivation.

Abstract

The commercial realization of microalgal biodiesel production necessitates substantial impulsion towards development of strategies to improve lipid yields upstream. Nitrogen stress is the most widely used lipid enhancement strategy; yet, it is associated with compromised biomass productivity. In this novel approach, combined effect of metals and EDTA on lipid productivity of Acutodesmus obliquus was investigated under nitrogen limited conditions. The effect of metal concentrations, individually and in combination, on microalgal lipids and biomass production is a scarcely exploited area. Combined metal stress alleviates the constraint of low biomass production under nitrogen limitation and improved the overall lipid productivity. Highest lipid productivity of 73.23 mg L⁻¹ d⁻¹ was achieved with a combination of iron 9 mg L⁻¹, magnesium 100 mg L⁻¹ and calcium 27 mg L⁻¹ at limited nitrogen (750 mg L⁻¹). This was 1.72 fold higher than nitrogen stress alone and 1.99 fold higher than BG11 medium. Iron was found to be most significantly influencing metal followed by magnesium in response surface methodology data analysis. The enhanced photosynthetic performance and chlorophyll content further confirmed the significant impact of iron and magnesium on the microalgal biomass. The addition of EDTA to the optimised metal combination further improved the lipid productivity to 80.23 mg L⁻¹ d⁻¹ (2.18 fold). At 3000 L open cultivation pond this strategy has resulted in an increase of 2.08 fold in lipid productivity. Higher biodiesel conversion rates were also observed with this easy, universally applicable and scalable lipid enhancement strategy.

Introduction

Biodiesel production from microalgae is potentially feasible as these tiny photosynthetic microbes present substantial biomass and lipid productivity compared to other edible and non-edible plant based feedstocks [1], [2], [3]. Furthermore, microalgal lipids are majorly constituted of suitable fatty acid composition for the biodiesel production [4], [5], [6]. Though there are several remarkable advantages of microalgae over other feedstocks, biomass production is still an expensive process due to a number of factors associated with microalgal cultivation [7], [8]. One possible solution for improving the economics of microalgal cultivation is to select a suitable strain and investigate its response to different cultivation conditions for enhancement of the overall lipid productivity [9], [10], [11]. It is well known that a number of factors could influence lipid accumulation in microalgae, such as nitrogen starvation or limitation [12], [13], phosphate limitation [14] high salinity, carbon source concentration, light intensity, and temperature [15], [16], [17]. Under the stress conditions, microalgae tend to accumulate energy in dense forms such as lipids [2], [10], [18]. Recent reports have shown the potential of combining different nutrients and abiotic stress factors to improve microalgal lipid productivity. To obtain maximum yields, it is imperative to have knowledge of the synergistic effects of factors as well as significance of each factor with regards to lipid accumulation [15], [19], [20], [21], [22]. Breuer et al. [15] investigated the effects of light, pH, and temperature on TAG accumulation under nitrogen deficient conditions and found pH and temperature to be major influencing factors for TAG accumulation. The highest TAG content (40%) was obtained at pH 7 and 27.6 °C which was independent of light variation. Ji et al. [23] studied the effects of variation in temperature, light intensity and photoperiod on Desmodesmus sp. and observed high biomass production at a combination of temperature: 30 °C, light intensity: 98 μmol m⁻² s⁻¹ and photoperiod: 14:10 (L:D Light:Dark photoperiod). The effects of environmental factors such as light intensity and temperature have been studied at laboratory scale in controlled environment [7], [10], [24]. However, controlling these conditions at commercial level are impractical and energy intensive thus incurring additional cost [10], [25], [26], [27]. Alteration of other nutrient concentrations could be the possible solution for tackling the drawbacks of the nitrogen limitation, as it is easy to undertake and scalable. Karpagam et al. [28] investigated the effects of different stress factors on the lipid accumulation of two microalgal strains Coelastrella sp. M-60 and Micractinium sp. M-13 by using response surface methodology. Combination of high concentrations of citric acid and effluent from sugar industry resulted in 1.35 fold increase in lipid yield.

Though nitrogen limitation is the widely applied strategy for lipid enhancement, it is associated with compromised biomass production which ultimately affects the overall lipid productivity [10], [12]. Hence, huge culture volumes or repeated runs are required to compensate for the loss of biomass at large scale, which will increase the overall production cost [29]. Thus, there is need for a strategy which can be coupled with nitrogen limitation to improve biomass and also enhance the lipid accumulation to accomplish attractive lipid productivity.

Trace metals such as iron, magnesium, copper, calcium, manganese are very important for the cellular mechanism of microalgae viz, photosynthesis, cell division, respiration, intra cellular transportation, protein and lipid biosynthesis and are reported to improve biomass and lipid yields in microalgae [30]. Liu et al. [31] studied the effect of iron on the lipid content of Chlorella vulgaris, and found that it can be improved up to 56% by addition of iron (1.2 × 10⁻⁵ mol L⁻¹) into the medium. A combination of low nitrogen and high iron concentration also reported to increase lipid productivity of up to 74.07 mg L⁻¹ d⁻¹ in Ankistrodesmus falcatus [8]. Huang et al. [32] reported 1.25 folds increase in the cell density of Monoraphidium sp. FXY-10 with supplementation of 100 μM magnesium into the medium. Similarly, Ethylene-diamine-tetra-acetic acid (EDTA) supplementation also reported to improve the lipid accumulation in the microalgae [10]. Ren et al. [33] reported an increase in the total lipid content (28.2%) and lipid productivity (29.7%) in microalgae Scenedesmus obliquus with an increase in EDTA concentration (0–1 mg L⁻¹) thereby revealing its potential as a lipid enhancer.

Effect of metals and EDTA on microalgae for enhanced lipid and biomass productivity is still a scarcely exploited area. Previous studies were focused on the effect of individual metals in improving biomass and lipid productivities of microalgae under sufficient nitrogen concentrations. To the best of our knowledge no other study investigated the combined impacts of these metals and EDTA under nitrogen limited conditions. A few studies have reported the actual physiological role of the metal on microalgae. Thus the aim of the present study was to develop an integrated strategy involving combined metals and EDTA stress to alleviate the compromised biomass production at nitrogen limited condition and enhance lipid accumulation for achieving the possible ceiling lipid productivity by microalgae. The photosynthetic physiology of microalgae under metal stress was examined for better understanding of role of these metals in overcoming the drawbacks of nitrogen limitation. Biodiesel conversion and impact of this strategy on fatty acid composition was also studied.