Elsevier

Bioresource Technology

Volume 281, June 2019, Pages 118-125
Bioresource Technology

Nitrogen modulation under chemostat cultivation mode induces biomass and lipid production by Chlorella vulgaris and reduces antenna pigment accumulation

https://doi.org/10.1016/j.biortech.2019.02.063Get rights and content

Highlights

Abstract

Algal growth limitation in large-scale cultivation mostly results from high level synthesis of photosynthetic pigments, owing to self-shading effects and attenuation of light distribution. To overcome this problem, here we investigated the influence of nitrogen modulation on changes in antenna pigments as well as biomass and lipid production by Chlorella vulgaris under a chemostat continuous cultivation mode. The production of algal antenna pigments, including chlorophylls and carotenoids, was promoted in a total nitrogen (TN) concentration-dependent manner. Maximum algal biomass and lipid production were obtained from 70 mg/L of TN concentration along with a significant increase in light transmittance and reduction in antenna pigments. Furthermore, the composition of polyunsaturated fatty acids remarkably augmented at low TN concentrations. These results suggest that the reduction in algal antenna pigment synthesis via modulation of nitrogen concentration may serve as an effective strategy to enhance algal biomass and lipid production.

Introduction

The demand for algal biomass has steadily increased owing to their applications for the production of alternative fuels, bioactive compounds, as well as nutraceuticals and pharmaceuticals (Odjadjare et al., 2017). Microalgal lipid production has attracted attention of researchers, as the extracted algal lipid may not only be converted into available biodiesel through trans-esterification reaction but may be used for bioactive fatty acid (FA) production (Chauton et al., 2015, Chisti, 2007, Odjadjare et al., 2017). In comparison with common terrestrial plants, microalgae show higher growth rate, and their oil production is estimated to be as high as 136,900 L/ha following a maximum of 70% oil accumulation (Chisti, 2007). Moreover, algal biomass does not compete with human food resources such as corn and soybean and excludes the requirement of wide arable land (Chisti, 2007, Odjadjare et al., 2017). Therefore, algal biomass has been considered as a promising sustainable resource for green technology. However, the high costs of algal lipid derived from low biomass and lipid productivity still hamper its industrial usage. Several studies have been conducted to improve algal biomass production via genetic engineering through mass cultivation facilities such as open raceway pond, photobioreactor (PBR), and conjugated system (Chagas et al., 2015, Chisti, 2007, Kim et al., 2018, Yun et al., 2018, Lee et al., 2018, Radakovits et al., 2010). The combination of mass cultivation facility and genetic engineering has promoted algal biomass and target metabolite production as well as flocculation activity required for harvesting using symbiotic phycospheric bacteria and different stress factors (Cho et al., 2015, Cho et al., 2019, Kakarla et al., 2018, Kim et al., 2014, Lee et al., 2013, Ramanan et al., 2016). However, the high antenna pigment accumulation in algal chloroplast may limit biomass production, owing to self-shading effect and limited light transmittance into culture medium, especially at stationary growth phase (Perrine et al., 2012). Furthermore, the fast photon energy capture by light-harvesting complex (LHC) and slow downstream electron transfer rate cause over 50% energy loss in algal cells; hence, reduction in algal antenna pigment production is essential to enhance light transmittance and biomass production (Perrine et al., 2012) and has emerged as one of the efficient strategies (Blankenship and Chen, 2013, Perrine et al., 2012). In a previous study of Jin et al. (2016), deficiency of HPE1, an uncharacterized gene, was shown to affect the expression of chlorophyll-related genes in nucleus and reduce the chlorophyll content of Arabidopsis thaliana. The loss of HPE1 gene function enhanced the photosynthetic quantum yield of A. thaliana. Perrine et al. (2012) reported that the reduction in chlorophyll b level in microalga Chlamydomonas reinhardtii efficiently increased algal growth. The results of these studies demonstrate that the reduction in antenna size may increase the photosynthetic efficiency per unit illumination by promoting light transmittance and enhance algal biomass production. Although mutant-based modulation of antenna size showed successful performance under laboratory settings, transient mutation still causes problems related to the maintenance of large scale cultivation system. Furthermore, other approaches for algal antenna pigment reduction, especially chemostat cultivation mode, are poorly understood.

Chemostat cultivation mode has been effectively applied to monitor the effect of light intensity on the growth of microalga Ettlia sp. (Seo et al., 2017). Chemostat cultivation is ideal for algal physiological study, as various nutrients are continuously maintained during the cultivation period and diverse factors are effectively controlled by the chemostat control system. Furthermore, chemostat culture assures continuous algal biomass production by maintaining stable conditions (Seo et al., 2017). However, over the past decades, most of algal physiological studies have been performed under batch cultivation mode and the results may be affected by changes in nutrient composition.

Nitrogen plays vital role in algal growth through the synthesis of essential cellular components, including proteins, nucleic acids, and chlorophyll molecules (Lourenço et al., 2004). Nitrogen dose-dependently decreased the chlorophyll content of Chlorella vulgaris in a 5 L cylindrical plexiglass PBR (Lv et al., 2010). Moreover, Dean et al. (2010) reported that the chlorophyll a content of microalgae C. reinhardtii and Scenedesmus subspicatus significantly decreased under low nitrogen concentration in all growth phases. Despite the inhibitory effects of low nitrogen concentration on cell growth, the results revealed that nitrogen regulation efficiently modulated antenna pigment production in microalgae. Thus, in the present study, we hypothesized that the reduction in antenna size through nitrogen modulation in a continuous chemostat cultivation system may mitigate the self-shading effect and eventually increase algal biomass productivity. Furthermore, as algal biomass shows improved lipid productivity in most algal cells under low nitrogen levels, we speculate an increase in algal lipid productivity in optimal light transmittance zone (OLTZ) (Zhang et al., 2013). To prove these hypotheses, we examined the effect of total nitrogen (TN) concentration on antenna pigment change and biomass production as well as lipid productivity in a freshwater microalga C. vulgaris OW-01 under chemostat cultivation mode.

Section snippets

Design of chemostat mode for algal cultivation

Microalga C. vulgaris OW-01 was previously isolated from swine wastewater (Cho et al., 2013). Algal cultivation was performed using BG11 medium (Stanier et al., 1971). To examine the effect of TN concentration on antenna pigment production, a chemostat continuous cultivation system was designed as previously described by Cho et al. (2016) (Fig. 1). Peristaltic pump was used for the supply of TN-modulated BG11 culture medium (50–250 mg/L) into a 170 mm × 150 mm glass PBT vessel (CNS, Korea) with

Fluctuation in algal growth under different TN concentrations

To estimate algal growth response under various TN concentrations, daily cell concentration and pH change were monitored under nitrogen-modulated chemostat cultivation mode. As shown in Fig. 1, BG11 media containing different levels of nitrogen (NaNO3) were continuously supplied into 3 L algal culture via silicon tube lines with peristaltic pump. This chemostat cultivation system was successfully applied in previous studies for monitoring biomass and lipid productivity of microalgae Ettlia sp.

Conclusions

In the present study, nitrogen-modulated microalgal chemostat cultivation was conducted to verify algal growth response and cellular antenna pigment accumulation as well as algal lipid production. The results revealed that nitrogen modulation under chemostat cultivation system significantly reduced algal antenna pigment accumulation, thereby increasing algal biomass and lipid productivity along with light transmittance. Our results suggest that the maintenance of well-modulated nitrogen level

Acknowledgements

This work was supported by a grant from Marine Biotechnology Program (20150184) funded by Ministry of Oceans and Fisheries, Korea; by the Advanced Biomass R&D Center (ABC) of Global Frontier Project funded by the Ministry of Science and ICT (ABC-2015M3A6A2065697); and by grant from the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (www.kribb.re.kr).

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