Advance Publication J. Gen. Appl. Microbiol. doi 10.2323/jgam.2017.06.001 „2017 Applied Microbiology, Molecular and Cellular Biosciences Research Foundation Full Paper The effects of abscisic acid, salicylic acid and jasmonic acid on lipid accumulation in two freshwater Chlorella strains (Received May 18, 2016; Accepted June 5, 2017; J-STAGE Advance publication date: December 30, 2017) Guanxun Wu,1,# Zhengquan Gao,1,#,* Huanmin Du,1 Bin Lin,1 Yuchen Yan,3 Guoqiang Li,1 Yanyun Guo,1 Shenggui Fu,2 Gongxiang Wei,2 Miaomiao Wang,1 Meng Cui,1 and Chunxiao Meng1, * 1 School of Life Sciences, Shandong University of Technology, Zibo 255049, China 2 School of Sciences, Shandong University of Technology, Zibo 255049, China 3 The Undergraduate Brigade, Second Military Medical University, Shanghai 200433, China Sustainable renewable energy is being hotly de- Key Words: biodiesel; Chlorella; lipid; bated globally because the continued use of finite phytohormones; renewable energy fossil fuels is now widely recognized as being un- sustainable. Microalgae potentially offer great op- portunities for resolving this challenge. Abscisic Introduction acid (ABA), jasmonic acid (JA) and salicylic acid (SA) are involved in regulating many physiologi- Due to diminishing reserves, the global petroleum sup- cal properties and have been widely used in higher ply is shrinking and rising in cost at a fast pace, and fur- plants. To test if phytohormones have an impact ther climate change will be enhanced by the continued use on accumulating lipid for microalgae, ABA, JA and of fossil fuels. These factors have increased the demand SA were used to induce two Chlorella strains in the for alternate fuels, thereby making the production of fuels present study. The results showed 1.0 mg/L ABA, from alternate sources more feasible. Microalgae, the larg- 10 mg/L SA, and 0.5 mg/L JA, led strain C. vul- est biomass producers, have a high neutral lipid content, garis ZF strain to produce a 45%, 42% and 49% outcompeting terrestrial plants for biofuel production, and lipid content that was 1.8-, 1.7- and 2.0-fold that of also have the ability to grow rapidly and synthesize and controls, respectively. For FACHB 31 (number 31 accumulate large amounts of neutral lipid stored in of the Freshwater Algae Culture Collection at the cytosolic lipid bodies (Mutanda et al., 2011). Biodiesel is Institute of Hydrobiology, Chinese Academy of Sci- produced from microalgal oil, thus crude fossil petroleum ences), the addition of 1.0 mg/L ABA, 10 mg/L SA, could be replaced by mass cultured biomass microalgal and 0.5 mg/L, JA produced 33%, 30% and 38% oil for eco-sustainable biodiesel production in the near lipid content, which was 1.8-, 1.6- and 2.1-fold that future (Rawat et al., 2013). However, the high cost of al- of controls, respectively. As for lipid productivity, gae production is the biggest obstacle to the adoption of 1.0 mg/L ABA increased the lipid productivity of an industrial and commercial process. A major conclusion C. vulgaris ZF strain and FACHB-31 by 123% and of the cost analyses for large-scale microalgae production, 44%; 10 mg/L SA enhanced lipid productivity by conducted at the end of the last century, is that, even with 100% and 33%; the best elicitor, 0.5 mg/L JA, aug- aggressive assumptions, project costs for biodiesel are two mented lipid productivity by 127% and 75% com- times higher than current petroleum diesel fuel costs pared to that of controls, respectively. The results (Sheehan et al., 1998). Therefore, the need to search for above suggest that the three phytohormones at low-cost and highly efficient ways to induce microalgae physiological concentrations play crucial roles in to produce lipids is urgent. inducing lipid accumulation in Chlorella. *Corresponding authors: Zhengquan Gao and Chunxiao Meng, School of Life Sciences, Shandong University of Technology, Zibo 255049, China. Tel: +86 533 2762265 Fax: +86 533 2781832 E-mail: [email protected] Tel: +86 533 2781329 Fax: +86 533 2781832 E-mail: [email protected] #These authors contributed equally to this work. None of the authors of this manuscript has any financial or personal relationship with other people or organizations that could inappropriately influence their work. 2 WU et al. Algae can modify their lipid metabolism efficiently and display tremendous diversity and sometimes unusual pat- terns of cellular lipids in response to changes in environ- mental conditions, and this is one of the most important reasons why algae can survive and proliferate over a wide range of environmental conditions (Guschina and Harwood, 2006; Hu et al., 2008). Lipid production, like the production of other carbohydrate-based storage com- pounds, is often dependent on environmental conditions, some of which await elucidation and development (US DOE, 2010). As stated in previous studies, most microalgae can alter their lipid accumulation and other secondary metabolic products under unfavorable cultiva- tion conditions as a protective mechanism to cope with stresses. In recent years, considerable attention has been focused on lowering biofuel costs, greenhouse gas emis- sions, land and water resource needs, and on improving compatibility with fuel distribution systems and vehicle engines (Demirbas, 2011). Growth conditions and the ge- netics of lipid production have been explored with the goal of enhancing the economic production of microalgae for bioproducts or lipids for biofuels recently (Tate et al., 2013). Various studies have focused on enhancing growth and metabolite production by varying culture conditions Fig. 1. Fluorescence (A, C, E, G, I, K, M, O, Q and S) and light microscopy (B, D, F, H, J, L, N, P, R and T) images of C. vul- of the algae, including pH, temperature, irradiation level, garis ZF strain and FACHB 31 in different statuses of the in- carbon source, aeration and concentrations of specific duction course. nutrients (Bhola et al., 2011; Hsieh and Wu, 2009). Other A and B, K and L indicate the beginning status of C. vulgaris ZF strain studies have focused on the genes involved in the biosyn- and FACHB 31 (on day 1 of cultivation) with dyeing of Nile red, re- thesis of fatty acids in microalgae (Merchant et al., 2011). spectively. C and D, M and N indicate the final status (on day 18 of However, thus far no reports have investigated using cultivation and the 3rd day of starvation) of the blanks of C. vulgaris ZF strain and FACHB 31, respectively. E and F, O and P indicate the phytohormones to induce microalgae to accumulate lipids final status of 10 mg/L ABA treatments; G and H, Q and R indicate the to the best of our knowledge, although they have been final status of 1.0 mg/L SA; I and J, S and T indicate the final status of widely used in higher plants to enhance production or 0.5 mg/L JA treatments of C. vulgaris ZF strain and FACHB 31, re- quality. spectively. In most cases, these phytohormones act as signal mol- ecules to promote and drive many physiological proper- ties in the cells. It has been reported that phytohormones Chlorella to heavy metal pollution and other abiotic are necessary for manipulating some physiological and stressors, which suggests that they might play important biochemical processes in algal cells, including growth and roles in responding to abiotic stressors and algal adapt- aging performance, metabolic product biosynthesis and ability to stresses (Bhola et al., 2011; Piotrowska- resistance to stresses (Tarakhovskaya et al., 2007). How- Niczyporuk et al., 2012). ever, knowledge of the algal hormonal system and infor- These phytohormones should possess a good potential mation about hormone metabolism and mechanisms of to facilitate microalgal lipid production, since they can action in algae are still rather fragmentary, since this in- promote metabolite production in C. vulgaris and C. formation is extremely scarce (Bajguz, 2009; pyrenoidosa (Tate et al., 2013). However, little attention Tarakhovskaya et al., 2007). Currently, the presence of a has been paid to the effects of plant hormones on full-value hormonal system in algae and the correspond- microalgal lipid production. ABA, SA and JA, three typi- ence of its biological activities with those of higher-plant cal stress hormones, are involved in the regulation of many hormones are debated; the metabolism and mechanisms physiological properties by acting as signal molecules. of action of phytohormones in microalgae are still unclear. Previous studies have also indicated that ABA, SA and JA Chlorella is a well-studied genus of green algae and are used widely in higher plants (Bajguz, 2009). However, some species are regarded as potential candidates for the relationship between lipid accumulation and hormone microalgae biodiesel production since they can make cer- stimulation in microalgae is still unknown. In this regard, tain amounts of lipids that are proposed for use in biofuel the purpose of the present study was to investigate the production (Bajguz, 2010; Hu et al., 2008). Chlorella has effects of ABA, SA, and JA, on lipid accumulation in two also been used as a model for plants and a useful tool for Chlorella strains (C. vulgaris ZF strain and FACHB 31, studying the influences of phytohormones on growth and C. pyrenoidesa). It was anticipated that the application of metabolite accumulation (Czerpak et al., 2006; Hu et al., ABA, SA, and JA, could lead to improved lipid accumu- 2008). There are reports that some phytohormones, includ- lation in the two Chlorella strains, so that a higher lipid ing auxins, cytokinins, ABA, polyamines, brassinosteroids, production could be achieved. JA and SA, can improve the adaptability and tolerance of The effects of abscisic acid, salicylic acid and jasmonic acid on lipid accumulation in two freshwater Chlorella strains 3 Fig.