Research article
Lipid production in aquatic plant Azolla at vegetative and reproductive stages and in response to abiotic stress

https://doi.org/10.1016/j.plaphy.2018.01.012Get rights and content

Highlights

  • In Azolla TAG containing lipid droplets accumulate in all organs and symbiont.

  • The highest level of lipids was detected in the male microsporocarps.

  • Stress reduces lipid level and increases production of anthocyanins in Azolla.

  • Azolla is feedstock for production of biodiesel.

Abstract

The aquatic plant Azolla became increasingly popular as bioenergy feedstock because of its high growth rate, production of biomass with high levels of biofuel-producing molecules and ability to grow on marginal lands. In this study, we analysed the contribution of all organs of Azolla to the total yield of lipids at vegetative and reproductive stages and in response to stress. Triacylglycerol-containing lipid droplets were detected in all (vegetative and reproductive) organs with the highest level in the male microsporocarps and microspores. As a result, significantly higher total yields of lipids were detected in Azolla filiculoides and Azolla pinnata at the reproductive stage. Starving changed the yield and composition of the fatty acid as a result of re-direction of carbon flow from fatty acid to anthocyanin pathways. The composition of lipids, in regard the length and degree of unsaturation of fatty acids, in Azolla meets most of the important requirements for biodiesel standards. The ability of Azolla to grow on wastewaters, along with their high productivity rate, makes it an attractive feedstock for the production of biofuels.

Introduction

The goal of reducing our dependency on fossil fuels, and to prevent further deforestation and competition with agriculture, has triggered an extensive search for domestication of new bioenergy feedstock which (i) can generate substantial renewable biomass over a short period; (ii) can grow on marginal lands; and (iii) are rich in bioenergy molecules which can be converted into biofuels using a set of well-established technologies (Long et al., 2013, Miranda et al., 2016). Search for the energy crops which can use wastewater as a source of key nutrients represents one of the most globally researched areas and is becoming a subject of intense public and scientific interest (Miranda et al., 2014, Miranda et al., 2016, Marmiroli et al., 2006, Salt et al., 1998, Dushenkov, 2003, Muradov et al., 2014). Most of the known terrestrial bioenergy crops cannot grow even in diluted wastewaters. Microalgae have been extensively investigated as the third generation of bioenergy feedstocks because of their high growth rates, substantial lipid concentration and ability to grow in wastewaters removing their primary pollutants (C, N, and P) (Borowitzka and Moheimani, 2013, Schenk et al., 2008, Rajkumar et al., 2014, Egede et al., 2016). However, the high cost of harvesting of microalgae (up to 30% of total cost) is still the major obstacle for the large-scale production of low-value products such as biofuels (Borowitzka and Moheimani, 2013, Schenk et al., 2008, Rajkumar et al., 2014, Aguirre et al., 2013).

Aquatic plants represented by submerged, emerged (rooted) and free-floating species colonise contaminated wetlands have attracted significant attention as a potential valuable feedstock for second and third generation biofuels because of their ability to produce a large amount of biomass, impressive bioremediation rates, as well as cheap and easy maintenance and harvesting (Miranda et al., 2014, Miranda et al., 2016, Dhir et al., 2009, Cui and Cheng, 2015). Among these species, free-floating plants have obvious advantages because of their low harvesting cost. A number of free-floating aquatic species which have been evaluated for both wastewater treatment and biofuel production include: water hyacinth (Eichhornia crassipes) (Ruan et al., 2016, Zhao et al., 2012a), water lettuce (Pistia stratiotes), (Viger et al., 2015, Robles-Pliego et al., 2015, Mukherjee et al., 2015), water ferns: Salvinia (Chandanshive et al., 2016, Mubarak et al., 2016) and Azolla species (Miranda et al., 2016, Muradov et al., 2014, Costa et al., 1999, Costa et al., 2009, Pereira et al., 2011, Pereira and Carrapico, 2009, Brouwer et al., 2014, Brouwer et al., 2016), and representatives of the Lemnaceae or duckweed (Appenroth et al., 2015, Lam et al., 2014, Zhao et al., 2012b, Verma and Suthar, 2015).

Azolla (also known as mosquito fern, duckweed fern, fairy moss and water fern) has become increasing popular because of its biomass production and bioremediation potential (Miranda et al., 2016, Muradov et al., 2014, Costa et al., 1999, Costa et al., 2009, Pereira et al., 2011, Brouwer et al., 2014, Brouwer et al., 2016, Carrapiço, 2010). Unlike most of the terrestrial and aquatic plants, Azolla can grow efficiently even in the absence of nitrogen in media utilising the nitrogen-fixing capacity of its natural symbiont, the endophytic cyanobacterium, Anabaena azollae Strasburger (A. azollae) (Zheng et al., 2009, Pereira and Vasconcelos, 2014, Calvert and Peters, 1981). This symbiosis is associated with the fixation of up to 1.1 t/ha-yr of nitrogen, which is significantly higher than the nitrogen fixation rate of legumes (0.4 t N/ha-yr) (Costa et al., 1999, Hall et al., 1995, Kollah et al., 2016).

Doubling its biomass every 4–7 days Azolla is one of the fastest growing plants, with a productivity varying between 2.9 and 5.8 g dw/m2-day (10.5–21.1 t dw/ha-yr) when grown on artificial media, wastewaters and maturation ponds (Fig. S1) (Kollah et al., 2016). Under optimal conditions in natural ecosystems, such as rivers, lagoons and irrigation channels Azolla can bloom with growth rates up to 300 g/m2-day of fresh biomass (1095 t/ha-yr) (Van Hove et al., 1987) and 25.6–27.4 g dw/m2-day of dry biomass (93.4–100 t dw/ha-yr) (Miranda et al., 2016, Costa et al., 1999, Peters et al., 1980a). Growth in wastewater is associated with the removal of key nutrients such as N and P with rates of up to 2.6 t N/ha-year and 0.434 t P/ha-yr, respectively (Muradov et al., 2014, Costa et al., 1999, Costa et al., 2009, Song et al., 2012).

Azolla is a relatively new feedstock for bioenergy production, and its promising potential is based on its unique chemical composition. Together with their evolutional symbiont, A. azollae, Azolla representatives contain three major types of energy molecules, starch (approx 6% dw) and cellulose/hemicellulose (up to 35% dw) and lipids (8% dw) which are found separately in known terrestrial feedstocks and microalgae (Miranda et al., 2016, Costa et al., 1999, Costa et al., 2009, Brouwer et al., 2016, Song et al., 2012, Peters et al., 1980b).

The energy accumulated in lipids/TAG is twice that of cellulose which makes them a desirable feedstock for bio-oil production (Winichayakul et al., 2013). In terrestrial plants, TAG are mainly accumulated in seeds, pericarps, and pollen, which can transfer carbon molecules from one generation to the next and allow seeds to germinate until photosynthesis becomes effective (Lin and Oliver, 2008, Baud and Lepiniec, 2010). The production of these oil-containing organs in terrestrial plants is seasonal. Moreover, in spite of the fact that oil plants can accumulate up to 50% dw of TAG in their seeds, just 0.06–0.5% dw of the TAG can be extracted from the vegetative organs, leaves and stems (Lin and Oliver, 2008, Yan et al., 2013). This triggers an intensive search for the plant species which can accumulate a substantial amount of lipids/TAG in their vegetative organs, such as leaves.

In this work, we continued research on the use of Azolla as the next generation bioenergy crop. To our knowledge, for the first time, we (i) analysed the contribution of each organ and cyanobacterial symbiont of A. filiculoides on its total lipid yield; (ii) showed developmental stage-specific changes in lipid's yield and composition triggered by the production of the lipid-rich male microsporocarps at the reproductive stage; (iii) showed changes in lipid yield and composition associated with up-regulation of the flavonoid pathway in stressed plants; (iv) assessed A. pinnata (the second most common Azolla species widely found in warm-temperate and tropical regions) as feedstock for lipid/bio-oil production; and (v) evaluated contributions of lipid composition, the length, and degree of unsaturation of fatty acids of Azolla for the key biodiesel properties, such as iodine number, cetane number, density, pour point and others. Duckweed representative, Landoltia punctata (L. punctata), which lipid content was analysed earlier was used as a control in this study (Verma and Suthar, 2015, Yan et al., 2013, Zhong et al., 2016).

Section snippets

Growing Azolla and duckweed

L. punctata, A. filiculoides, and A. pinnata were collected from RMIT University's collection of aquatic plants. Plants were grown in 10 L containers with Hoagland nutrient solution (for L. punctata) and H40 media (for Azolla species) (Pereira and Carrapico, 2009). The plants were grown in a glasshouse covered with shade cloth, with natural photoperiod and at 23-26 °C. The photosynthetic photon flux density was 50–70 μmol/m2/s. The solution in each container was mixed every day. Three

Characterization of A. filiculoides, A. pinnata and L. punctata plants

Fig. 1 shows red colouration of A. filiculoides and A. pinnata leaves in response to different abiotic stresses, such as cold and starvation (Fig. 1). Earlier we showed these stresses are triggering the accumulation of flavonoids and anthocyanins molecules in these plants (Muradov et al., 2014). L. punctata contains red pigments mainly on the lower, abaxial side of the fronds. Similar to terrestrial flowering plants seasonal changes in temperature and photoperiod triggers reprogramming of

Conclusions

  • In Azolla lipids and a TAG containing LD were detected in all (vegetative and reproductive) organs with the highest level in the male microspores.

  • Azolla's response to the changing developmental and environmental conditions triggering re-programming of the fatty acid pathways. Exposure to the stress re-directs carbon flow from the fatty acids to the flavonoid and anthocyanin pathways, which leads to a reduction of the total lipid's level.

  • Non-stressed Azolla growing at the reproductive stage can

Funding

Australia: this work was funded by the School of Science of RMIT University.

Authors’ contributions

AM coordinated this project and designed experiments related to organ- and developmental stage-specific production of lipids; involved in the interpretation of data, made substantial contributions in writing the manuscript, and approved the final version for publication. AFM involved in growing of aquatic plants, a collection of samples, statistical analysis, writing of the manuscript; ZL and SR conducted a biochemical analysis of lipids. All authors were involved in final approval of the

Competing interests

The authors declare that they have no competing interests.

Acknowledgements

This work was supported by the College of Science, Engineering and Health and the School of Science of RMIT University.

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