Floridoside production by the red microalga Galdieria sulphuraria under different conditions of growth and osmotic stress

Abstract

Floridoside is a compatible solute synthesized by red algae that has attracted considerable attention due to its promising antifouling and therapeutic properties. However, research on industrial applications of floridoside is hampered by limited compound availability and the development of a production process yielding high amounts of this glycoside has not been explored yet. In the present work, floridoside accumulation by the red microalgae Galdieria sulphuraria under different conditions was investigated in order to optimize the production of this glycoside in this microalgae. G. sulphuraria shows consider advantages over other red algae as potential industrial producer of floridoside due to its unicellular nature, its ability to grow heterotrophically in complete darkness and its acidophilic lifestyle. The main compatible solute accumulated by G. sulphuraria under salt stress was purified, identified as floridoside by ¹H-NMR and used as standard for quantification. Our results showed that applying the osmotic stress after the cells had grown first in medium with no salt resulted in higher floridoside yields compared to those obtained in cells growing under osmotic stress from the beginning. Among several parameters tested, the use of glycerol as carbon source for cell growth showed the most significant impact on floridoside accumulation, which reached a maximum of 56.8 mg/g dry biomass.

MartinezGarcia and van der Maarel AMB Expr (2016) 6:71 DOI 10.1186/s1356801602446

Floridoside production bythe redmicroalga Galdieria sulphuraria underdierent conditions ofgrowth andosmotic stress

Marta MartinezGarcia and Marc J. E. C. van der Maarel*

Introduction

Compatible solutes are small organic molecules synthesized by cells under various stress conditions that can be accumulated at high intracellular concentrations without interfering with the normal functioning of the metabolism (da Costa etal. 1998; Roberts 2005; Hagemann and Pade 2015). Floridoside (2-O--d-galactopyranosylglycerol) is a compatible solute synthesized by almost all red algae species, except the members of the class Ceramidiales, under high osmotic pressure conditions (Kirst and Bisson 1979; Reed 1985; Ekman etal. 1995). This glycoside also constitutes the major soluble pool of carbon xed by photosynthesis and is a precursor for cell wall polysaccha-rides in some species (Li etal. 2001, 2002). Apart from its

invivo role as osmolyte, oridoside has been described to have certain properties that have raised the interest in this molecule. Hellio etal. (2004) reported that oridoside is able to inhibit the settlement of cryptid larvae on the surface of underwater devices, suggesting its application as non-toxic, natural compound for preventing biofouling, a worldwide problem estimated to cause a loss of billions of dollars to the marine industry (Callow and Callow 2002). Moreover, oridoside is a potential therapeutic agent with the ability to modulate the immune response (Courtois et al. 2008; Kim et al. 2013) and to promote bone formation (Ryu et al. 2015). Floridoside shares structural similarity with 2-O--d-glucopyranosylglycerol (GG), a compatible solute accumulated by cyanobacteria that is considered a promising moisturizing agent (Thiem etal. 1997), a non-cariogenic, low calorie sweetener (Takenaka and Uchiyama 2000) and a protein stabilizer (Sawangwan etal. 2010). The structural similarity suggests that orido-side might also be functional in these applications.

*Correspondence: [email protected] Biotechnology and Bioproduct Engineering, Engineeringand Technology Institute Groningen (ENTEG), University of Groningen, Groningen, The Netherlands

2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/

Web End =http://creativecommons.org/licenses/by/4.0/ ), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

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The development of industrial applications of oridoside is hampered by limited compound availability. Chemical synthesis of oridoside has been reported, albeit with insufficient yields and requiring a sequence of steps to direct the reaction towards the stereochemically pure product (Wewer and Linhardt 2008). To date, there are no studies describing the enzymatic production of oridoside, but this strategy has been used for the synthesis of the related compounds 3-O--dgalactopyranosylglycerol and GG (Takenaka and Uchiyama 2000; Goedl etal. 2008; Wei etal. 2013; Jeong etal. 2014). Although the use of glycosidases takes advantage of the stereospecicity of these enzymes when forming a linkage in a single step, it suers from a lack of regioselectivity towards a specic hydroxyl group, leading to the product being a mixture of regioisomers (Scigelova etal. 1999) that can complicate the downstream processing. Extraction of oridoside from the natural producers, i.e. red algae, is a promising alternative but requires the optimization of the cultivation conditions to increase the production of this glycoside by the cells.

In the present study, we opt for the extremophilic red microalgae Galdieria sulphuraria as oridoside producer. This unicellular rhodophyta is one of the most primitive eukaryotes on earth (Yoon et al. 2006) and thrives in acidic environments with pH values from 0 to 4 and temperatures up to 56 C. G. sulphuraria is also a metabolically exible species, being able to grow in complete darkness using a wide range of carbon sources (Gross and Schnarrenberger 1995) and displaying tolerance to various stresses (Schnknecht etal. 2013; Minoda etal. 2015; Pade etal. 2015). Its unicellular nature would confer G. sulphuraria an advantage over other multicellular red algae species for large scale cultivation and would allow to avoid seasonal variations in oridoside production reported for seaweed harvested from marine habitats (Kasrten etal. 1993; Meng and Srivastava 1993; Kerjean et al. 2007). Moreover, its acidophilic lifestyle would considerably reduce the risk of microbial contamination during large-scale fermentations.

In this work, we analyse oridoside accumulation under dierent cell cultivation and osmotic stress conditions in order to optimize the production of this glyco-side in G. sulphuraria.

Materials andmethods

Strain andcultivation conditions

Galdieria sulphuraria strain SAG 108.79 was purchased from the culture collection of the University of Gttingen (Sammlug von Algenkulturen, Gttingen, Germany). Cells were maintained growing on plates of Allens mineral medium (Allen 1959) at pH 4 with 1.5% (w/v) agar at 40C and constant illumination of 100E/m2s. Colonies

were transferred to a fresh plate once a month. For liquid cultures, G. sulphuraria was grown at 40C in complete darkness on a rotary shaker at 150rpm in Allen medium at pH 2 supplemented with 1% (w/v) glycerol and, when applicable, NaCl at a concentration of 0.5, 1 or 1.5M. Cell growth was monitored by measuring the OD at 800nm.

In order to purify oridoside that could be used as standard, cells were grown until late exponential phase and then salt-stressed with 1M NaCl for 24h at 40C. To construct the time-course of oridoside and glycogen content after salt addition, cells were grown until late exponential phase and then salt-stressed with 1 M NaCl for 0, 4, 8, 16, 24 and 48h at 40C. To determine the eect of the carbon source and type and concentration of the osmotic agent on oridoside production, cells were grown on 1% (w/v) carbon source (glycerol, galactose or glucose) until late exponential phase, harvested and washed with ultra-pure water and resuspended in 100mL of the osmotic agent (NaCl, KCl, CaCl2 or sorbitol) at dierent concentrations (when applicable, 0.5, 1 or 1.5M) for 24h at 40C. To test the eect of temperature on oridoside production, cells were grown until late exponential phase on 1% glycerol, salt-stressed with 1M NaCl and incubated at 20, 30 or 50C for 24h. For the experiment with stepwise osmotic stress conditions, cells were grown until late exponential phase and then salt-stressed with NaCl added in 2, 5 or 10 steps until a nal concentration of 1M for 24h at 40C.

Obtention ofthe low molecular weight compounds (LMW) fraction andoridoside purication

Osmotically stressed cells were harvested by centrifugation at 5000g for 5min, washed twice with ultra-pure water and freeze-dried. The dry cell pellet was mixed with 20 mL of 80 % ethanol and low molecular weight compounds were extracted from the cells by two rounds of 15min stirring plus 15min incubation in an ultrasonic bath (Elma) at room temperature, followed by a nal incubation in a waterbath at 70C for 5min Cell debris was removed by centrifugation at 10,000g for 10 min and the supernatant was mixed with one volume of ultra-pure water and 0.5 volumes of chloroform. After separation of the two phases by centrifugation at 10,000g for 10min, the upper (hydroalcoholic) phase was transferred to a new tube and mixed with ionic resin Amberlite MB20 (DOW) overnight. The supernatant was concentrated under vacuum on a rotary evaporator and freeze-dried. The dry residue was resuspended in 1mL of ultra-pure water. This was denominated the LMW fraction. Floridoside was puried from this fraction by preparative thin layer chromatography (TLC) on silica gel 60 plates (Merck-Millipore) using isopropanol:ethylacetate:water (3:1:1 by volume) as mobile phase. The identity and purity

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of oridoside was conrmed by 1H-NMR analysis. The sample was dissolved in 600L of deuterium oxide (D2O, 99.9 %atom, Sigma-Aldrich), freeze-dried and exchanged in the same solvent one more time. 1H-NMR spectra were recorded on a Varian 500 spectrometer (NMR centre, University of Groningen, The Netherlands) at a probe temperature of 25C. Acetone ( 1H 2.225ppm in D2O)

was used as internal reference for chemical shift assignment and data were analysed using MestReNova 9.1.0 (Mestrelab Research S.L).

Floridoside quantication

Floridoside fractions were analysed by high pH anion exchange chromatography coupled with pulsed ampero-metric detection (HPAEC-PAD) on a ICS3000 workstation equipped with a CarboPac PA-1 column (2250mm) and a ICS3000 ED detector (Dionex) using isocratic elution in 50mM NaOH. A standard series of puried oridoside in a concentration range of 5500M was used to construct a calibration curve for quantication. Floidoside yields were expressed relative to the dry biomass.

Glycogen extraction andquantication

Glycogen was extracted from G. sulphuraria cells as previously described (Martinez-Garcia etal. 2016) and was quantied relative to the dry biomass. For the comparison of glycogen content at dierent growth phases, all cell suspensions were prepared for disruption at the same concentration of 50mg of dry cells/mL water.

Results

Galdieria sulphuraria was grown heterotrophically in medium containing 1% glycerol and dierent NaCl concentrations and cell growth was monitored by measuring OD values at 800nm. NaCl had an eect on both the duration of the lag phase prior exponential growth and the maximal OD value reached by the culture (Fig. 1). In cultures containing 0.5 and 1M NaCl, the lag phase had a similar duration (4856h) than that of the culture without salt. Cultures containing 1.5M NaCl showed a remarkably longer lag phase when compared to the others (144 h), after which cells could still grow exponentially. The maximum OD values were aected by NaCl in a concentration-dependent manner. Nonetheless, all salt-stressed cultures reached OD values that were close to or higher than 7.

In order to identify the major compatible solute in G. sulphuraria, the low molecular weight compounds of osmotically stressed cells were extracted with 80% ethanol. The major constituent of this fraction was puried by preparative TLC and analysed by 1H-NMR (Fig.2). The compound was identied as oridoside according to the

chemical shifts reported by Simon-Colin et al. (2002). The purity of oridoside was conrmed by the absence of a signal at 4.9ppm, characteristic of the anomeric proton in isooridoside (1-O--d-galactopyranosylglycerol) (Bondu etal. 2007). This puried oridoside was used to prepare a calibration curve to quantify the production of the glycoside by G. sulphuraria under dierent growth and osmotic stress conditions.

Because cell growth was delayed by NaCl addition, especially at high concentrations, we decided to analyse oridoside content in cells that were osmotically stressed only after pre-growing in medium without salt and compare it to that of cells growing under osmotic stress. With this strategy, higher biomass yields could be obtained and the duration of the production process would be shortened. In order to determine the time-point of the growth curve at which the osmotic stress should be applied to obtain the highest oridoside yields, we quantied the amount of biomass, glycogen and oridoside at dierent growth phases (Table1). In both late exponential and stationary growth phases, the amount of biomass (4.15 and 4.94g dry cells/L, respectively) and the amount of glycogen accumulated by the cells (36.76 and 35.40 % of the dry biomass, respectively) were very similar. However, the amount of oridoside was 3 times higher in late exponential phase than in stationary phase. Consequently, in subsequent experiments the osmotic stress was applied once the cells had reached late exponential growth phase in medium with no salt.

A time-course of oridoside accumulation after osmotic stress application was performed in order to identify the moment at which the amount of glycoside was maximal. Floridoside content showed almost a ve-fold increase during the rst 8h after salt addition and then a more moderate increase during the following

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Table 1 Biomass, glycogen andoridoside yields atdierent phases ofG. sulphuraria growth inmedium with1% glycerol andno salt

Growth phase Biomass (g dry cells/L) Glycogen (% dry biomass) Floridoside (% dry biomass)

Early exponential 0.69 0.09 20.07 1.39 0.52 0.02

Middle exponential 2.68 0.51 29.39 2.50 1.20 0.04

Late exponential 4.15 0.19 36.76 2.03 1.41 0.14

Stationary 4.94 0.17 35.40 5.79 0.47 0.06

Values represent the average of three independent measurementsstandard deviation

hours until reaching a maximum of 5.4% of the dry bio-mass after 24h (Fig.3). Maintaining the osmotic stress over a longer time (48h) did not result in higher orido-side accumulation. Glycogen content decreased slightly during the rst 8h, concomitant to the increase in oridoside during that period, and then showed variable values for the rest of the time-points, reected by the high standard deviations obtained.

We then compared the accumulation of oridoside in late exponential phase cultures that were growing under osmotic stress on dierent NaCl concentrations with that of cultures pre-grown on medium with no salt and then osmotically stressed for 24h. The latter strategy resulted in signicantly increased oridoside yields, which were around 10 times higher in the case of cultures osmotically stressed with 1 and 1.5 M NaCl (Fig. 4). In cells growing under osmotic stress, oridoside accumulation did not correlate with the increase in salt concentration, since the content of glycoside was higher in cultures containing 0.5M NaCl than in cultures with greater amounts of salt. In cells osmotically stressed after reaching late

exponential growth phase, an increase in NaCl from 0.5 to 1M resulted in a twofold increase in oridoside content, but a higher salt concentration did not yield more glycoside.

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Finally, we tested the inuence of dierent parameters on the accumulation of oridoside by G. sulphuraria, such as the carbon source used for cell growth, the type of compound causing the osmotic stress, the temperature of incubation and the way of applying the osmotic stress. Cells that had grown on glycerol as carbon source accumulated around 2.5 times more oridoside when subjected to osmotic stress compared to those grown on galactose or glucose (Fig.5a). The use of sorbitol (as an example of non-ionic solute) and KCl as osmotic agents resulted in oridoside yields that were not signicantly dierent (p>0.05) to those obtained with NaCl (Fig.5b). CaCl2 had a negative inuence on oridoside accumulation, the content being at least 2.5 times lower than that obtained with the other types of solutes. The addition of the osmotic agent to the culture at once or in a stepwise manner and the temperature of incubation during the osmotic stress did not cause remarkable dierences in oridoside yields (Fig.5c, d), although cells incubated at 50C accumulated somewhat lower amounts of glyco-side. Cultures incubated at 60 and 70C during osmotic stress yielded very low amounts of oridoside (data not shown).

Discussion

Floridoside is a compatible solute known to be accumulated by marine red seaweeds (Kirst 1980; Reed et al. 1980; Ekman etal. 1995) that has attracted considerable attention for its potential antifouling and therapeutic properties (Hellio et al. 2004; Courtois et al. 2008; Kim et al. 2013; Ryu et al. 2015). However, industrial applications for oridoside have not been developed yet due

to limited compound availability. A production process yielding high amounts of this glycoside would facilitate this task. With this idea in mind, we analysed oridoside production by the thermoacidophilic red microalgae G. sulphuraria under dierent conditions in order to assess its potential as industrial producer for this glycoside.

Although this microalgae species does not inhabit marine environments, it is reported to be tolerant to high concentrations of dissolved substances in the medium (Schmidt et al. 2005), including NaCl (Gross et al. 2002), and to accumulate oridoside as compatible solute (De Luca and Moretti 1983; Pade etal. 2015). Accordingly, we found that G. sulphuraria growth was not inhibited by NaCl, although it was considerably slowed down. Therefore, we investigated the possibility of shortening the process of oridoside production by inducing its accumulation in late-exponential cells grown in medium with no salt. With this strategy, cultures reached higher cell densities in less time and cells accumulated substantial amounts of glycogen. The storage glucan constitutes an easily accessible intracellular reserve of glucose from which the precursors of oridoside (UDP-galactose and glycerol-3-phosphate) can be synthesized (Hagemann 2016). Other red algae species have been reported to synthesize oridoside under hyperosmotic conditions from carbon obtained by degradation of the intracellular storage glucan rather than from newly assimilated carbon (Reed 1985; Ekman etal. 1995; Simon-Colin etal. 2004; Bondu etal. 2009). Interestingly, we observed that G. sulphuraria accumulated glycogen already at early stages of the growth curve, diering from other microorganisms where glycogen accumulation is triggered by macronutrient limitation (Lillie and Pringle 1980; Preiss 1984). G. sulphuraria also produced oridoside in quantiable levels before being osmotically stressed, which is in accordance with the fact that this glycoside is not only a compatible solute but also a transient carbon reservoir (Li etal. 2001). This dual role of oridoside could be responsible for the dramatic dierences in glycoside content between cells growing under osmotic stress and cells stressed only after reaching late exponential phase in medium with no salt. During prolonged (96 h) incubation under high osmotic pressure, other compounds known to be synthesized by G. sulphuraria under salt stress [e.g. betaine (Schnknecht et al. 2013)] could have taken over the role of compatible solute, allowing oridoside to function as carbon reservoir and to be degraded to sustain exponential cell growth. For cells that were osmotically stressed after growing rst in medium with no salt, the shorter incubation time with salt (only 24h) could result in a preferential accumulation of oridoside over other osmolytes.

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Floridoside accumulation following an osmotic shock was a fast response. 70% of the maximal oridoside yield was reached within the rst 8 h, a similar rate to that observed for the marine red seaweed Porphyra purpurea (Reed etal. 1980). The decrease in glycogen content did not mirror completely the increase in oridoside, suggesting that cells might use glycogen as substrate for oridoside production only during the rst hours of osmotic stress. Afterwards, compounds released from surrounding dying cells might have been the main source of precursors for oridoside synthesis, therefore eliminating the need to degrade glycogen. This seems plausible if we consider that G. sulphuraria possesses a high number of membrane sugar transporters to assimilate a wide range of substrates (Schnknecht etal. 2013).

Floridoside accumulation in G. sulphuraria cells osmotically stressed for 24 h was mainly dependant on the carbon source used for cell growth, with glycerol inducing the highest accumulation. All osmotic

stress-causing agents, regardless of being ionic salts or a non-ionic solute, induced similar oridoside production, suggesting that oridoside synthesis in G. sulphuraria is not directly regulated by specic ions but is dependent on the external osmotic pressure, as Reed etal. (1980) described for the red macroalga P. purpurea. The temperature independence of oridoside accumulation would represent an advantage when considering G. sulphuraria as potential industrial producer for oridoside because the osmotic stress step could be performed without heat supply, thereby reducing process costs.

In conclusion, in this study we have described the culture conditions that promote the highest oridoside accumulation in G. sulphuraria. Further optimization of the cultivation conditions and the extraction procedure for increased biomass and oridoside yields could turn G. sulphuraria into an efficient industrial producer of oridoside, a promising antifouling and therapeutic compound.

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Authors contributions

MMG and MM designed the experiments, analysed the results and wrote the manuscript; MMG performed the experiments. Both authors read and approved the nal manuscript.

Acknowledgements

The authors wish to thank prof. dr. Lubbert Dijkhuizen (Microbial Physiology, University of Groningen) for kindly allowing us to use the HPAECPAD equip ment and dr. Hans Leemhuis for critical reading of the manuscript.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was nancially supported by Avebe (Veendam, The Netherlands).

Received: 15 August 2016 Accepted: 7 September 2016

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