Algal Research 49 (2020) 101931

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Algal Research
journal homepage: www.elsevier.com/locate/algal

Characterization and comparison of Porphyridium sordidum and Porphyridium T

purpureum concerning growth characteristics and polysaccharide production
⁎
Edilberto Vicente Medina-Cabreraa, Broder Rühmanna, Jochen Schmida,e, Volker Siebera,b,c,d,
a
Chair of Chemistry of Biogenic Resources, Technical University of Munich, Campus for Biotechnology and Sustainability, 94315 Straubing, Germany
b
Catalysis Research Center, Technical University of Munich, 85748 Garching, Germany
c
Fraunhofer IGB, Branch BioCat, 94315 Straubing, Germany
d
The University of Queensland, School of Chemistry and Molecular Biosciences, 68 Cooper Road, St. Lucia 4072, Australia
e
Norwegian University of Science and Technology, Department of Biotechnology and Food Science, Sem Sælands vei 6-8, 7491 Trondheim, Norway

A R T I C LE I N FO A B S T R A C T

Keywords: Microalgae as photosynthetic microorganisms are the source of valuable compounds such as proteins, car-
Porphyridium sordidum otenoids, lipids and carbohydrate polymers. Among the different phyla, Porphyridium as part of the red micro-
Porphyridium purpureum algae (Rhodophyta) are of high importance as producers of sulfated polysaccharides. As the name suggests these
Growth behavior microalgae are typically red; however, Porphyridium sordidum has an olive-green color and was recently de-
Carbohydrate monomers
scribed as a putative exopolysaccharide producer. Exopolysaccharides (EPS) are sugars polymers excrete to the
Sulfate
culture medium. In this study P. sordidum is evaluated for the first time in detail as newly described EPS producer
Rheological measurements
in direct comparison to the already characterized EPS producer Porphyridium purpureum. The evaluation was
performed on several aspects, such as morphological differences between the two strains followed by the
comparison of the growth behavior, nitrate and phosphate uptake during cultivation of both strains, and a
detailed analysis of the EPS. P. purpureum consumed 51 ± 4.6% of nitrate and 95 ± 2.1% of phosphate
compared to P. sordidum which utilized 17 ± 0.1% of nitrate and 68 ± 0.1% of phosphate at day 30 of
cultivation. The produced EPS were analyzed and revealed the major constitutional carbohydrate monomers of
the EPS to be xylose, galactose, glucose and glucuronic acid. In addition, other substituents that decorate the EPS
were identified as sulfate and methyl groups. Finally, rheological measurements (viscosity curves and amplitude
sweeps) were performed to analyze the physicochemical properties.

1. Introduction properties [8,9]. Unlike most bacterial and fungal EPSs, microalgae
EPSs can bear specific functional substituents like methyl (CH3) and
Among the different products generated by microalgae, the poly- sulfate (SO4−2) groups. These substituents confer unique properties to
saccharides represent important bio-macromolecules. Basically, there the EPS for broader applications as antiviral, anticoagulation and
are two types of polysaccharides that are produced by microalgae, the bioremediation agents [10–12].
storage polysaccharides in the cytoplasm and the secreted ones, which Currently, several cyanobacteria (prokaryotic algae) are known as
can be found in the culture media, also known as exopolysaccharides EPS producers. The most important ones are Nostoc, Phormidium,
[1,2]. EPSs are produced by various microbes, with the xanthan gum Arthrospira and Cyanothece genus [13,14]. On the other hand, several
producing bacteria Xanthomonas campestris as the best known com- phyla of eukaryotic microalgae are also reported to produce EPSs, such
mercial representative [3]. Other bacteria described to produce EPS are as Chlorophyta (green microalgae), Bacillariophyta (diatoms), Crypto-
Azotobacter vinelandii (alginate), Sphingomonas paucimobilis (gellan), phyta, Miozoa (dinoflagellates) and Rhodophyta (red microalgae)
and Bacillus subtilis (levan) [4–6]. Additionally, some fungi have been [2,13,15]. Several genuses from the Rhodophyta such as Rhodella,
described as EPS producer, such as Aureobasidium pullulans (pullulan) Flintiella, Rhodosorus and Porphyridium have been described as EPS
and Sclerotium glutanicum (scleroglucan) to name just two [4,7]. These producers [16–20].
EPSs have multiple applications in the pharmaceutical, cosmetic and In this work, Porphyridium sordidum was characterized in detail as
food industries because of their rheological and health promoting an EPS producer for the first time. Although, this strain is a

⁎
Corresponding author at: Chair of Chemistry of Biogenic Resources, Technical University of Munich, Campus for Biotechnology and Sustainability, 94315
Straubing, Germany.
E-mail address: [email protected] (V. Sieber).

https://doi.org/10.1016/j.algal.2020.101931
Received 8 October 2019; Received in revised form 22 April 2020; Accepted 22 April 2020
2211-9264/ © 2020 Elsevier B.V. All rights reserved.
E.V. Medina-Cabrera, et al. Algal Research 49 (2020) 101931

Rhodophyta, it has a distinctive olive-green color [21–24]. In addition, 2.4. Harvesting of exopolysaccharides (EPS)
P. sordidum was compared to the better characterized EPS producing
Rhodophyta, P. purpureum, by analyzing growth behavior, EPS pro- At the end of the culturing process, EPS was harvested by alcohol
ductivity and composition [25]. precipitation. In brief, the microalgae cells were pelleted by cen-
trifugation at 4000 xg for 10 min at RT. The supernatants (370 mL)
2. Material and methods were separated from the cell pellets and mixed by use of an overhead
stirrer (Heidolph Instruments GmbH & Co. KG, Schwabach, Germany)
2.1. Chemical and reagents with two volumes of 2-propanol to precipitate the EPS. Finally, the EPS
fibers were sieved to be separated from the alcohol mixture. Then, the
All reagents were of analytical or microbiological grade and were collected EPS was re-dissolved overnight in 250 mL of ultra-pure water,
purchased from Carl Roth GmbH (Karlsruhe, Germany) or Merck KGaA centrifuged at 75,000 xg for 15 min to eliminate remaining con-
(Darmstadt, Germany) unless stated otherwise. All the experiments taminants, and then re-precipitated with 2 volumes of 2-propanol, to be
were performed with ultra-pure water obtained from a PURELAB collected by sieving the EPS with an organdy cotton cloth and dried at
system. RT for 3 days to evaporate the 2-propanol.

2.2. Microalgae strains and culture conditions 2.5. Determination of the EPS monomer composition

The two microalgae used in this work, Porphyridium sordidum (SAG The carbohydrate monomers of the P. sordidum and P. purpureum
114.7) and Porphyridium purpureum (SAG 1380-1a) were initially cul- EPSs were analyzed as described by Rühmann et al., 2014 [29]. In brief,
tivated in different media such as Bold's Basal Medium (BBM), BG-11, a 0.1% solution of each EPS sample was prepared in ultra-pure water.
modified Provasoli (Pm) and modified Artificial seawater (ASW) The evaluations were performed as technical triplicates. A 20 μL vo-
medium, all the media compositions are given in the supplementary lume of aliquots was hydrolyzed with 20 μL of 4 M trifluoroacetic acid
data, Annex 1. The media were autoclaved at 121 °C for 20 min. (TFA) at 121 °C for 90 min, centrifuged at 2000 ×g, 2 min, 20 °C and
Samples were maintained in conical flasks for the conducted studies neutralized with an appropriate volume of 3.2% ammonium hydroxide.
in modified ASW media. To evaluate growth behavior, both strains Then, 25 μL of the neutralized EPS solution was transferred to a mi-
were cultured in a cylinder system designed in our group using the crotiter plate and mixed with 75 μL of a PMP reagent (0.1 M methanolic
following parameters. Illumination of the system was done by an ex- 1-phenyl-3-methyl-5-pyrazolone solution: 0.4% ammonium hydroxide
ternal white fluorescent lamp (LUXLINE PLUS, F18W, Germany) and an solution 2:1), incubated at 70 °C for 99 min in a PCR cycler. A 20 μL
additional blue fluorescent lamp (AQUA-GLO, 20 W T8, Japan) in a 12/ volume of aliquots of the samples was mixed with 130 μL of 1:26 di-
12 day-night cycle. Air was provided to the cylinder system at a fixed luted 0.5 M acetic acid, and filtrated (0.2 μm Supor, Pall Corporation)
flow of 100 mL min−1 controlled by a flowmeter (Read-y for gas flow, into a microtiter plate. The settings used for the HPLC were the fol-
Switzerland). The set-up of the entire system is displayed in the sup- lowing: pure acetonitrile (eluent A) and 5 mM ammonium acetate
plementary data, Annex 2. The inoculum was cultured in a 250 mL buffer (pH 5.6) with 15% acetonitrile (eluent B); the Macherey-Nagel
conical flask for 15 days at room temperature (RT), 170 rpm and 12/ Gravity C18 column, 100 mm length, 2 mm i.d., 1.8 μm particle size,
12 h light/dark cycle with a white LED light (1000 lm, 15 W, Germany). was used in this test at a fixed temperature of 50 °C and a flow rate of
Each strain was cultured in biological triplicate in a final volume of 0.6 mL min−1 coupled to a mass-spectrophotometer. The chromato-
0.5 L, daily; 2 mL samples were taken and centrifuged at 27,000 xg for grams were evaluated using the Chromeleon™, Bruker Hystar and
5 min, the free-cell media were stored at −20 °C for further analysis. Quant Analysis software.
The growth was evaluated by measuring the OD at 750 nm, which was
reported to be used for different microalgae strains as it allows the 2.6. Glucose assay
evaluation of the cell growth without the interference of the pigments
that are naturally produced by microalgae in Griffiths et al., 2011 and The glucose content was measured before and after the hydrolysis
Gaignard et al., 2019 [26,27]. The specific growth rate (μmax) Eq. (1) and neutralization steps using the method described by Rühmann et al.,
and doubling time (td) Eq. (2) were calculated for the microalgae used 2016 [30]. 1:10 dilutions of the hydrolyzed samples from PMP were
in this study (supplementary data, Annex 3) [28]. But it must be taken used in this evaluation. Briefly, 50 μL of standard solutions (0, 2.5, 5,
into account, that these values are apparent, as factors that affect mi- 10, 25, 50, 100, 250 and 500 μM) and samples were placed in 96-well
croalgae growth (photoperiod, light shadowing in high concentrated plates (technical triplicate). Followed by the addition of 50 μL of master
culture, etc.), were not considered for their calculation. mix (40 mM potassium phosphate at pH 5.7, 1.5 mM 2.2-azinobis-(3
The Zeiss Axio Observer Z1 microscope was used to measure the ethylbenzthiazoline)-6-sulfonic acid, 4 U glucose oxidase and 0.2 U
diameter of 12 random microalgae cells and the t-test was used to horseradish peroxidase) into the samples and standards. An incubation
evaluate the data. step was required at 400 rpm for 30 min at 30 °C to measure the
samples at 418 nm and at 480 nm for background correction.
2.3. Evaluation of nitrate and phosphate consumption
2.7. Pyruvate assay
The nitrate (NO3−) uptake was determined by a NO3− electrode
(PRONOVA Analysentechnik GmbH & Co. KG). Standards of sodium This assay was performed according to Rühmann et al., 2016 [30].
nitrate (1, 2, 4, 6, 8, 10 and 12 mM) were added to ASW medium 100 μL of a 1:10 diluted samples (before and after hydrolysis and
without nitrate and the supernatant from these samples were measured neutralization) and standards (0, 0.5, 1, 2.5, 5, 10, 25, 50 and 100 μM)
in technical triplicate. were placed in a microtiter plate and mixed with 100 μL of master mix;
The phosphate (PO43−) uptake was tested in technical triplicate by 0.1 mM N-(carboxymethylamino‑carbonyl)-4.4′-bis(dimethylamino)-di-
use of the colorimetric assay Phosphate-test PO43−(Merck). 100 μL phenylamine sodium salt (DA-64), 0.1 mM thiamine pyrophosphate,
monopotassium phosphate standards solutions (0.01, 0.02, 0.04, 0.08, 0.2 mM magnesium chloride, 40 mM potassium phosphate buffer at
0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 mM) were prepared in phosphate free pH 5.7, 0.1 U pyruvate oxidase and 0.4 U horseradish peroxidase.
ASW medium, and supernatants free of microalgae cells were mixed Followed by incubation at 37 °C, 400 rpm for 30 min; and measured at
with 25 μL of the reactant and measured at 400 nm in 96 flat bottom 727 nm and 540 nm (correction wavelength) for the pyruvate quanti-
microtiter plates (MTP). fication. All measurements were performed as technical triplicate.

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E.V. Medina-Cabrera, et al. Algal Research 49 (2020) 101931

Fig. 1. Microscopic pictures were taken with the Zeiss Axio

Observer Z1 with the ×100 objective on samples at day 20 of
the culturing process. (A) Porphyridium sordidum showing
olive-green color and a diameter of around 10 μm (B)
Porphyridium purpureum with the typical red color of
Rhodophyta. Compared to P. sordidum it has a smaller dia-
meter (around 8 μm). The scales denote 10 μm for both
strains. (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this
article).

2.8. Determination of the EPS sulfate content Data were collected and analyzed with Rheoplus V.3.61 software.

The sulfate content was quantified and compared using the two
methods described in the following lines. An enzymatic method de- 3. Result and discussion
scribed by Ortiz-Tena et al., 2018 [31]. In which, 50 μL of hydrolyzed/
neutralized samples (1:20 dilution), magnesium sulfate standards (2.5, 3.1. Evaluation of the most suitable media composition and morphological
5, 10, 25, 50, 100, 150, 200 and 250 μM), positive control (carrageenan characterization
κ) and negative control (gellan) were mixed with 50 μL of master mix
(12.5 mM of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) Different prevalent media were tested to identify the most suitable
buffer at pH 7.8, 1 mM guanosine triphosphate, 1 mM adenosine tri- one for both Porphyridium strains due to the different sources of where
phosphate, 3 mM magnesium chloride, 0.46 μM adenosine triphosphate this genus can be found (fresh and salty water) [33]. The use of BBM
sulfurylase and 5.3 μM adenylyl-sulfate kinase), incubated at RT for and BG-11 resulted in poor growth of P. purpureum. The modified Pm
45 min. Then, 100 μL of a second master mix (100 mM dipotassium and ASW medium presented cell growth for P. purpureum, but a higher
phosphate at pH 6.5, 100 μM N-(carboxymethylamino‑carbonyl)-4.4′-bis growth was detected in the modified ASW medium (data not shown).
(dimethylamino)-diphenylamine sodium salt (DA-64), 50 μM thiamine Therefore, this medium was chosen to perform the following experi-
pyrophosphate, 100 μM magnesium chloride, 500 μM phosphoe- ments. Even more, the ASW medium was previously reported to be used
nolpyruvate, 500 μM adenosine monophosphate, 0.05 U pyruvate for culturing P. purpureum [34,35]. For P. sordidum, the only medium
phosphate dikinase, 0.05 U pyruvate oxidase and 0.2 U horseradish that could sustain cell growth was the modified ASW, although it was
peroxidase) was added and incubated at 37 °C for 30 min; measured at reported that P. sordidum was isolated from the botanical greenhouse
727 nm and 540 nm. The second method used was a modified version of samples and not from a marine water environment [21,22]. The mod-
the turbidimetric method described by Lundquis et al., 1980 [32]. In ifications performed on the ASW media for this study are the following.
brief, 200 μL of standards (0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9 and The nitrate source was changed from potassium (used in previous in-
10 mM), as well as 1:10 hydrolyzed samples were placed in measuring vestigations) to the sodium salt at the same concentration of 9.88 mM
triplicate (96 well plates), 100 μL of a master mix at 300 rpm, which because sodium nitrate is most commonly used in other microalgae
contained 2.4 mM barium chloride and 15 mM PEG 6000, to be mea- media [36]. Additionally, TRIS-HCl was excluded from the ASW
sured at 600 nm. All evaluations were performed in technical triplicate. medium, given that it has no nutritional effect and other ASW media
reportedly do not have this compound in their composition [35]. Fi-
2.9. Determination of protein content nally, the microelement solution was prepared separately from the
other compounds from the media (supplementary data, Annex 1.1).
EPSs solutions of 0.1% were used to measure the protein content of The morphological evaluations of both Rhodophyta strains were
the samples by use of the Roti®-Quant Kit (Carl Roth). For this, 50 μL of performed with the Zeiss Axio Observer Z1 with the ×100 objective
sample and 0, 20, 40, 60, 80 and 100 mg L−1 of BSA standards were (Fig. 1). The phenotypical characteristic as reported, the red color of the
placed in technical triplicates in 96-well MTP and mixed with 200 μL of P. purpureum and the olive-green color of the P. sordidum could be
the Bradford reagent at 100 rpm for 30 s and incubated at 37 °C for confirmed [21,22]. The morphological evaluation allowed the com-
5 min to be measured at 590 nm. parison of the mean cell-size, which could affect growth rate and EPS
production. P. sordidum cells have a larger diameter (9.8 ± 0.8 μm)
2.10. Rheological measurements compared to the P. purpureum (7.9 ± 0.7 μm) cells, therefore P. sor-
didum has a lower doubling time because of its bigger dimension
1% solutions from P. sordidum and P purpureum EPS were dissolved compared to P. purpureum. This effect was previously described in other
in ultra-pure water overnight for rheological characterization. In short, microalgae (for example diatoms), showing that larger cell size had a
the measurements were performed in an air-bearing MCR300 con- lower growth rate when they were compared to other smaller cells [37].
trolled-stress rheometer, using cone plate geometry (50 mm diameter, 1 The quantification of the microalgae diameter is presented in the sup-
°cone angle, 0.05 mm gap) at a constant temperature of 20 °C. plementary data, Annex 4. A summary of the morphological char-
Two rheological measurements were performed as technical tripli- acteristics, such as diameters, calculated surface and volumes are pro-
cate, the viscosity curves which were obtained during a logarithmic vided in Table 1. The growth parameters, which were calculated in this
shear-rate ramp (γ̇ = 0.1–1000 s−1), and the shear stress amplitude investigation, are applicable to this particular culture system.
sweep (logarithmic ramp from 0.1–100 Pa) at a constant frequency of
1 Hz by recording measurements at 20 points per decade (log scale).

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E.V. Medina-Cabrera, et al. Algal Research 49 (2020) 101931

Table 1 data, Annex 5.2.

The diameter of the two Rhodophyta strains was calculated by measuring Moreover, the approximated doubling time (td) of P. purpureum
random cells (n = 12), and the surface and volume were calculated. A summary (3.2 ± 0.2 days) was shorter than that of P. sordidum
of the calculated growth parameters (td and μmax), the final values of the (4.1 ± 0.1 days). Despite the difference in the OD750nm response, the
OD750nm at the end of 30 day culture in the modified ASW medium. The growth
calculated doubling time of both strains differ only by one day. The
parameters are approximation as their calculation was done with OD750nm. The
bigger P. sordidum had a lower response compared to the smaller P.
data present denote the average and standard deviation as variability (n = 3).
purpureum in the entire visible spectra (supplementary data, Annex 5).
Characteristics Strain
3.3. Comparison of nutrient uptake for P. sordidum and P. purpureum
P. sordidum P. purpureum

Diameter [μm] 9.8 ± 0.8 7.9 ± 0.7 The nutrients uptake was analyzed for a detailed evaluation of
Surface [μm2] 306 ± 55 197 ± 33 growth characteristics. Nitrate was quantified as it is a key nutrient for
Volume [μm]3 509 ± 142 264 ± 64
cell proliferation and protein generation of microalgae [44]. It is known
μmax, [day−1] 0.17 ± 0.01 0.22 ± 0.01
td, days 4.1 ± 0.1 3.2 ± 0.2
that low concentrations of nitrate favor the shift to the stationary phase,
OD750nm 0.47 ± 0.04 3.2 ± 0.5 thus inducing EPS production as response to starvation [45,46]. For P.
purpureum, the nitrate uptake was 0.22 ± 0.0 mM day−1 in contrast to
P. sordidum which only consumed 0.06 ± 0.0 mM day−1. After
3.2. Comparison of the growth behavior of P. sordidum to P. purpureum 30 days of cultivation P. purpureum metabolized (68 ± 0.1%) and P.
sordidum (17 ± 0.1%) of the total nitrate. Thus, it can be concluded
The two Rhodophyta strains were compared in biological triplicate that the nitrate uptake affects growth of P. purpureum much more than
to evaluate their growth behavior in cylinder reactors. Two types of for P. sordidum due to its higher uptake as is shown in Fig. 3A.
light (white and blue light) were used for the culturing process, since it As second nutrient phosphate consumption was evaluated, for
is reported that blue light enhances cell growth as well as EPS pro- which it is known that its depletion is strongly related to a decrease of
duction in different microalgae strains and especially in the microalgae cell generation [44]. In this respect, it was observed that in
Porphyridium genus [38,39]. Daily samples were taken and the optical the first 10 days phosphate was consumed by P. purpureum, reaching
density (OD) at 750 nm was used to evaluate the growth rate. In a 0.04 ± 0.00 mM day−1. At the end of the cultivation process (day 30)
preliminary work, other wavelengths (OD440nm and OD680nm) which 95 ± 2.1% of phosphate was consumed (Fig. 3B). P. sordidum con-
were reported to be used to analyze microalgae growth, were tested and sumed 0.02 ± 0.00 mM day−1 of phosphate in the first 10 days. After
compared to OD750nm [40–42]. It was corroborate that the OD750nm was that, no significant phosphate uptake was observed until the end of the
the most suitable wavelength to measure cell growth in microalgae in cultivation process (51 ± 4.6%). Thus, it could be concluded that
addition it was previously reported to be used for P. purpureum [34]. phosphate greatly triggers cell growth as it was better metabolized than
Both Porphyridium strains grew best in modified ASW medium, with the nitrate.
P. purpureum showing significantly faster growth than P. sordidum
(Table 1). This might be explained by the different origin of the strains, 3.4. Gravimetrical determination of EPS titers
since P. purpureum was isolated from salty water and grew well in en-
riched media, such as the modified ASW medium [43]. The use of the The first precipitations of P. purpureum as well as P sordidum su-
OD750nm allowed an indirect comparison between the growth of both pernatants resulted in gravimetrically determined EPS titers of
strains (Fig. 2). The difference in the OD750nm detected between the P. 0.69 ± 0.04 g L−1 and 0.51 ± 0.02 g L−1 respectively. The sub-
sordidum and P. purpureum were mediated by the different cell size. This sequent analysis of the monomer composition showed that only a very
behavior was observed in the standard curve shown in supplementary low recovery of 14% could be realized, indicating a quite low purity of
the EPS, contaminated with e.g. cell debris, proteins and salts. By those
observations, an optimized purification protocol was established, re-
sulting in a carbohydrate recovery of 44% for P. purpureum and 42% for
P. sordidum, which corresponds to recoveries described for other mi-
crobial EPSs quantified by the HT-PMP method [47–49].
The titers of the purified EPSs were thus determined to be
0.24 ± 0.03 g L−1 for the P. purpureum, and 0.16 ± 0.01 g L−1 of EPS
for the P. sordidum. The final titers and the volumetric productivity are
summarized in Table 2. The volumetric productivity of P. purpureum
(8.1 ± 1.1 mg L−1 day−1) seems to be higher compared to P. sordidum
(5.4 ± 0.3 mg L−1 day−1),
Low N/P ratios (below 4.9) seem to induce EPS production, while
high N/P ratios (35–50) are described to induce cell growth, which,
depending on the strain, can also be associated with high EPS pro-
duction. The N/P ratio of around 16 is considered an equilibrated
medium, where N and P will be depleted simultaneously until ideally
both are limiting at the same time, leading to high cell growth as well as
high EPS production [50,51]. During the cultivation process due to the
lower nitrate consumption compared the phosphate uptake, the N/P
Fig. 2. Comparison of the growth curves (n = 3) of P. sordidum (green) and P. ratio increased in both strains, being more drastic in P. sordidum than in
purpureum (red), measured at OD750nm. The main difference of the measure- P. purpureum. This behavior opens the possibility to modify the culture
ments is the lower response of P. sordidum compared to P. purpureum at the medium to enhance the growth and EPS production in future in-
wavelength of 750 nm. The graph is based on the average including the stan- vestigations, as it was shown for other Porphyridium strains [50,51].
dard deviation as variability (n = 3). (For interpretation of the references to Nevertheless, individual evaluation of nutrients consumption showed,
color in this figure legend, the reader is referred to the web version of this that the phosphate consumption was higher compared to nitrate, and
article). this might have a higher impact on the EPS production for both

4
E.V. Medina-Cabrera, et al. Algal Research 49 (2020) 101931

Fig. 3. Comparison of nutrients uptake

(n = 3), nitrate (A) and phosphate (B) for
both Rhodophyta strains; P. purpureum (red)
and P. sordidum (green) over a 30 days
cultivation process. P. purpureum consumed
more nitrate and phosphate than P. sor-
didum, during the cultivation process. The
graph is based on the average including
standard deviation as variability (n = 3).
(For interpretation of the references to color
in this figure legend, the reader is referred
to the web version of this article).

Table 2 by the glucose assay, because monomeric glucose was only detected in
EPS titers obtained after the 30 days culture of both strains based on the op- the hydrolyzed samples, indicating that no monomeric glucose origi-
timized purification protocol. The volumetric productivity of EPS is given based nates from the culture medium. The polymeric structure of the EPSs in
on the calculations. Values are based on the average with the standard devia- both strains was corroborated by the glucose assay (supplementary
tion as variability (n = 3). data, Annex 7).
Strain EPSa [g L−1] Volumetric productivityb [mg L−1 day−1] Apart from the carbohydrate structure, the microalgae EPSs con-
tained other molecules as decoration [13]. In this sense, various
P. sordidum 0.16 ± 0.01 5.4 ± 0.3
methodologies were used to detect those substituents. Pyruvate was the
P. purpureum 0.24 ± 0.03 8.1 ± 1.1
first substituent to be analyzed in the microalgae EPS, because several
a
Concentration: EPS weight by volume. microbial and some microalgae EPS are to have it in their structure
b
Volumetric productivity: EPS weight by culture time and volume. [56,57]. A very sensitive enzymatic assay that uses pyruvate oxidase
(POX) was applied [30]. It revealed that the EPSs from both Porphyr-
Porphyridium strains. This behavior has also been observed in some idium strains did not contain any pyruvate. The protein content of the
cyanobacteria and diatoms [52–54]. EPS samples, as quantified with the Bradford assay, showed a protein
content of 8.3 ± 0.6 mg L−1 for the P. sordidum EPS, which was
0.84 ± 0.01% of the total EPS weight and 7.5 ± 1.5 mg L−1 for the P.
3.5. Evaluation of the EPS chemical composition purpureum EPS which was 0.77 ± 0.28% of the total EPS sample. Thus,
both EPSs contain a small amount of protein, which could be con-
The EPSs produced by P. sordidum were analyzed to consist of the sidered contamination.
following carbohydrate monomers expressed in mass percentage: The main difference in substituents between the EPSs was the sul-
44 ± 2.2% (196 ± 11 mg L−1) xylose, 31 ± 2.2% fate content, which is described to be a prominent substituent in mi-
(136 ± 5.5 mg L−1) galactose, 21 ± 0.83% (93 ± 3.4 mg L−1) croalgae EPS [13,58]. For the EPS of P. sordidum, a content of
glucose, and 4.1 ± 0.03% (18 ± 0.26 mg L−1) glucuronic acid. 179 ± 38 mg L−1 was detected, which correlates to 18 ± 4.2% of the
Moreover, the presence of uronic acids was corroborated by identifying total EPS content and for the EPS of P. purpureum, 122 ± 29 mg L−1
an uronic acid hexose dimer peak (m/z = 687). The uronic acid hexose were detected, which represent 13 ± 3.2% of the total weight. The
dimer was detected at 4.1 min; the fragmentation pattern is presented sulfate content was validated by obtaining similar results by the two
in the supplementary data, Annex 6.1. Also, another uronic acid hexose methods used in this study (supplementary data, Annex 7). The mea-
dimer was detected at 3.8 min, but the concentration was too low to sured 18% of sulfate for the P. sordidum EPS, is higher than the sulfate
obtain a fragmentation peak. These uronic acid hexose dimers can be content reported for other microalgae EPSs (6% to 10%) [58]. Fur-
explained in two ways. Since two hexoses were detected in the P. sor- thermore, a higher concentration of sulfate (around 13%) was detected
didum EPS, each one can generate a dimer with the glucuronic acid, in this study compared to the 9.1% reported for P. purpureum EPS by
thus two different dimers were detected. The monomer composition of Roussel et al., 2015 [25]. The complete chemical compositions of the
the P. sordidum EPS is shown in Fig. 4A. Recently, Nikolova et al., 2019 EPSs are summarized in Table 3.
provided a brief description of P. sordidum EPS, which was composed of However, the most prominent difference between the P. sordidum
xylose, galactose, glucose, glucuronic acid, mannose and rhamnose and P. purpureum EPS, evidenced in the mass spectra chromatogram
[55]. The more noticeable difference between the compositions of both (Fig. 4) and in the mass fragmentation pattern (supplementary data,
EPSs is the presence of mannose in the composition of the EPS recently Annex 6), were the methylation patterns for the P. sordidum EPS, the
reported in contrast with the P. sordidum EPS in this study. On the other presence of a methyl-pentose and a methyl-hexose was detected at
hand, the monomer composition of the EPS from P. purpureum was 8.8 min of retention time in the chromatogram of the P. sordidum EPS
previously described by Roussel et al., 2015, wherein the molar ratios (Fig. 4A). The methylated carbohydrates were identified by the parti-
calculated were: xylose 26.3%; galactose 44.2%; glucose 26.3%; glu- cular fragmentation pattern of m/z 525 (hexose m/z 511 + methyl m/z
curonic acid 2.3% and fucose 1.0% [25]. 14) and m/z 495 (pentose m/z 495 + methyl m/z 14) (supplementary
In contrast, a different composition was identified for the P. pur- data, Annex 6A). Quantification of these methylated carbohydrates was
pureum EPS in this study which is the following: 46 ± 1.4% not possible due to the lack of specific standards. Methylated carbo-
(207 ± 9.1 mg L−1) xylose, 30% ± 0.5 (132 ± 3.3 mg L−1) ga- hydrates are not very common in microbial polysaccharides, but they
lactose, 20 ± 0.7% (92 ± 2.2 mg L−1) of glucose and 3.8 ± 0.22% are described to be present in some polysaccharides of microalgae and
(17 ± 0.86 mg L−1) glucuronic acid. Fucose was not detected in the specifically in the polysaccharides of the Porphyridium sp. [59,60].
polymer. In addition, two uronic acid hexose dimers (at 3.8 and These methylated carbohydrates are of high interest, because they can
4.1 min) were identified, which are also present in the polysaccharide confer novel properties to the polysaccharide. Methyl carbohydrates
produced by P. sordidum. The monomers detected in the hydrolysates of have never been described for P. sordidum EPS [55].For P. purpureum no
the P. purpureum EPS are shown in the following chromatogram methyl pentose or methyl hexose was detected. However, an uronic
(Fig. 4B). The polymeric nature of microalgae EPSs were underpinned

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E.V. Medina-Cabrera, et al. Algal Research 49 (2020) 101931

Fig. 4. Overlay of UV 245 nm (black) and MS extracted ion (color) chromatograms (3–9 min), for P. sordidum (A) and P. purpureum (B); the EIC colors are the
following: pink m/z 687, purple m/z 701, yellow m/z 525, green m/z 511, magenta m/z 481, red m/z 525 and yellow m/z 495. (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this article).

acid hexose dimer was eluted at 4.8 min with an m/z = 701 (uronic The chemical compositions determined for the P. purpureum and P.
acid hexose dimer m/z = 687 + methyl m/z = 14), which an offset to sordidum EPSs in this study were different from the ones previously
the other previously mentioned dimers due to a hydrophobic group, reported [25,55]. This could be explained by differences of the culti-
such as methyl group is part of this dimer. However, the position of the vation media, cultivation process and analytics used to unravel the
methyl group and the dimer could not be identified due to the lack of an chemical composition of the EPS. This phenomenon has been reported
appropriate standard. The methyl group could be linked to the hexose for various EPS producing microalgae and cyanobacteria
as it was previously reported in other microalgae EPS or the methyl [13,59,61–69]. The amount of harvested EPS for both Rhodophytas
group could be linked to the uronic acid [13,59,61]. Remarking that decreased dramatically due to the applied intensified down-stream
this is the first time that a methyl pattern was detected in the P. pur- processing (2 x precipitations with 2-propanol, 2 x centrifugation and
pureum EPS, whose monomer composition was described before sieving steps). This decreased the overall EPS yields, but at the same
[13,25]. time increased the content of the carbohydrate fraction, due to the

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E.V. Medina-Cabrera, et al. Algal Research 49 (2020) 101931

Table 3 Table 4
Summary of the monomer composition and decorations as analyzed for both Consistency and flow index were obtained by fitting the viscosity curve data of
Rhodophyta EPSs (mg L−1). The table denotes the average and standard de- both EPSs into the power-law. The R2 was calculated to corroborate the cor-
viation as variability (n = 3). rected fitting of the three biological replicate used in this evaluation. The table
denotes the average and standard deviation as variability (n = 3).
Components P. sordidum P. purpureum
Strain Consistency Index (K) Flow index (n) R2
Quantifiable Glucose 93 ± 3.4 92 ± 2.2
Galactose 135 ± 5.5 132 ± 3.3 P. sordidum EPS 8.45 ± 0.31 0.08 ± 0.01 0.9998
Xylose 196 ± 11 207 ± 9.1 P. purpureum EPS 8.62 ± 1.03 0.08 ± 0.00 0.9998
Glucuronic acid 18 ± 0.26 17 ± 0.86
Protein 8.3 ± 0.6 7.5 ± 2.8
Sulfate 179 ± 38 122 ± 0.29
4. Conclusions
Sample concentration 972 ± 15 979 ± 17
% Recovery 65 ± 4.9% 59 ± 4.4%
Unquantifiable Pyruvate – – P. sordidum, as relative new EPS producer, was compared to the
Methyl pentose + – already described EPS producer P. purpureum in detail. The phenoty-
Methyl hexose + –
pical differences were mainly the bigger sized cells and the olive-green
Methyl uronic acid hexose dimer – +
color of P. sordidum, compared to the red colored and smaller sized cells
of P. purpureum. Uptake of nitrate and phosphate was higher for P.
elimination of salts and other co-precipitates. purpureum, which led to better growth, corroborating that an enriched
medium (modified ASW) enhanced the growth of P. purpureum in
contrast to P. sordidum. The better uptake of nutrients by P. purpureum
3.6. Rheological characterization of the microalgae EPS lead to higher EPS titers compared to P. sordidum. The carbohydrate
fraction was similar in both EPS, being xylose the most abundant car-
To analyze the physico-chemical properties of both EPSs and the bohydrate, follow by the galactose, glucose and glucuronic acid in that
putative influence of the different decorations with substituents, basic order. The most noticeable differences in the chemical structures were
rheological measurements were performed. The first evaluation was observed in the EPS decorations such as the sulfate content, which was
based on the determination of viscosity curves by increasing the shear higher in the P. sordidum EPS compared to P. purpureum EPS.
rates to detect the change in viscosity. Other EPSs such as xanthan, Furthermore, the main difference was the methylation pattern on the
Porphyridium sp. EPS and Porphyridium cruentum EPS showed a shear EPSs. In the P. purpureum EPS, a methylation was identified in a hexose
thinning behavior as indicated by lowered viscosity at increased shear glucuronic acid dimer for the first time, in contrast the P. sordidum EPS,
rates [17,49,70]. This behavior is explained by a decrease in the in- which had a methyl pentose and methyl hexose in its structure. Due to
teractions between the single EPS fibers due to increased shear rates, the similarities in the chemical compositions, the rheological properties
thus decreasing the viscosity. The viscosity curves for both EPSs were were quite similar, only the amplitude sweeps showed noticeable dif-
very similar, which might be due to the similar basic chemical com- ferences, possibly because of the distinct sulfate content and methyl
positions (Fig. 5A). The flow curve data fit the power-law (η = Kγ̇ n − 1 ), pattern of the carbohydrate polymers. A deeper physico-chemical
obtaining the consistency (n) and flow index (K), as presented in characterization can be achieved by applying further rheological eva-
Table 4. luations. Productivity might be enhanced via optimized cultivation
The second measurement was based on amplitude sweeps to eval- conditions. For this, intensified optimization of the media composition
uate the viscoelastic behavior by determination of the storage (G') and and a scale up of the cultivation system can be investigated; next steps
the loss modulus (G"). As the shear stress increased, both moduli are to enhance the EPS production and in that way continue in depth
maintained until they reached the yield stress where the values of the investigation of both EPSs.
moduli dramatically changed. In this evaluation a difference between
the P. sordidum EPS and P. purpureum EPS was observed. The yield stress
of the P. sordidum EPS was reached at a lower shear rate compared to Author contributions
the P. purpureum EPS. Both amplitude sweeps are presented in Fig. 5B.
The rheological evaluation of Porphyridium sp. and P. cruentum EPSs EVMC performed the experiments. JS VS and EVMC designed the
was reported in previous investigations. Although, each study had a study. EVMC, JS and BR analyzed and interpreted the data. EVMC JS
different focus; the flow curve was determined, showing a similar shear and BR wrote the manuscript. All authors revised it critically for sci-
thinning behavior to the EPS produced by P. sordidum and P. purpureum entifically as well as technically soundness. All authors approve of the
[17,70,71]. final version to be submitted.

Fig. 5. Both rheological evaluations (n = 3)

were performed at 20 °C with a 1% solution
of each of the EPSs. (A) Viscosity curves, for
the EPS of P. sordidum + and P. purpureum
x. (B) Amplitude sweeps, with storage
modulus G' (● P. sordidum and ■ P. pur-
pureum) and loss modulus G" (○ P. sordidum
and □ P. purpureum). The graph is based on
the average including the standard devia-
tion as variability (n = 3). (For interpreta-
tion of the references to color in this figure
legend, the reader is referred to the web
version of this article).

7
E.V. Medina-Cabrera, et al. Algal Research 49 (2020) 101931

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