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ORIGINAL ARTICLE
Enriching Rotifers with “Premium”Microalgae: Rhodomonas lens
Paula Coutinho
1,2
&Martiña Ferreira
1,3
&Isabel Freire
1
&Ana Otero
1
Received: 24 June 2019 / Accepted: 20 November 2019
#Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
The nutritional value of the marine cryptophyte Rhodomonas lens for the filter feeder Brachionus plicatilis as well as its
biotechnological potential as a source of phycoerythrin (PE) and polyunsaturated fatty acids (PUFA) were evaluated in semi-
continuous cultures maintained with different daily renewal rates (RR), from 10% (R10) to 50% (R50) of the total volume.
Steady-state cell density decreased from 22 to 7 × 10
6
cells mL
−1
with increasing RR, with the maximum cell productivity, nearly
0.4gL
−1
day
−1
, observed with R40. PE cell content attained the highest values with the highest RR (circa 9 pg cell
−1
). All
treatments of R. lens maintained under nitrate-saturated conditions (R20-R50) showed a similar high content of PUFAs, > 60% of
total fatty acids (FA), with linolenic acid (18:3n-3) and 18:4n-3, representing 12 and 29% of total FA respectively. The PUFA
level in the nitrogen-limited R10 cultures was significantly lower (37%). R. lens promoted higher weight gain in the rotifer
B. plicatilis than Tisochrysis lutea (T-ISO), a species commonly used for rotifer culture and enrichment. Significant differences
were found in the protein content and in the ratio n-3/n-6 fatty acids among rotifers fed with R. lens from different RRs, with
higher values being found in those fed with R. lens from higher RRs. The enrichment of the rotifers for short periods of 3 h was
sufficient to modify the biochemical composition of the rotifers, but it was evidenced as too short for the accumulation of PUFAs,
when compared to long-term (24 h) enrichment. The rotifers reflected the higher protein and PUFA content of R. lens cultivated
with nutrient sufficient microalgae (R40) after only 3 h of enrichment. These results demonstrate that semi-continuous culture of
R. lens under appropriate conditions can strongly enhance the nutritional value of this species, being reflected in the growth and
biochemical composition of the filter feeder, even in short exposure periods.
Keywords Rhodomonas lens .Semi-continuous cultures .Phycoerythrin .PUFA .Brachionus plicatilis .Rotifer
Introduction
Microalgae are produced worldwide for many purposes in-
cluding aquaculture applications, as raw material for animal
feeds, as food supplement or healthy food for human con-
sumption, nutraceuticals, pharmaceuticals and cosmetics
(Borowitzka 1997;PulzandGross2004;Spolaoreetal.
2006; Sathasivam et al. 2019). Despite the wide biotechnolog-
ical potential of microalgae, only a few species have been
explored as source of fine chemicals (Spolaore et al. 2006)
or for aquaculture applications (Muller-Feuga 2000).
Improved cultivation technologies have been developed in
recent years as a result of the increasing interest in microalgae
as source of biodiesel (Wijffels and Barbosa 2010). These
new, improved technologies, including fermentation technol-
ogies, enable the production of novel species with great bio-
technological potential that require a more controlled environ-
ment and are difficult to cultivate in primitive culture systems
(Phwan et al. 2018).
Cryptophyceae comprises more than 30 genera with 220
marine and freshwater unicellular species (Novarino 2012;
Guiry and Guiry 2019) that are provided with flagella. Their
chloroplasts are believed to be derived from a photosynthetic
eukaryotic endosymbiont (Dunstan et al. 2005)thatjustifies
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s10126-019-09936-4) contains supplementary
material, which is available to authorized users.
*Ana Otero
anamaria.otero@usc.es
1
Instituto de Acuicultura and Dpto. de Microbiología y Parasitología,
Fac. Biología/CIBUS, Campus Vida, Universidade de Santiago de
Compostela, 15782 Santiago de Compostela, A Coruña, Spain
2
Present address: CPIRN-IPG - Center of Potential and Innovation of
Natural Resources, Polytechnic Institute of Guarda,
6300-559 Guarda, Portugal
3
Present address: Department of Aquaculture,
ANFACO-CECOPESCA, Ctra. Colegio Universitario 16,
36310 Vigo, Spain
Marine Biotechnology
https://doi.org/10.1007/s10126-019-09936-4
their unique pigment profile. In addition to chlorophyll, a
number of carotenoids and phycobiliproteins (PBPs) are also
present in the plastids that confer them the typical blue-green
or red colour. PBPs—phycocyanin and phycoerythrin (PE)—
are antennae-protein pigments involved in light-harvesting
which are present in cyanobacteria, rhodophytes,
cryptomonads and cyanelles (Glazer 1994) and have applica-
tions in the food, cosmetic and pharmaceutical industries
(Glazer and Stryer 1984; Pulz and Gross 2004; Sekar and
Chandramohan 2008; Chaloub et al. 2015; Raghav Sonani
et al. 2016).
Several works refer the potential of different cryptophytes
for PE production under different temperatures, irradiances
and nutrient levels (Eriksen and Iversen 1995; Bartual et al.
2002; Chaloub et al. 2015;Vuetal.2016). Cryptophytes have
a great potential for aquaculture applications due to their high
content of protein and PUFAs and constitute a high-quality
diet for brine shrimp, copepods and scallop larvae (Koski et al.
1998; McKinnon et al. 2003; Knuckey et al. 2005; Tremblay
et al. 2007; Ohs et al. 2010; Wenzel et al. 2012; Zhang et al.
2013) improving the ingestion and food conversion rate in
bivalves and the survival, growth, lipid content and reproduc-
tion of mollusks when used in co-feeding with other “stan-
dard”microalgal species (Brown et al. 1998; Tremblay et al.
2007), besides showing excellent results for the growth and
survival of filter feeders such as Artemia (Seixas et al. 2009).
In contrast, the potential of Cryptophyceae for rotifer pro-
duction has barely been evaluated (Guevara et al. 2011;Li
et al. 2015). The availability of rotifers with high nutritional
and microbiological quality is crucial for the success of fish
larvae culture, and research is constantly providing new in-
sight on strategies to culture and enrich rotifers efficiently,
balancing costs, convenience and rotifer quality, e.g. through
the combination of microalgae and artificial diets (Li et al.
2015), use of probiotic bacteria associated to microalgal cul-
ture (Mejias et al. 2018), use of processed microalgae (Sales
et al. 2019) or tuning the duration of the enrichment period
(Estévez and Giménez 2017;Eryalçın2018). Different studies
have shown that both the performance of rotifer cultures and
their biochemical composition can be strongly improved
through the use of protein- and PUFA-rich microalgae
(Ferreira et al. 2011,2018; Thépot et al. 2016), a requirement
that can be fulfilled by Cryptophyceae. Among cryptophytes,
species of the genus Rhodomonas show optimal ratios of ste-
rols, amino acids and PUFAs, constituting excellent candi-
dates for aquaculture diets (Brown et al. 1998; Seixas et al.
2009; Guevara et al. 2016;Peltomaaetal.2017; Yamamoto
et al. 2018). Nevertheless, its cultivation in standard culture
systems presents some difficulties due to its sensitivity to en-
vironmental conditions. Incident light during initial cultiva-
tion phases must be strictly controlled, to avoid photo-inhibi-
tion, especially when the inoculum has been produced under
strong light-limited conditions. Nitrogen availability and pH
are also critical factors. In adverse culture conditions,
Rhodomonas spp. show immediate photo-inhibition, cell ag-
gregation and in some cases cell lysis, as discussed in previous
works for Rhodomonas lens (Seixas et al. 2009)andfor
R. salina (Thoisen et al. 2018).
In the present work, we tested the biotechnological and
nutritional potentials of R. lens for improving rotifer produc-
tion and enrichment. Semi-continuous cultures of this species
were carried out at different daily RRs in order to improve the
nutritional profile of the microalga, and the harvested biomass
was used in cultivation and enrichment experiments of
Brachionus plicatilis, in order to test the effect of the culture
conditions of R. lens on the biochemical profile generated in
this filter feeder that is used as live-feed for marine fish larvae.
Materials and Methods
Rhodomonas lens Culture
The marine microalgae Rhodomonas lens Pascher et Ruttner
(CCMP 739) and Tisochrysis lutea (formerly Isochrysis aff.
galbana, strain T-ISO, CCMP1324) were cultured in 30-mm-
diameter glass tubes containing 80 mL volume under a circa-
dian light/dark cycle of 12 h:12 h, laterally illuminated with an
irradiance of 242 μmol photon m
−2
s
−1
, and at a temperature
of 21 ± 1.5 °C. Cultures were started in batch mode using
sterilized seawater (salinity of 3.5%) enriched with Algal-1
culture media (NaNO
3
8 mM, NaH
2
PO
4
·2H
2
O0.4mM,Na-
EDTA 0.10 mM, C
6
H
5
FeO
7
·5H
2
O 0.08 mM, ZnCl
2
4μM,
MnCl
2
·4H
2
O4μM, Na
2
MoO
4
·2H
2
O4μM, CoCl
2
·6H
2
O
0.4 μM, CuSO
4
·5H
2
O0.4μM, thiamine 0.14 mg L
−1
,biotin
20 μgL
−1
,vitaminB1212μgL
−1
) (Fabregas et al. 1984).
Continuous aeration was provided (200 mL min
−1
) which was
supplemented with periodic injection of CO
2
during the light
period, in order to provide a source of inorganic carbon and
keep pH below 8.0. Cultures were started with a cell density of
5.6 × 10
6
cells mL
−1
and once late logarithmic phase was
reached, the semi-continuous regime was started, with daily
RRs of 10%, 20%, 30%, 40% or 50% of the total volume of
cultures (R10 to R50). In T-ISO cultures, only R10 and R30
were applied. Three replicates were set for each condition.
Cell density was determined by means of a Neubauer
haemacytometer. Cultures were maintained for at least 3 days
in steady-state before being used for the rotifer experiments.
The daily harvested cultures were directly used for the enrich-
ment of the rotifers. Samples of 5 to 10 mL were also taken for
biochemical analysis, centrifuged, the culture media was re-
moved and the pellets were immediately frozen at −18 °C
until being analysed. In some experiments, low steady-state
cell density and instability were observed with R50. This in-
stability of R. lens under higher RRs submitted to higher irra-
diance per cell in some of the experiments was the result of
Mar Biotechnol
initial photo-inhibition in the cultures due to lower initial cell
densities. This fact was corroborated in several experiments,
in which the success of the semi-continuous culture strongly
depended on the previous history and concentration of the
inoculum and the fact that cultures had not entered into severe
nitrogen limitation when the RR is initiated.
Brachionus plicatilis Experiments
The rotifer B. plicatilis was maintained routinely in 6-L flasks
with sterilized seawater (salinity 3.5%), being fed with a mix-
ture of different marine microalgal species. Previous to the
experiment, rotifers were filtered and deprived of food for
24 h in order to avoid interferences of the gut content with
the final results. Three different experiments were performed.
In a first assay, rotifers were fed with R. lens and T-ISO from
nutrient-limited and nutrient-saturated cultures (R10 and R30
respectively) during 7 days, in order to evaluate the nutritional
value and the nutrient delivery efficiency of R. lens in com-
parison with T-ISO, a species commonly used in aquaculture.
Rotifers were transferred to 1 L flasks containing autoclaved
seawater, being the final volume and the final rotifer density
700 mL and 50 individuals mL
−1
, respectively. Three repli-
cates of rotifer cultures were set for each microalgal diet. Food
ration was calculated on a cell number basis, and considering
the equivalent in weight to 2000 Tetraselmis suecica-
rotifer
−1
day
−1
(80 pg cell
−1
for R. lens and 15 pg cell
−1
for
T-ISO), regardless the culture conditions (Patiño 1995).
Therefore, 6000 cells of R. lens or 33,000 cells of T-ISO were
supplied per individual daily. After feeding, the volume of the
rotifer cultures was adjusted with sterilized seawater in order
to maintain the same volume in all cultures. In a second ex-
periment, R. lens from semi-continuous cultures maintained
from R10 to R40 was used to enrich the rotifers for 24 h.
Rotifers were transferred to 1-L glass bottles containing
600 mL of sterilized seawater, at a density of
150 rotifers mL
−1
and supplied with 5000 R. lens
cells rotifer
−1
, enough to feed the rotifers during 24 h, as
previously observed with equivalent food doses of other
microalgae (Patiño 1995;Ferreiraetal.2008,2009). Rotifer
cultures were set in triplicate for each microalgal treatment
(R10 to R40) plus an unfed control group toevaluate the effect
of enrichment. The R50 diet was not tested due to the low cell
density obtained for this RR in this particular assay due to
culture instability. Samples of 150 mL of rotifers cultures were
collected after 24 h on a 45-μm mesh, rinsed with distilled
water and frozen at −20 °C for fatty acids analysis, whereas
the remaining culture volume was collected and freeze-dried
to carry out the gross composition analysis. Rotifer density
was measured by direct counting of 1 mL samples under a
stereoscope in triplicate. In the third assay, short-term (3 h)
and long-term (24 h) enrichment periods were compared with
R. lens cultures maintained at R10 and R40 in order to
evaluate the nutritional value and the nutrients delivery effi-
ciency of R. lens under nutrient-limited and nutrient-saturated
conditions. Similar conditions for microalgae and rotifers cul-
tures were maintained for all the experiments.
Sampling Procedures and Biochemical Composition
Analyses
Dry weight was determined by collecting 5 mL samples of
microalgal culture on previously weighed pre-combusted
Whatman GF/C fibreglass filters. Microalgal samples were
washed three times with 5 mL of 0.5 M ammonium formate
in order to remove salts (Utting and Helm 1985). Rotifer sam-
ples (100 mL of rotifer culture) were also collected on
Whatman GF/C filters and rinsed with distilled water. Filters
were dried overnight at 80 °C and dry weight determined
gravimetrically. Culture medium from microalgal cultures
was collected after centrifugation for absorbance registration
at 220 nm (Helios Omega UV-Vis, Thermo Scientific) and
NO
3−
determination (Collos et al. 1999). A standard curve
was generated with pure NaNO
3
at different concentrations
(0.05, 0.10, 0.15, 0.30, 0.40 and 0.50 mM) dissolved in sea-
water. Protein content was determined by the Folin-phenol
method (Lowry et al. 1951) after alkaline hydrolysis with
NaOH 1.0 M at 95 °C, whereas carbohydrates were quantified
by the phenol-sulphuric acid method (Kochert 1978)insam-
ples treated with NaOH 1.0 M. Lipids were quantified by the
charring method (Marsh and Weinstein 1966) after extraction
of total lipids (Bligh and Dyer 1959). For C-H-N determina-
tion, freeze-dried microalgal samples were analysed with an
autoanalyser Flash EA 1112 de Thermo Finnigan. For the
extraction of PE from R. lens cultures, 3 to 5 mL culture
samples were centrifuged and resuspended in distilled water,
being frozen at −20 °C for cell disruption. Determination of
PE was carried out using the formulas proposed by Bennett
and Bogorad (1973) after reading pigment concentrations in a
spectrophotometer at wavelengths of 565 nm, 620 nm and
650 nm (Bryant et al. 1979). Chlorophyll content was deter-
mined spectrophotometrically after extraction of 3-mL centri-
fuged microalgal culture sample with 3 mL of acetone (90%
v/v) with 1 min of sonication. Samples were then centrifuged
(15 min at 4 °C at 3000 rpm). Absorbance was read at wave-
lengths 630 and 664 nm for chlorophyll “a”and “c2”. Jeffrey
and Humphrey (1975) and Humphrey (1979) equations were
used for determining the concentration. Fatty acids were iden-
tified and quantified using a gas chromatograph-mass spectro-
graph (GC-MS PerkinElmer 800-8000 Series), equipped with
an Omegawax TM 250 column 30 m× 0.25 mm (Supelco,
Inc.), after methanolysis of the lipid extracts with 5% HCl in
methanol at 85 °C during 2:30 h and extraction with hexane
(Sato and Murata 1988). Triheptadecanoin (Sigma®, St.
Louis, MO) was used as internal standard. All the analyses
were carried out in triplicate.
Mar Biotechnol
Statistical Analysis
Statistical analyses were done using the software SPSS for
Windows v.14. After verifying that data met the requirements
of normality (Kolmogorov-Smirnov test), comparisons be-
tween groups were done by the analysis of variance
(ANOVA) followed by Tukey HSD test for posthoc multiple
comparisons or T Wilcoxon for pair comparisons.
Significance was accepted for pvalues lower than 0.05.
Results
Semi-continuous Cultures of R. lens
The semi-continuous cultures were started from a logarithmic
phase culture with a density of 18.8 × 10
6
cells mL
−1
(Supplementary data). Cell densities as high as 23 ×
10
6
cells mL
−1
could be obtained in stationary phase in differ-
ent experiments. Cultures were not allowed to achieve station-
ary phase for the initiation of the semi-continuous regime,
since previous experiments indicated that the batch cultures
easily collapse once nitrogen is depleted. The steady-state cell
density with R10 was slightly higher than the initial cell den-
sity, achieving 22.16 × 10
6
cells mL
−1
and decreased to
7.17 × 10
6
cells mL
−1
R50 (Fig. 1). Productivity expressed
in grams per litre per day increased with RR, from 0.18 to
0.38 g L
−1
day
−1
with R10 and R40, respectively. Although
in this experiment a high steady-state cell density could be
achieved with R50, small differences in growth and stability
of microalgal cultures were recorded in the different indepen-
dent experiments at this high RR that were reflected in small
differences in steady-state cell density and biochemical pro-
file. Therefore, for the rotifer enrichment experiments, only
the cultures maintained with R10 to R40 were used.
The cell content of the antenna phycobiliprotein PE in-
creased linearly with RR, attaining maximum values in the
R50 (circa 9 pg cell
−1
) despite the decrease in cell density.
This PE cell content corresponded to a threefold increase in
the PE content in comparison with the R10 condition. Since
PE content increased continuously with RR despite the higher
light availability at high RR derived from the lower cell den-
sity, the results confirm that PE could act as nitrogen reservoir
in nutrient-saturated conditions (Fig. 2). Cellular chlorophyll
content increased slightly from 1.70 ± 0.03 pg cell
−1
in R10 to
2.04 ± 0.06 pg cell
−1
inR40andsufferadecreaseatthe
highest R50 (Fig. 2). Similarly, individual cell dry weight
increased with RR, from 79 pg cell
−1
with R10 to 116 pg
cell
−1
with R40 and decreased slightly with R50 (100 pg
cell
−1
) (data not shown), mainly dependent of the cellular
protein content.
The C:N ratio decreased from R10 (8.53) to R20 (5.08),
being stable for higher RRs (Fig. 3), indicating that cultures
were nutrient-limited at R10 and nutrient-saturated for higher
RRs. Protein cell content increased from 36.09 ±
1.65 pg cell
−1
with R10 to 63.73± 1.63 pg cell
−1
with R40
and decreased thereafter (Fig. 3). This increase in protein cell
content is the main responsible for the increase in cell dry
weight. Regarding the organic composition of R. lens
(Fig. 4), protein levels increased with increasing RR, becom-
ing stable after the R40 condition and attaining nearly 64% of
the total organic fraction. Relative lipid content in the biomass
decreased with increasing RRs up to R40, increasing
thereafter.
Significant differences were found in the fatty acid (FA)
composition of the different cultures (Table 1), especially in
the proportion of certain PUFAs, between nitrogen-limited
and nitrogen-saturated cultures. The main FAs found in
R. lens were tetradecanoic acid (14:0), palmitic acid (16:0),
linolenic acid (18:3n-3), octadecatetraenoic acid (18:4n-3),
eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid
Fig. 1 Steady-state cell density (× 10
6
cells mL
−1
) (bars) and productivity
(g l
−1
day
−1
)(line)ofR. lens cultured semi-continuously at different daily
renewal rate. Symbol (asterisk) indicates statistically significant groups.
The data are shown as means ± standard deviations (n=3)
Fig. 2 Chlorophyll content (a + c2) (pg cell
−1
) (bars) and PE (pg cell
−1
)
(line) of R. lens cultured semi-continuously at different daily renewal
rates. Letters indicate statistically significant groups. The data are shown
as means ± standard deviations (n=3)
Mar Biotechnol
(22:6n-3). The most striking difference among the different
conditions was observed in the total level of PUFAs, with
similar values being found in nutrient-sufficient cultures (~
65% of total FA) in comparison with a much lower value
obtained with R10 (37.5%), clearly indicating the nutrient-
limitation in this RR. The sum of saturated FA was high in
the nutrient-limited culture (circa 55% in R10), decreasing
considerably to ~ 30% in the other RRs. Monounsaturated
FA followed the same trend than saturated FA, decreasing
from 8 to 2% of total FA.
Cultivation and Enrichment of B. plicatilis with R. lens
In a first experiment, R. lens was compared to T. lutea T-
ISO, a species commonly used in aquaculture for the
culture and enrichment of rotifers. Both microalgae were
cultured at R10 and R30, corresponding to N-limited and
N-saturated conditions, respectively, to study the effect
of this parameter on the nutritive value of the two spe-
cies of microalgae for Brachionus plicatilis. As observed
in R. lens, the increase in RR produced an increase in
protein cell content derived from the change from
nutrient-limited to nutrient-saturated conditions, as previ-
ously observed by Ferreira et al. (2008). The rotifer and
egg numbers after 7 days of cultivation were higher with
R. lens when compared to T-ISO (Fig. 5a, b). Differences
between microalgal treatments were more relevant for
egg production. Moreover, important differences were
found in the biochemical composition of rotifers depend-
ing on the microalga used as feed. As expected, a higher
RR resulted in a higher organic weight and protein con-
tent of rotifers for both microalgal species (Table 2).
Furthermore, when the total food provided was calculat-
ed on the basis of organic weight, the total organic
weight provided with T-ISO was significantly higher
than with R. lens, especially in the nutrient limited cul-
tures maintained at R10, but resulted in rotifers with a
clearly lower organic content, independently of the RR
applied. Although a similar composition of the organic
fraction in the rotifer was obtained, the lower organic
weight of the diet provided and the higher weight of
the rotifers obtained resulted in a better food conversion
rate for R. lens in comparison with T-ISO, especially
regarding the protein content. The higher nutritional val-
ue of R. lens for the cultivation of B. plicatilis in com-
parison with T-ISO was also demonstrated in semi-
continuous cultures of the rotifer, in which the growth
rate of the rotifer was twice as high with R. lens
(Supplementary Figure 2).
In the second assay, the effect of the modification of
the biochemical composition of R. lens cultured semi-
continuously with different RRs (from 10 to 40%) on
the rotifer B. plicatilis wastestedina24-henrichment.
An increase in the RR applied to microalgal cultures re-
sulted in an increase in the organic weight of the enriched
rotifers, which increased from 126 to 134 ng rotifer
−1
in
R10andR40,respectivelyinonly24h,incomparison
with the initial unfed group (80 ng rotifer
−1
,p< 0.05)
(Fig. 6). The protein content of rotifers increased with
the RR applied to the R. lens culture used to fed them,
from 74 ng rotifer
−1
withR10to93ngrotifer
−1
with R40,
reflecting the increase in the protein content of the
ingested microalgae (Figs. 4and 6). The rotifers enriched
during 24 h modified its fatty acid profile, reflecting that
of the microalga used, producing a positive correlation
(r
2
= 0.85) between the ratio n-3/n-6 of the microalgae
and of the rotifers. The levels of n-3 FA found in rotifers
apparently followed the same trend as the ingested diet,
increasing from R10 to R30 and showing a slight decrease
in the R40 condition (Fig. 7). As for n-6 PUFAs in roti-
fers, a decrease from diet R10 to R40 was observed, but
in this case, no apparent correlation between dietary levels
and rotifer body composition was found (Fig. 7).
Fig. 4 Organic fraction composition of R. lens cultured semi-
continuously at different daily renewal rates (%) (black-filled column,
% protein; grey-filled column, % lipids; white-filled column, %
carbohydrates)
Fig. 3 Protein content (pg cell
−1
) (bars) and C:N ratio (atoms) (line) of
R. lens cultured semi-continuously at different daily renewal rates. Letters
indicate statistically significant groups. The data are shown as means ±
standard deviations (n=3)
Mar Biotechnol
Short-Term Versus Long-Term Enrichment
of Brachionus plicatilis
Finally, a short-term enrichment (3-h) was compared with the
conventional 24-h enrichment with R. lens cultivated at R10
and R40, corresponding to nutrient-limited and nutrient-
saturated conditions, respectively. The 3-h enrichment with
microalgae from R10 did not result in a significant increase
in rotifer density or eggs concentration (203 ± 16 rotifer and
39 ± 6 eggs) in comparison with the initial density (200 rotifer
+ 40 eggs), while a significant increase in rotifer and egg
numbers was obtained with R40 (217 ± 8 rot. + 40± 5 eggs)
(test Wilcoxon, p< 0.1) even after such a short enrichment
period (Fig. 8a). The 3-h enrichment improved the protein
content of the rotifers, although this increase was clearly
higher with R. lens from R40. Differences between rotifers
fed nutrient-limited and nutrient saturated R. lens were much
more marked after a 24-h enrichment. This difference was
mainly due to the number of eggs produced; 147 ±
7eggsmL
−1
with R40 and 95 ± 6 eggs mL
−1
with R10
(Fig. 8a). A longer enrichment was positive for the nutrient-
limited microalgae, further increasing protein content (Fig.
8b), while no further beneficial effect on gross biochemical
composition could be observed in the long-term enrichment
for the nutrient saturated R40 microalgae.
In the 24-h enrichment, significant changes on organic
fraction occurred with the RR applied, mainly due to the in-
crease of lipids content from the R10 and R40. Rotifers main-
tained their saturated fatty acids content around 30% in all
conditions, while the portion of monounsaturated decreased
from 30 to 16% and PUFA increased proportionally, from
36% with the R10 to 54% with the R40 (Table 3). It should
be noted that the highest polyunsaturated fatty acid content,
54.53%, was reached with the R 40% and 24 h of enrichment,
while the PUFA level obtained for R40 with 3 h enrichment
was the same than that obtained with R10 cultures both at 3
and 24-h, confirming the higher nutritional value of
microalgae maintained with high RRs (Table 3).
Discussion
The steady-state cell densities of R. lens obtained with R10
(22.27 ± 0.32 × 10
6
cells mL
−1
) were much higher than the
maximum values reported by other authors (less than 2 ×
10
6
cells mL
−1
)forRhodomonas sp. batch cultures (Renaud
et al. 1999,2002; Valenzuela-Espinoza et al. 2005;Dunstan
et al. 2005; Lafarga-De la Cruz et al. 2006; da Silva et al.
2009; Thoisen et al. 2018). Even the values obtained with
R50 (7.17 ± 0.6 × 10
6
cells mL
−1
) were much higher than the
stationary-phase cell densities reported in the literature. The
maximum cell densities observed in the different experiments
during the stationary phase of batch cultures (23 ×
10
6
cells mL
−1
) were similar to those reported for the R10
condition. In previous works with R. lens cultured semi-
continuously in 6-L flasks, Seixas et al. (2009) described a
steady-state density of 3.7 × 10
6
cells mL
−1
using ALGAL-1
medium (4 mM NO
32−
) and a daily RR of 30%, which is more
Table 1 Fatty acid (FA) compo-
sition (% of total FA) of
Rhodomonas lens cultured semi-
continuously at different daily re-
newal rates (from 10% to 50% of
total volume)
Renewal rate
Fatty acid R10 R20 R30 R40 R50
12:0 1.06 ± 0.50 0.44 ± 0.38 0.41 ± 0.08 0.30 ± 0.01 0.58 ± 0.08
13:0 0.14 ± 0.03 0.32 ± 0.01 0.31 ± 0.03 0.33 ± 0.01 0.24 ± 0.33
14:0 26.14 ± 5.05 13.55 ± 3.76 15.53 ± 1.54 12.59 ± 0.75 16.47 ± 3.20
15:0 0.25 ± 0.02 0.25 ± 0.02 0.26 ± 0.05 0.28 ± 0.01 0.16 ± 0.23
15:1 0.18 ± 0.02 0.39 ± 0.02 0.33 ± 0.01 0.29 ± 0.00 0.24 ± 0.34
16:0 26.10 ± 1.72 15.92 ± 2.33 16.85 ± 0.91 17.48 ± 0.84 18.52 ± 1.65
16:1 0.66 ± 0.06 0.57 ± 0.03 0.53 ± 0.21 0.59 ± 0.01 0.62 ± 0.11
18:0 0.84 ± 0.19 0.72 ± 0.19 0.59 ± 0.05 2.08 ± 0.01 0.87 ± 0.24
18:1n9 7.04 ± 1.11 0.80 ± 0.09 1.03 ± 0.38 1.05 ± 0.05 1.31 ± 0.44
18:2n6 0.58 ± 0.51 0.85 ± 0.19 0.73 ± 0.15 0.69 ± 0.02 0.69 ± 0.02
18:3n3 15.67 ± 1.55 24.90 ± 2.78 22.38 ± 2.72 23.13 ± 0.82 21.18 ± 0.66
18:4n3 12.46 ± 0.98 27.46 ± 1.22 28.74 ± 1.75 28.93 ± 2.43 27.27 ± 0.64
20:4n3 0.40 ± 0.11 0.94 ± 0.10 0.83 ± 0.10 0.88 ± 0.16 0.70 ± 0.00
20:5n3 5.21 ± 1.47 8.90 ± 0.25 7.89 ± 0.25 7.93 ± 0.10 7.59 ± 0.10
22:6n3 3.18 ± 1.33 4.00 ± 0.99 3.60 ± 0.11 3.44 ± 0.14 3.56 ± 0.05
∑Saturated 54.53 ± 7.51 31.20 ± 6.71 33.94 ± 2.65 33.07 ± 1.62 36.84 ± 5.73
∑Monounsaturated 7.88 ± 1.18 1.76 ± 0.14 1.89 ± 0.60 1.93 ± 0.06 2.18 ± 0.89
∑PUFAs 37.50 ± 5.95 67.04 ± 6.75 64.18 ± 5.08 65.00 ± 3.66 60.99 ± 1.49
Mar Biotechnol
in accordancewith the values obtained here with the same RR
(11 × 10
6
cells mL
−1
for R30). It should be noted that several
preliminary experiments demonstrated the importance of the
initial development of the cultures for the achievement of high
cell densities in batch mode and stable semi-continuous cul-
tures of R. lens. Nutrient sufficient, log-phase inoculums are
required for obtaining robust growth and low dilutions should
be maintained during the up-scale steps in order to maintain
optically dense cultures, resilient to photoinhibition during the
delicate initial 24 h after inoculation. Due to the high nitrogen
demand of this species, the considerable differences in cell
densities in comparison with data reported by other authors
can be certainly explained by differences in the nutrient con-
centration. Whereas f/2 or fmedia are generally used in the
literature (0.88 to 1.7 mM NO
3−2
), in our case, the concentra-
tion of NO
3−2
was 8 mM. In addition, we have observed that
nitrogen limitation results in a fast collapse of batch cultures
once stationary phase is reached. Other factors that could ex-
plain the observed differences with other reports could be
related to the higher irradiance and short light-path (3 cm
diameter) conditions used. In the present work, maximal pro-
ductivity was obtained at the intermediate R30-R40, as report-
ed for other microalgal species such as Tetraselmis suecica,
Isochrysis galbana,Chroomonas sp. and Isochrysis aff.
galbana T-ISO; Nannochloropsis gaditana cultured semi-
continuously in similar conditions (Otero and Fábregas
1997; Otero et al. 1997a,b;Bermúdezetal.2004; Ferreira
et al. 2008,2018).
Demonstrating its independence of light availability, the
content of PE in R. lens cells increased sharply with the RR
applied, with a maximum value of 8.44 pg cell
−1
obtained for
R50. It should be noted that effective light availability in the
cultures increased continuously with the RR due to the de-
crease in cell density. The C:N ratio indicates that cultures
were nutrient sufficient at R20 and higher, but both chloro-
phyll and PE cell content increased with RR in nutrient satu-
rated conditions, despite the higher light availability, indicat-
ing a unique behaviour of pigment content in this species,
since a decrease in chlorophyll cell content with RR would
be expected under nutrient-saturated conditions (Otero and
Fig. 5 Density of rotifers Brachionus plicatilis (a) and eggs production (b) during 7 days of culture with Rhodomonas lens (black) cultures and T-ISO
(grey) from the semi-continuous culture at renewal rates of 10% (solid) and 30% (hashed)
Table 2 Biochemical composition of the Brachionus plicatilis in culture with Rhodomonas lens cultures and T-ISO from semi-continuous culture at
renewal rates of 10% and 30% (R10, R30, T10 and T30 respectively)
R. lens T-ISO
R10 R30 T10 T30
Rotifer
Protein (ng/rotifer) (p≤0.05) 134.27 ±13.68 a 177.60± 14.41 b 102.35 ±23.23 c 132.46± 22.87 a
Carbohydrates (ng/rotifer) (p≤0.05) 16.88 ± 4.14 a 21.20 ± 4.48 a, b 27.31 ± 4.50 c 24.75 ± 5.79 a, b, c
Lipids (ng/rotifer) (p≤0.05) 16.70 ± 4.01 a 20.87 ± 4.35 a, b 26.82 ± 4.37 c 24.32 ± 5.60 b, c
Diet
Protein (ng/dose) 222.52 253.68 173.51 244.66
Carbohydrates (ng/dose) 36.55 12.35 65.56 22.76
Lipids (ng/dose) 76.33 56.81 212.98 137.24
Mar Biotechnol
Fábregas 1997;Fábregasetal.1998; Otero et al. 1998).
Although a protective role of PE could be hypothesised, the
concomitant increase of chlorophyll content excludes this hy-
pothesis, except for the extreme R50 treatment, where PE
content continued increasing while chlorophyll content de-
creased. In the cryptophytes, the phycobiliproteins play a ma-
jor role in harvesting light for photosynthesis, with not only a
greater ability to capture light across the spectrum, but also a
greater efficiency at transferring this energy to the photosys-
tems (Greenwold et al. 2019). Since PE is part of the antenna,
its increase may have forced an increase the number of active
centres, resulting in an increase in chlorophyll content. The
observed increase in PE content seems therefore to be corre-
lated to the increase of nitrogen availability.
The obtained values for PE content are much higher than
the 1.5 pg cell
−1
described by Thomas and Lonsmann (1995)
for Rhodomonas sp. and also than those reported by Bartual
et al. (2002) and da Silva et al. (2009) for other species of
Rhodomonas (nearly 5 pg cell
−1
) cultured in higher volume
flaks (2 L and 0.5 L, respectively). These lower values are
probably derived from the lower nitrogen concentration in
the culture media used in these experiments. Previous reports
have already indicated that PE accumulation in Rhodomonas
sp. serves as a nitrogen storage compound as well as light-
trapping pigment (Ludwig and Gibbs 1989; Eriksen and
Iversen 1995) and light-limited conditions combined with N-
saturated conditions tend to favour the accumulation of PE in
Rhodomonas sp. (da Silva et al. 2009). Seixas et al. (2009)
observed an almost 200% increase of PE content per cell (up
to 7.8 pg cell
−1
)inR. lens cultured in 6-L glass bottles under
light-limited conditions when compared to light-saturated
conditions. In our case, an increase in nitrogen availability
caused an increase in PE content, independently of light
availability.
Regarding the FA composition of R. lens, the highest
percentages of PUFAs were found in nutrient-saturated
conditions, with a biochemical profile similar to that re-
ported by other authors for different species of
Rhodomonas or in R. lens itself (Renaud et al. 1999;
Dunstan et al. 2005;Seixasetal.2009;Peltomaaetal.
2017; Thoisen et al. 2018). In general, under conditions
of N limitation, an increase in growth rate in semi-
continuous cultures of microalgae, derived from higher
RR, results in an increase in the amount of polyunsatu-
rated fatty acids, accompanying an increase in photosyn-
thetic pigments, to the detriment of the content of satu-
rated and monounsaturated fatty acids, while the percent-
age of PUFAs remained stable with RR under nutrient
saturated conditions (Otero et al. 1997a,b;Oteroand
Fábregas 1997;Seixasetal.2009; Ferreira et al. 2011).
This trend was also observed in R. lens,withastep
increase in PUFAs from the N-limited R10 (37.5%) to
the N-saturated R20 (67.04%), remaining stable thereaf-
ter (Table 1). Saturated fatty acids decreased from
54.53% ± 7.51 with R10 to 31.20% ± 6.71, indicating a
decrease in reserve lipids. Logarithmic-phase cultures of
Rhodomonas sp. and Rhodomonas baltica presented a
wide variation in the values of saturated fatty acids,
ranging between 27 and 60%, monounsaturated between
2 and 18% and polyunsaturated between 21 and 55%
(Tremblay et al. 2007;Costardetal.2012; Parrish
et al. 2012). The huge range of variation in fatty acid
composition reported here and in the literature for R. lens
depending on culture conditions underlines the impor-
tance of the application of continuous or semi-
continuous culture systems for the strict control of the
biochemical composition of microalgae. The values ob-
tained in this work for the percentage of PUFAs (65%)
with the higher RRs are higher than those reported for
logarithmic phase cultures by other authors. The use of
higher nutrient concentrations andirradianceinthepres-
ent work, which allowed the achievement of high cell
densities, has also probably contributed to the achieve-
ment of such high PUFA content.
Based on the growth rate, egg production and general
biochemical composition, especially highlighting the
Fig. 7 Fatty acid composition (% total) of rotifers fed for 24 h with
R. lens cultured at different renewal rates. Black-filled column, ω3(%
total); grey-filled column, ω6 (% total) monounsaturated; line: ω3/ω6
ratio
Fig. 6 Biochemical composition of rotifers after 24 h enrichment with
R. lens cultured at different renewal rates and of the control group
(unfed).(black-filled column, protein (ng); grey-filled column, lipids
(ng); white-filled column, carbohydrates (ng))
Mar Biotechnol
protein and PUFAs content, culture of Brachionus
plicatilis with Rhodomonas lens was confirmed as an
interesting alternative to other microalgal species more
commonly used, such as T-ISO. Rhodomonas lens pro-
duced in semi-continuous regime was confirmed as very
appropriate food for rotifers, with high protein content
and high levels of n-3 fatty acids and interesting Sat +
Mon/PUFAs ratios. In nutrient-saturated cultures, the in-
corporation of protein and lipids from the microalgae to
the rotifer increases continuously with RR even though
the content of these components in the microalgae has
already stabilized or even decreased.
As for short- versus long-term enrichment experiment, sig-
nificant differences in the growth and proximate composition
of rotifers could be observed after only 3 h enrichment with
R. lens from the nutrient-saturated conditions in comparison
with the low RR, nutrient-limited cultures. This difference
was more marked after 24 h of enrichment. The higher protein
content and lower C:N ratios of the high RR microalgae
should be responsible for the higher digestibility and lower
Fig. 8 aDensity of the rotifer Brachionus plicatilis and eggs
concentration (black-filled column, rotifers; white-filled column, eggs).
bComposition of the organic fraction (%) of Brachionus plicatilis (black-
filled column, protein; grey-filled column, lipids; white-filled column,
carbohydrates) enriched for 3 and 24 h with R. lens cultured semi-
continuously at renewal rates of 10% and 40%. The data are shown as
means ± standard deviations (n=3)
Table 3 Fatty acid (FA) compo-
sition (% of total FA) of
Brachionus plicatilis rotifers
enriched for 3 and 24 h with
Rhodomonas lens cultured semi-
continuously at different daily re-
newal rates (from 10% to 40% of
total volume) (R10 and R40 re-
spectively). The data are shown as
means ± standard deviations
(n=3)
Fatty acid Initial 3 h 24 h
R10 R40 R10 R40
14:0 4.80 ± 0.51 9.61 ± 1.12 10.60 ± 0.14 5.60 ± 0.60 5.03± 1.19
16:0 24.05 ± 0.27 21.47 ± 1.02 19.58 ± 1.07 19.76 ± 0.52 21.61 ± 2.44
16:1 11.67 ± 1.45 5.90 ± 0.67 4.34 ± 0.52 5.25 ± 0.91 7.36 ± 0.70
16:4n3 1.35 ± 0.08 0.71 ± 0.11 0.86 ± 0.12 ––
18:0 5.90 ± 0.58 4.30 ± 0.79 3.55 ± 0.52 4.34 ± 0.51 5.71 ± 1.47
18:1n9 17.53± 0.35 14.08 ± 0.25 16.71 ± 1.46 17.07 ± 1.18 7.19 ± 0.45
18:2n6 4.51 ± 0.13 3.50 ± 0.11 2.76 ± 0.01 0.84 ± 1.46 3.71 ± 0.33
18:3n6 0.58 ± 0.01 ––1.71 ± 0.48 0.42 ± 0.03
18:3n3 4.20 ± 0.10 12.81 ± 0.18 14.97 ± 0.75 14.29 ± 0.99 13.63 ± 0.68
18:4n3 1.25 ± 0.69 6.14 ± 0.28 6.05 ± 0.42 7.23 ± 0.26 8.83 ± 1.60
20:1n9 1.66 ± 0.45 1.40 ± 0.23 1.38 ± 0.02 1.88 ± 0.27 1.71 ± 0.22
20:3n6 1.03 ± 0.03 0.60 ± 0.04 0.35 ± 0.02 –0.73 ± 0.06
20:3n3 3.42 ± 0.19 1.93 ± 0.11 1.03 ± 0.10 –2.25 ± 0.21
20:4n6 0.49 ± 0.00 0.53 ± 0.08 1.12 ± 0.10 –0.71 ± 0.06
20:4n3 2.99 ± 0.10 2.68 ± 0.13 3.68 ± 0.37 5.82 ± 0.80 3.37 ± 0.38
20:5n3 9.44 ± 0.33 8.58 ± 0.42 6.86 ± 0.58 7.82 ± 0.17 10.60 ± 1.35
22:5n3 2.96 ± 0.26 1.78 ± 0.10 1.19 ± 0.13 2.04 ± 0.35 2.27 ± 0.25
22:6n3 0.89 ± 0.02 3.08 ± 0.16 3.95 ± 0.42 4.26 ± 0.13 4.31 ± 0.18
SAT 34.75 35.38 33.73 29.71 32.35
MONO 30.87 21.39 22.43 24.20 16.26
PUFAs 36.28 45.12 44.72 44.85 54.53
(SAT + MONO)/PUFAs 1.81 1.26 1.26 1.20 0.89
Mar Biotechnol
food conversion rate observed in the rotifers. It should be
noted that the different diets were calculated on the basis of
cell number, and since microalgae from higher RR cultures
have a lower cell weight than those cultured at low RR, the
total dry weight ad organic weight provided with the diet was
lower in the high RR cultures, as reported previously with
other microalgal species (Ferreira et al. 2008,2009,2011,
2018). Interestingly, while the increase of n-3 levels in rotifers
could be attributed to the diet, as values followed the same
trend of the corresponding ingested R. lens, the decrease of n-
6 levels observed in rotifers from diet R10 to R40 was not
linked to dietary levels. This fact may have crucial implica-
tions for the rearing of marine larvae with live prey as the ratio
n-3/n-6 FA plays important physiological and nutritional roles
(Rodriguez et al. 1994;Izquierdo1996; Coutteau et al. 1997;
Eryalçın2019). The enrichment of the rotifers for short pe-
riods of 3 h was sufficient to modify the biochemical compo-
sition of the rotifers, but it was evidenced as too short for the
accumulation of polyunsaturated fatty acids. The enrichment
during 24 h modifies the profile of fatty acids of the rotifer,
reflecting the biochemical profile of the microalga used, pro-
ducing positive correlations between the percentages of the n-
3 fatty acids and for the ratio n-3/n-6 of microalgae and
rotifers.
In conclusion, results demonstrate the feasibility of
maintaining stable semi-continuous cultures of the
cryptophyte R. lens, even at high RRs. Considerable
changes in the productivity and composition of this bio-
technologically interesting species could be observed
when applying different daily RRs, which are useful to
maximize the production of biotechnologically valuable
compounds such as PE and PUFAs. Despite showing a
more delicate response to culture conditions than other
species commonly used in biotechnological or aquacul-
ture applications, such as Nannochloropsis sp.,
Isochrysis spp., Tetras e l m i s sp. or Porphyridium sp., es-
pecially during the scale-up procedure, R. lens was
foundtoattainsimilarproductivities than those species
when cultured in similar conditions of daily RR, photo-
period and N availability (Otero and Fábregas 1997;
Fábregas et al. 1998;Ferreiraetal.2008,2009,2011,
2018). As long as N-sufficient concentrations are main-
tained and irradiance is far from photo-inhibition levels,
R. lens can be easily cultured, with a relevant produc-
tion of PE and an interesting profile of C18 polyunsat-
urated FA. The food results obtained for the cultivation
and enrichment of the filter feeder B. plicatilis,anor-
ganism often used for the rearing of larvae of marine
organisms, indicate the excellent nutritional value of this
species.
Funding Information This work was supported by the grant “Axudas do
Programa de Consolidación e Estructuración de Unidades de
Investigación Competitivas (GPC)”from the Consellería de Cultura,
Educación e Ordenación Universitaria, Xunta de Galicia
(ED431B2017/53).
Compliance with Ethical Standards
Conflict of Interest The authors declare that there is no conflict of
interest.
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