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\begin{document}

\title{Media engineering in marine diatom Phaeodactylum tricornutum employing
cost-effective substrates for sustainable production of high value
renewables}



\author[1]{Mohammed Rehmanji}%
\author[1]{Asha Nesamma}%
\author[2]{Nida Khan}%
\author[2]{Tasneem Fatma}%
\author[1]{Pannaga Jutur}%
\affil[1]{International Centre for Genetic Engineering and Biotechnology New Delhi}%
\affil[2]{Jamia Millia Islamia Central University}%


\vspace{-1em}



  
  \date{June 14, 2024}


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\selectlanguage{english}
\begin{abstract}
Phaeodactylum tricornutum is a marine diatom, and well-studied model of
unicellular microalga. This diatom contains a wide range of high-value
renewables (HVRs) with high commercial relevance owing to their
importance in human nutrition and health. In this study, we screened P.
tricornutum for biomass, eicosapentaenoic acid (EPA) and fucoxanthin
production under photoautotrophic and mixotrophic condition with various
substrate combinations. Results highlights that culture supplemented
with glycerol and urea lead to enhanced biomass, biochemical and HVR
production. Further continuous feeding of urea in glycerol supplemented
medium results in an increase in biomass yield (0.77 g L-1) by
\textasciitilde{} 2-fold. Additionally, continuous feeding of urea
channelizes the carbon flux towards biosynthesis of fatty acids
increasing FAME content by \textasciitilde{}2-fold as compared to the
control conditions. Overall EPA and fucoxanthin production was 27 mg L-1
and 11 mg L-1 (\textasciitilde{}2 \& 4 fold) in urea fed cultures
respectively. Present study demonstrates efficient valorization of
cost-effective substrates such as glycerol and urea for the production
of high-value renewables in P. tricornutum.%
\end{abstract}%



\sloppy


\textbf{1. Introduction}

The transition from biofuels to bio-product based economy, demands a
suitable feedstock with capability of producing multiple high-value
renewables (HVRs). Microalgae are by far the most abundant primary
producers responsible for photosynthetic conversion of light energy and
carbon dioxide (CO\textsubscript{2}) into sustainable
renewables\textsuperscript{{[}1{]}}. Recent advances in bioprocess
technology supports the development of microalgal cell factories for
establishing environmentally sustainable manufacturing of HVRs. In this
regard \emph{Phaeodactylum tricornutum} , a unicellular, marine pennate
diatom, is considered as a potential feedstock for the production of
biofuel and HVRs such as\textsuperscript{{[}2, 3{]}}, eicosapentaenoic
acid (EPA), docosahexaenoic acid (DHA)\textsuperscript{{[}4{]}},
chrysolaminarin\textsuperscript{{[}5{]}},
fucoxanthin\textsuperscript{{[}6, 7{]}}, etc., and can be considered as
a suitable microalgal cell factory for sustainable biorefinery
processes\textsuperscript{{[}8{]}}.

One of the major fatty acid in \emph{P. tricornutum} is the \selectlanguage{greek}ω-\selectlanguage{english}3 fatty
acid EPA (C20:5), having a significant commercial importance in
pharmaceutical and nutraceutical industries\textsuperscript{{[}9,
10{]}}. These long chain polyunsaturated fatty acids (LC-PUFAs) have a
bioactive role against a variety of disorders, including coronary heart
disease, thrombosis, and recently in prospective adjuvant therapy in
COVID-19-related cardiovascular problems\textsuperscript{{[}11-14{]}}.
EPA content in \emph{P. tricornutum} is reported upto 3-5 \% of DCW (dry
cell weight) with an average productivity of 56 mg
L\textsuperscript{-1}D\textsuperscript{-1}, highlighting a potential
alternate vegan source of \selectlanguage{greek}ω-\selectlanguage{english}3 fatty acid
production\textsuperscript{{[}15{]}}. Commercially EPA from \emph{P.
tricornutum} is available in global market \emph{via}brand name SIMRIS\selectlanguage{ngerman}®
ALGAE OMEGA-3, containing 50 mg of EPA per capsule
(www.simris.com/pages/ingredients).

Additionally, fucoxanthin (1\% - 6\% DCW) an important HVR, is the
primary carotenoid produced in
\emph{P.tricornutum}\textsuperscript{{[}16{]}{[}6{]}}. During
photosynthesis, xanthophylls acts as a light harvesting pigment
connected to fucoxanthin-chlorophyll a/c-proteins (FCP), which are an
integral part of the thylakoids\textsuperscript{{[}17{]}}. Furthermore,
due to its unique structure, fucoxanthin has various major bioactivities
such as anti-oxidant, anti-obesity, anti-cancer properties, and it has
been found to be an effective treatment for chronic disorders such as
Alzheimer's\textsuperscript{{[}18-21{]}}. Commercial source of
fucoxanthin is primarily brown seaweeds, which are difficult to meet
market demands due to low productivity, low quality, and high cost. The
amount of fucoxanthin generated by \emph{P. tricornutum} is
substantially higher than brown seaweeds, making it a potential choice
for commercial production\textsuperscript{{[}22{]}}.

Media engineering strategies for microalgae cultivation have recently
acquired appeal as feasible techniques for achieving high HVR
output\textsuperscript{{[}7, 23{]}}. Though the high costs of
substrates, is a major impediment for generating commercially viable
product \textsuperscript{{[}24{]}}. Mixotrophy, on the other hand,
represents an innovative methodology for HVRs production in \emph{P.
tricornutum}\textsuperscript{{[}25, 26{]}}. It appears challenging to
attain high biomass, EPA, and fucoxanthin content simultaneously; hence,
designing an appropriate strategy for the mixotrophic cultivation
of\emph{P. tricornutum} is critical for the commercial co-production of
HVRs.

\emph{P. tricornutum} grows mixotrophically and has been reported to
grow efficiently on glucose, fructose, mannose, lactose and
glycerol\textsuperscript{{[}27{]}}. Furthermore, various nitrogen
sources such as nitrate, nitrite, ammonia, and urea has been employed
for its cultivation\textsuperscript{{[}28{]}}. It has been reported that
cultivation of \emph{P. tricornutum} on glycerol supplemented medium
yields 0.4 g L\textsuperscript{-1}D\textsuperscript{-1} of biomass and
8.5 mg L\textsuperscript{-1}D\textsuperscript{-1} of
EPA\textsuperscript{{[}29{]}}\emph{.}Furthermore, utilizing urea as sole
nitrogen source resulted in a considerable enhancement in EPA content
(26 mg g\textsuperscript{-1})\textsuperscript{{[}28-30{]}}. Compared to
the conventional nitrogen source i.e. sodium nitrate
(NaNO\textsubscript{3}), urea is the cost effective and environmental
friendly substrate for the cultivation of microalgae and thus the
production of HVRs\textsuperscript{{[}28, 31{]}}. The nutrient costs for
the large scale cultivation of \emph{P. tricornutum} on F/2 medium (with
NaNO\textsubscript{3}) approximately to be USD 0.15
kg\textsuperscript{-1} biomass, whereas its cost decreased to half i.e.
USD 0.07, kg\textsuperscript{-1} biomass when grown on a modified medium
(with urea) (Cui et al., 2021). Whereas crude glycerol, a by-product of
biodiesel processing, reported for the production of \selectlanguage{greek}β-\selectlanguage{english}carotene and DHA
from \emph{Schizochytrium limanicum}and \emph{Blakeslea
trispora}\textsuperscript{{[}32{]}}. As a result, using these substrates
might be a viable method for sustainable production of biomass, and HVRs
from \emph{P. tricornutum} .

The aim of this study is to examine the effect of glycerol, urea,
NaNO\textsubscript{3} and their various combinations for the production
of HVRs in \emph{P. tricornutum} , with a primary focus on EPA along
with fucoxanthin. Our preliminary screening revealed that glycerol
(0.1M) and urea (441 M) could be used as low-cost substrate for the
generation of biomass, lipids, carbohydrates and EPA. Moreover, feeding
additional urea to the culture supplemented with glycerol led to
significant enhancement of biomass, EPA, and fucoxanthin production. In
this context, we highlight an alternate strategy, beneficial for the
sustainable co-production of various HVRs from \emph{P. tricornutum} .

\textbf{2. Experimental section}

\textbf{2.1 Microalgal strain and pre-culture conditions}

The marine diatom, \emph{Phaeodactylum tricornutum} UTEX 646, was
obtained from the Microbial Culture Collection at the National Institute
for Environmental Studies (NIES) in Tsukuba, Japan. The microalgae were
grown in Erlenmeyer flasks (1000 mL) containing 400 mL medium (minimal
medium F/2)\textsuperscript{{[}33{]}} for 4 days, under a light regime
of 16:8 h and an illumination of 30 \selectlanguage{greek}μ\selectlanguage{english}mol m\textsuperscript{-2}
s\textsuperscript{-1} photosynthetically active radiation (PAR) at 24
\selectlanguage{ngerman}°C. The F/2 medium was prepared in artificial seawater as described
previously\textsuperscript{{[}34, 35{]}}.

\textbf{2.2 Experimental design}

Different substrates and various combinations along with concentrations
used for initial experiments are mentioned in \textbf{Table S1
(Supporting information)} . Glycerol and urea concentrations were
selected based on previous literature\textsuperscript{{[}29, 30{]}}.
Inoculation was carried out using the inoculum in linear growth phase as
described earlier with a biomass of 0.1 g L\textsuperscript{-1}. The
organic nutrients (urea and glycerol) were sterilized by filtration
through 0.2 \selectlanguage{greek}µ\selectlanguage{english}m membrane filters and added to the autoclaved medium in
sterile conditions. Subsequently the effect of urea feed was
investigated by cultivating \emph{P. tricornutum} on glycerol + urea or
M3. Urea was added as feed according to the consumption profile, before
its complete exhaustion from the medium (\textbf{Table S2, Supporting
information)} . Sampling was carried out at regular time intervals of 2
days for further analysis and growth was monitored by cell count using
hemocytometer\textsuperscript{{[}36{]}} and dry weight analysis (DCW).
Parameters such as specific growth rate and doubling time were
calculated as described previously\textsuperscript{{[}37, 38{]}}.

\textbf{2.3 Estimation of organic and inorganic substrates}

Residual sugars quantified using high-performance liquid chromatography
(HPLC) which includes the Aminex HPX-87H Ion Exclusion column (300 \selectlanguage{ngerman}× 7.8
mm) attached with a refractive index (R.I.) detector (Agilent
Techologies, USA)\textsuperscript{{[}39{]}}. The supernatant was
collected every 48 h and was diluted to 100m× with 4 mM sulphuric acid.
Sample of 10 uL injected into the column maintained at 40 ºC using 4 mM
H\textsubscript{2}SO\textsubscript{4} as the mobile phase with a flow
rate of 0.3 mL min\textsuperscript{-1} and a run time of 50 min
\textsuperscript{{[}40{]}}. The peak area of standard glycerol (Sigma
Aldrich Pvt. Ltd., USA) used as reference. Nitrate uptake by the cells
was measured spectrophotometrically\textsuperscript{{[}41{]}}. Briefly,
1 mL of culture was pelleted down at room temperature (RT), and
supernatant was diluted 50-fold with deionized water. The residual
nitrate content was determined by measuring the absorbance at 220 nm.
Urea estimation was done as described in Jung et
al\textsuperscript{{[}42, 43{]}}. Briefly supernatant was used directly
to perform the assay, 50 \selectlanguage{greek}μ\selectlanguage{english}L of supernatant was transferred into a clear
flat-bottom 96-well plate. Then 200 \selectlanguage{greek}μ\selectlanguage{english}L of freshly prepared working
reagent was added and mixed quickly by gently rocking the plate. The
reaction was incubated for 1 h at room temperature. Optical densities
(OD) at 430 nm were measured on the plate reader for measuring the urea
content in the medium.

\textbf{2.4 Biochemical characterization}

Biochemical analysis of all the samples were done for analyzing the
changes in composition, i.e., total proteins, carbohydrates, and lipids
content subjected to various growth conditions. The
sulfo-phospho-vanillin test was used to estimate the total lipid
content\textsuperscript{{[}44{]}}. Briefly, 0.5 mg cells were pelleted
and resuspended into 100 \selectlanguage{greek}µ\selectlanguage{english}L of distilled water. The cells were treated
with 2 mL of 98 \% H\textsubscript{2}SO\textsubscript{4} and incubated
at 100 \selectlanguage{ngerman}°C for 10 minutes. After cooling for 5 minutes on ice, 5 mL of
phosphovanillin reagent (0.6 g vanillin in 10 mL ethanol and 90 mL
distilled water and 400 mL of 85 \% phosphoric acid) was added, and the
samples incubated for 10 minutes at 37 °C, absorbance was measured at
530 nm. Total carbohydrate was estimated using a modified
phenol-sulfuric acid technique \textsuperscript{{[}45{]}}. Around 0.25
mg cells were hydrolyzed; with 0.2 mL of 98 \%
H\textsubscript{2}SO\textsubscript{4} at RT for 1 hour; following that,
5 \% of phenol was added along with 1 mL of
H\textsubscript{2}SO\textsubscript{4} and incubated at RT for 20 minutes
and the absorbance was measured at 490 nm. Protein content was
quantified using a modified biuret technique. After pelleting 0.5 mg
cells, 1 mL of extraction buffer (25 \% NaOH in 1N methanol) was added.
The reaction mixture was incubated at 80 °C for 15 minutes. To eliminate
debris, the sample was cooled to RT and centrifuged. The supernatant was
then treated with CuSO\textsubscript{4} solution (0.21 \%
CuSO\textsubscript{4} in 30 \% NaOH) and kept at RT for 10 minutes
before being measured at 310 nm\textsuperscript{{[}46{]}}.

\textbf{2.5 Quantification of total fatty acid methyl esters (FAMEs)}

A modified Bligh-dyer technique was adapted from shaikh et
al\textsuperscript{{[}35{]}} for extracting total fatty acid methyl
esters (FAMEs). The hexane layer was injected into an Agilent 6890 gas
chromatograph (GC) instrument, which was connected to a triple
quadrupole outfitted with an OMEGAWAX 250 column (30 m x 0.25 m x 0.25
m). The GC-MS running conditions were as stated by Kareya et al
\textsuperscript{{[}47{]}}. Concisely, 2 L of the sample was run in the
split mode (1:10) for 25 minutes, with the beginning oven temperature
set at 150 °C and a simultaneous escalation of 10 °C
min\textsuperscript{-1} to 240 °C. For quenching, helium (He) and
nitrogen (N\textsubscript{2}) gas were employed at flow rates of 2.25 mL
min\textsuperscript{-1} and 1.5 mL min\textsuperscript{-1},
respectively. The data was collected using the Agilent 7000D triple
quadrupole selective mass detector, which has a scan range of 30 to 550
amu. The produced peaks were compared to NIST libraries for
identification and alignment based on retention indices as well as mass
spectral similarity (cut-off hits with R-values greater than 600 were
selected).

\textbf{2.6 Quantification of fucoxanthin in \emph{P. tricornutum}}

Fucoxanthin was quantified using high-performance liquid chromatography
(HPLC), and the extraction was performed as reported in Paliwal et
al\textsuperscript{{[}48{]}}. Briefly, 0.5 mg cells were centrifuged and
resuspended in 1 ml of 100\% methanol. For pigment extraction, the cell
suspension briefly vortexed with glass beads for 20 minutes. The
supernatant was collected and analyzed by HPLC-UV (Agilent Infinity
series 1,260 HPLC, Agilent Technologies, Santa Clara, CA, United
States). Running condition were as follows, C30 column (4.6 x 250 mm, 5
mm) was used to run the samples, at 35 °C with the binary solvent system
as the mobile phase, which included methanol as primary solvent A and
methyl tert-butyl ether (MTBE) as solvent B. The run conditions were as
follows: 2--20 \% B for the first 10 minutes, then 20 \% B (10--12
minutes), 20--80 \% B (12--30 minutes), 80 \% B (30--32 minutes), and
80--2 \% B (32--35 minutes)\textsuperscript{{[}49{]}}. Pigments were
measured at 437 nm and identified by comparing the retention duration of
DHI standards from Hrsholm, Denmark.

\textbf{2.7 Statistical Analysis}

Experimental runs were conducted in triplicate \textbf{(} the mean
values were presented as ±SE), comparing each condition in the time
course for a period of 10 days. Statistical analyses such as ANOVA and
t-test were performed using Microsoft excel for determination of
significance.

\textbf{3. Results}

\textbf{3.1. Biomass production of \emph{P. tricornutum}}

Initial screening for measuring the growth and nutrient consumption in
photoautotrophic and mixotrophic conditions was performed. \emph{P.
tricornutum} was grown with an initial biomass concentration of 0.1 g
L\textsuperscript{-1}and attained a maximum biomass i.e. 0.77 g
L\textsuperscript{-1} (\textasciitilde{}2-fold higher) in M4 condition
in 10 days. Additionally higher growth rates were observed in the
mixotrophic cultures (\textbf{Table 1)} . Overall, the combined effect
of mixotrophy and urea feeding clearly show a higher growth rate and
biomass in \emph{P. tricornutum} . Previous reports have shown the
effect of mixotrophy on growth of \emph{P. tricornutum} which correlates
with our results \textsuperscript{{[}25, 27, 50, 51{]}}.

Substrate consumption reflects biomass productivity, thus nutrient
uptake rates were measured (\textbf{Figure S1, S2, S3, S4, Supporting
information)} . \emph{P. tricornutum} consumed around 3 g
L\textsuperscript{-1} glycerol (10\textsuperscript{th} day), 75 mg
L\textsuperscript{-1} NaNO\textsubscript{3} (6\textsuperscript{th} day),
and 26 mg L\textsuperscript{-1} urea (4\textsuperscript{th} day)
(\textbf{Figure S1, Supporting information} ). Further, in the presence
of glycerol, consumption rate of nutrients increased resulting in the
exhaustion of NaNO\textsubscript{3} by 4\textsuperscript{th} day (M1)
and urea by 2\textsuperscript{nd} day. Mixotrophic mode of \emph{P.
tricornutum} enhanced the nutrient uptake rate was previously reported
by Villanova et al.,\textsuperscript{{[}51{]}}. Interestingly, urea is
completely consumed in the medium by 4\textsuperscript{th} day (P2, P3,
M2, M3), whereas NaNO\textsubscript{3} is exhausted by
6\textsuperscript{th} (P1) day of cultivation (\textbf{Figure S2} ,
\textbf{S3, Supporting information} ). This also justifies decrease in
biomass and growth rate in P3 culture conditions as compared to P1and
P2. Hence, a strategy based on feeding urea was designed to maintain a
high biomass rate on a cost-effective substrate (M4). Urea was fed into
the medium at the early log phase at 36\textsuperscript{th} hour (before
urea depletion) according to the 12-hour urea consumption profile
(\textbf{Table S2} , \textbf{Figure S4, Supporting information)} .
Growth profile in M4 medium improved as compared to the P1 and M3,
highlighting it as effective nitrogen source for the growth of \emph{P.
tricornutum} . Effect of different nitrogen sources on growth of
\emph{P. tricornutum} , highlighted urea as the best source due to the
increase in biomass yield and lower substrate
cost\textsuperscript{{[}28{]}}. It can be observed that \emph{P.
tricornutum} prefers urea as nitrogen source and consumes it more
efficiently than NaNO\textsubscript{3} (\textbf{Figure S2, S4,
Supporting information} ).

\textbf{3.2. Biochemical analysis}

The volumetric concentration of biochemical constituent were estimated
to understand the influence of various growth conditions on cellular
constituents of \emph{P. tricornutum} (\textbf{Table 1} ). Biochemical
constituents were significantly enhanced in M4 condition. Protein titer
in M4 condition was maximum i.e., \textasciitilde{}362 mg
L\textsuperscript{-1} (\textasciitilde{}2.5-fold increase) and with
higher biomass productivity (\textbf{Figure 1)} . In addition, increased
protein concentrations were observed in photoautotrophic cultures with 2
nitrogen sources and combined mixotrophic medium supplemented with mixed
nitrogen source (P2 and M2) (\textbf{Table 1)} . Microalgae under the
influence of nitrogen rich environment (feed cultures) channelize the
photosynthetically fixed carbon to protein synthesis to support growth
and division, however, exactly an opposite trend is seen in nitrogen
deprived conditions, where cell division ceases and fraction of carbon
allocation to storage molecules increases at the expense of protein
synthesis \textsuperscript{{[}28{]}}.

The maximum lipid titer was 227 mg L\textsuperscript{-1} and 208 mg
L\textsuperscript{-1} in M4 and M3 cultures respectively (\textbf{Table
1)} . Our study highlights that glycerol and urea supplementation
enhanced lipid productivity which is quite interesting, as many
microalgae are known to accumulate lipids in response to N
limitation\textsuperscript{{[}52{]}}. This can be justified by the
addition of glycerol; \emph{P. tricornutum} in lipid accumulation phase
uses glycerol as a carbon source and hence channelize the carbon
directly towards lipid production, moreover feeding nitrogen leads to
more biomass, which in turn reflects in increasing the productivity of
biochemical constituents. The result clearly indicates that the strategy
of urea recharge \emph{via} feeding in glycerol supplemented medium can
effectively produce biomass and lipids.

Cultures supplemented in M3 condition showed higher carbohydrate
production i.e., 164 mg L\textsuperscript{-1}, and was enhanced to 231
mg L\textsuperscript{-1} (\textasciitilde{}1.5-fold increase) in M4
(\textbf{Table 1)} . Carbohydrate are primary storage reserves of
diatoms, one the first photosynthetic product of the Calvin--Benson
cycle and serves as a precursor for various cellular
components\textsuperscript{{[}53{]}}. Therefore, carbohydrate
accumulation and conversion into other metabolites is necessary for cell
survival. It was observed that addition of glycerol switches the
cellular metabolism towards carbohydrate production in\emph{P.
tricornutum}\textsuperscript{{[}52{]}}. Compared to lipid production,
the carbohydrate content was less in glycerol supplemented cultures,
which clearly demonstrates the channeling of carbon flux towards TAG
production\textsuperscript{{[}54{]}}. In this context, feeding \emph{P.
tricornutum} with a cost-effective nitrogen source like urea, resulted
in higher protein, lipid, carbohydrate and biomass production.
Highlighting the potential of our substrates to enhance the overall
productivity of \emph{P. tricornutum} and to further use in a
biorefinery-based system.

\textbf{3.3 FAME analysis}

FAME profile showed \textasciitilde{} 2-fold increase in glycerol
supplemented cultures (M1, M2, M3, M4), where \textasciitilde{}97 mg
L\textsuperscript{-1} of lipid was obtained in the M4 condition, which
is \textasciitilde{} 40 \% higher as compared to P1 and 8 \% as compared
to M3 cultures (\textbf{Table 2)} . Glycerol in the form of reduced
carbon source affects lipid metabolism by mimicking most of the effects
of nitrogen limitation\textsuperscript{{[}52, 55{]}}, hence the storage
molecules i.e. triacylglycerol (TAGs) are accumulated in M3 culture.
Moreover, significant variations were observed in FAME profile (\% total
fatty acid) in different culture conditions (\textbf{Table 2)} .
Saturated and monounsaturated fatty acid percentage were higher in P1,
P3, M1, M2 and M3 supplemented cultures, whereas P2 and M4 cultures
showed a higher polyunsaturated fatty acid percentage. EPA being the
major long chain fatty acid in \emph{P. tricornutum} was highest among
all other fatty acid species in all the conditions but
\textasciitilde{}2-fold increase of EPA \% of total fatty acid (TFA) was
observed in M4, accounting for 28\% of TFA (\textbf{Table 2).} These
results imply that feeding with cost effective nitrogen source can be
used as an effective strategy to enhance the omega-3 production in
\emph{P. tricornutum.}

\textbf{3.4. Enhancement of EPA and fucoxanthin production in \emph{P.
tricornutum}.}

Total EPA and fucoxanthin content is represented in \textbf{Figure 2A \&
B.} The EPA content increased from 9 mg L\textsuperscript{-1} to 27 mg
L\textsuperscript{-1} in M4 condition within 10 days \textbf{(Table 3)}
, which accounts for a \textasciitilde{}3-fold increase compared to P1
control and \textasciitilde{} 2-fold increase in compare to M3
conditions. Content wise on 6\textsuperscript{th} day, EPA was higher in
initial log phase accounting to 37 mg g\textsuperscript{-1} in the M2
condition, whereas M1 and M3 condition showed 34 mg
g\textsuperscript{-1} of EPA (\textbf{Figure 2A)} . Furthermore, EPA
content was higher in culture supplemented with only urea (P3) when
compared to NaNO\textsubscript{3} (P1) on 4\textsuperscript{th} and
6\textsuperscript{th} day (\textbf{Figure 2A)} . Increase in EPA content
in the initial growth phase was reported earlier in the presence of
urea\textsuperscript{{[}28{]}} and glycerol along with urea in \emph{P.
tricornutum}\textsuperscript{{[}29, 30, 56{]}}.

Along with EPA, fucoxanthin content was measured in various culture
conditions as depicted in \textbf{Figure 2B.} Higher fucoxanthin
production was observed in M4 conditions i.e., 11 mg
L\textsuperscript{-1} on 10\textsuperscript{th} day of experiment
(\textbf{Table 3)} . This showed a \textasciitilde{} 4-fold increase in
fucoxanthin productivity compared to P1 and M3 conditions. Fucoxanthin
production was 6.62 mg L\textsuperscript{-1} in medium supplemented with
P2 on 10\textsuperscript{th} day, whereas highest content was 21.9 mg
g\textsuperscript{-1} on 6\textsuperscript{th} day of cultivation in the
P1 condition (\textbf{Figure 2B)} . Addition of glycerol limited the
nitrogen content in the medium, and thus a decrease in the fucoxanthin
content is observed in glycerol supplementation (M1 and M3)
(\textbf{Figure 2B)} , although higher production is observed in M2
condition compared to M1 and M3 conditions, which can be justified by
the presence of two nitrogen source and residual N in the medium until
10\textsuperscript{th} day (\textbf{Figure S1, S2, S3, Supporting
information)} . Fucoxanthin being a primary carotenoid is highly
influenced by nitrogen content in the medium\textsuperscript{{[}57{]}}.
Previous reports highlights higher fucoxanthin content in
\emph{P.tricornutum}cultivated in nitrate-enriched
medium\textsuperscript{{[}7, 16{]}}, thus feeding cultures with urea,
increased biomass and the biomass associated metabolites i.e. EPA and
fucoxanthin.

\textbf{4. Discussion}

Microalgae derived HVRs are progressively playing a significant role in
the production of cosmeceuticals, medicinal alternatives and high-value
foods \textsuperscript{{[}58{]}}.\emph{P. tricornutum} as a cell factory
for marketable products which majorly includes omega 3 PUFAs like EPA
and light harvesting pigment fucoxanthin, \textsuperscript{{[}8{]}}. Due
to their high pharmaceutical and nutritional relevance, these HVRs
currently have a high market value for example the market size of omega
3 ingredients in 2019 exceeded USD 2.3 billion and is estimated to grow
at over 7.2\% compound annual growth rate (CAGR) between 2020 and 2026
(www.gminsights.com). Moreover, revenue generated from fucoxanthin was
USD 100 million in 2019, and is expected to reach USD 123 million by
2025, with a CAGR of 3.5\% (www.wboc.com).

Sustainable co-production of these HVRs is a challenging task in terms
of economic feasibility. Therefore, to enhance production, modification
in the cultivation parameters can be employed as an effective strategy.
Our preliminary analysis highlighted the effect of different substrates
(glycerol, urea, and NaNO\textsubscript{3}) and their different
combination on growth and biochemical constituents. Optimization of
medium with glycerol and urea (M4) addition is a suitable, sustainable
and economic approach, due to its beneficial effect on cellular
metabolism, biomass and HVRs production enhanced. Furthermore, since the
nitrate is depleted from the medium in the early phases of growth,
employing a strategy to feed urea in culture medium before exhaustion
proved to be more feasible in terms of enhancing HVR production.

Screening highlighted M4 as a suitable condition to achieve higher
biomass and better growth rate in \emph{P. tricornutum} . A combined
effect of mixotrophy and urea feeding resulted in higher biomass
compared to cultures without urea feeding. Mixotrophy metabolism
reportedly engages both respiration and photosynthesis at the same
time\textsuperscript{{[}26{]}}. Transport activities between the
chloroplast and mitochondria, as well as the physical connection between
the two organelles, have an intense energetic exchange during mixotrophy
in \emph{P. tricornutum}\textsuperscript{{[}52{]}}. Moreover, mixotrophy
enhances the nutrient uptake rate as observed in glycerol-supplemented
(M1 and M3) medium, which enhances nitrogen uptake (\textbf{Figure S1,
S2, S3, Supporting information)} .

The increase in biomass reflects the assimilation of C and N in cellular
biochemical components. Higher protein content was obtained in the
medium supplemented with M2 and M4 combinations depicting conversion of
excess nitrogen into cellular proteins. Currently microalgal proteins
are in demand as functional foods due to its high nutritional value, and
health benefits. Additionally utilization of whole algal biomass as
super foods or healthy foods is promoted worldwide in order to maintain
a balance diet\textsuperscript{{[}59{]}}. Therefore higher protein
production in \emph{P. tricornutum} makes it a suitable candidate for
nutraceutical applications. Moreover, carbohydrate productivities
reached to 231 mg L\textsuperscript{-1} in M4 condition (\textbf{Table
1)} . Anticancer activity of polysaccharides derived from\emph{P.
tricornutum} along with antibacterial, antioxidant, and antiviral
properties is well demonstrated\textsuperscript{{[}60, 61{]}}.
Polysaccharides from P\emph{. tricornutum} have a high commercial
potential and a wide range of applications in different industrial
sectors.

Biodiesel is considered as a desirable energy source, an exceptional
alternative to fossil fuels. According to the previous reports the FAME,
profile of \emph{P. tricornutum} meets the requirements of international
biodiesel standards, showing that it could be a good alternative for
biodiesel production\textsuperscript{{[}2{]}}. Our results are in
correlation with these studies showing a high percentage of C16, C16:1,
C18:1 (\%TFA). Moreover, higher FAME yields obtained in \emph{P.
tricornutum} on cost effective substrates, indicates \emph{de novo}fatty
acid synthesis in presence of glycerol. These fatty acids are main
components of storage lipids, which justifies its higher accumulation in
the form of TAGs in the mixotrophic conditions\textsuperscript{{[}26,
56{]}}. Feeding additional nitrogen shifted the fatty acid metabolism
towards membrane lipid synthesis i.e., EPA showing a higher percentage
of PUFAs in feed conditions (\textbf{Table 2} ).

The expense of nutrients is an inevitable liability in the production of
algal biomass (4-8 \% of total cost)\textsuperscript{{[}62{]}}. As a
result, the usage of recovered nutrients from secondary streams, such as
glycerol from the biodiesel sector and urea, ammonia from wastewater,
may be included into the biorefinery framework for the long-term
synthesis of HVRs. Although previous reports highlights the use of
glycerol as a C source for microalgae
production\textsuperscript{{[}63{]}} and the recovery of nutrients (N
and P) by digestate pretreatment\textsuperscript{{[}64{]}}, lesser data
is available on the combined effect of replacing both C and N with
secondary streams. The validity of such a strategy has been introduced
in our study by applying it on production of HVRs.

In M4 condition, higher EPA (27 mg L\textsuperscript{-1}) was
accumulated along with TAG (92 mg L\textsuperscript{-1}) (\textbf{Figure
2A, Table 2).} Indicating that glycerol triggers metabolic changes
resembling not only nitrogen depletion but also promote
growth\textsuperscript{{[}56{]}}. An increase in EPA content on initial
growth days (\textbf{Figure 2A)} indicate the necessity of structural
lipids in log phase but as nitrogen depletes in the medium EPA content
did not change (\textbf{Figure 2A)} in the respective conditions, though
urea-fed cultures maintain the EPA pool throughout the experiment. EPA
being one of the main structural lipids required during the growth
supporting conditions, whereas is incorporated into TAGs when the cells
are under nitrogen stress. Hence, to maintain high EPA content in stress
condition use of glycerol as carbon source can be ideal, additionally
feeding with the nitrogen source can enhance the productivity \emph{via}
., higher biomass yields.

Along with EPA, fucoxanthin content highlights a significant increase in
the productivity especially during feed conditions. Fucoxanthin is
associated with photosynthetic machinery and is enhanced as the growth
increases. Nitrogen on the contrary in the form of urea positively
regulates the photosynthetic machinery in feed condition (M4) reflected
by the higher biomass production. Hence, in the condition supplemented
with nitrogen substrates (P1, P2, and P3) fucoxanthin content was higher
(\textbf{Figure 2B)} as compared to glycerol-supplemented cultures (M1,
M2, and M3). Glycerol is found to be directly entering
glycolysis\emph{via} glycerol kinase producing dihydroxyacetone
phosphate and thus channelizing flux towards lipid synthesis rather than
carotenoid production \textsuperscript{{[}52{]}}.

Our findings highlight the effect of feeding nitrogen to the
glycerol-supplemented cells (M4), higher nitrogen content results in
increasing the biomass and thus enhancing growth related metabolites
like EPA and fucoxanthin. Presence of glycerol in the medium upregulates
the EPA biosynthesis \emph{via} regulating enzyme like stearoyl
desaturase \textsuperscript{{[}56{]}} and the effect of feeding
strategies of various nutrients on biomass, and lipid
productivities\textsuperscript{{[}65-67{]}} is mentioned in previous
reports. In conclusion, this study successfully highlights an
alternative application of feeding nutrients in mixotrophic mode to
increase HVRs like EPA and fucoxanthin. Further, the ability of \emph{P.
tricornutum} to grow mixotrophically using glycerol as the main carbon
source and urea as an additional nitrogen source can be applied in the
biorefinery approach for recovering carbon and nitrogen from waste
effluents to produce HVRs.

\textbf{Statements and Declarations}

\textbf{Declaration of Competing Interest}

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
influence the work reported in this paper.

\textbf{Acknowledgements}

This work was supported by grants from Department of Biotechnology
(BT/PB/Center/03/2011-Phase II), Ministry of Science and Technology,
Government of India (GoI), New Delhi, India. Fellowship(s) for MR, and
AAN are supported from Department of Biotechnology (DBT), New Delhi,
India, is duly acknowledged.

\textbf{Data availability statement:} All the data within the main
article and supplementary material are provided.

\textbf{Authors Contributions}

\textbf{Mohammed Rehmanji} : Conceptualization, Methodology,
Investigation, Analysis, Writing - Original Draft. \textbf{Asha Arumugam
Nesamma:} Methodology, Investigation, Analysis, Writing - Original
Draft, Writing - review \& editing. \textbf{Tasneem Fatma} :
Supervision, Writing - review \& editing. \textbf{Nida Jamil Khan:}
Supervision, Writing - review \& editing\textbf{. Pannaga Pavan Jutur} :
Conceptualization, Funding acquisition, Supervision, Methodology,
Validation, Project administration, Writing - review \& editing.

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\textbf{Figure Legends}

\textbf{Figure 1.} Time course profile of \emph{P. tricornutum} biomass
concentration in \textbf{Photo1 (P1):} Photoautotrophic control culture
with additions of 882 \selectlanguage{greek}µ\selectlanguage{english}M sodium nitrate
(NaNO\textsubscript{3});\textbf{Photo 2 (P2):} Photoautotrophic control
culture with additions of 441 \selectlanguage{greek}µ\selectlanguage{english}M urea and 882 \selectlanguage{greek}µ\selectlanguage{english}M sodium nitrate
(NaNO\textsubscript{3});\textbf{Photo 3 (P3):} Photoautotrophic control
culture with additions 441 \selectlanguage{greek}µ\selectlanguage{english}M of urea; \textbf{Mixo 1 (M1):} Mixotrophic
culture with additions of 0.1M glycerol and 882 \selectlanguage{greek}µ\selectlanguage{english}M sodium nitrate
(NaNO\textsubscript{3});\textbf{Mixo 2 (M2):} Mixotrophic culture with
additions of 0.1M glycerol, 441 \selectlanguage{greek}µ\selectlanguage{english}M of urea and 882 \selectlanguage{greek}µ\selectlanguage{english}M sodium nitrate
(NaNO\textsubscript{3}); \textbf{Mixo 3 (M3):} Mixotrophic culture with
additions of 0.1M glycerol and 441 \selectlanguage{greek}µ\selectlanguage{english}M of urea; \textbf{Mixo 4
(M4):}Mixotrophic culture with additions of 0.1M glycerol, feeded with
441 \selectlanguage{greek}µ\selectlanguage{english}M of urea post 36 hours of cultivation. Values indicate mean (n =
3) with standard error; * indicate statistical significance by one-way
analysis of variance (ANOVA), p-value \textless{} 0.05.

\textbf{Figure 2A.} Quantification of EPA content (mg
g\textsuperscript{-1}) of \emph{P. tricornutum} cultivated in different
conditions, \textbf{Photo1 (P1):} Photoautotrophic control culture with
additions of 882 \selectlanguage{greek}µ\selectlanguage{english}M sodium nitrate (NaNO\textsubscript{3});\textbf{Photo
2 (P2):} Photoautotrophic control culture with additions of 441 \selectlanguage{greek}µ\selectlanguage{english}M urea
and 882 \selectlanguage{greek}µ\selectlanguage{english}M sodium nitrate (NaNO\textsubscript{3});\textbf{Photo 3 (P3):}
Photoautotrophic control culture with additions 441 \selectlanguage{greek}µ\selectlanguage{english}M of urea;
\textbf{Mixo 1 (M1):} Mixotrophic culture with additions of 0.1M
glycerol and 882 \selectlanguage{greek}µ\selectlanguage{english}M sodium nitrate (NaNO\textsubscript{3});\textbf{Mixo
2 (M2):} Mixotrophic culture with additions of 0.1M glycerol, 441 \selectlanguage{greek}µ\selectlanguage{english}M of
urea and 882 \selectlanguage{greek}µ\selectlanguage{english}M sodium nitrate (NaNO\textsubscript{3}); \textbf{Mixo 3
(M3):} Mixotrophic culture with additions of 0.1M glycerol and 441 \selectlanguage{greek}µ\selectlanguage{english}M of
urea. Values indicate mean (n = 3) with standard error; * indicate
statistical significance by one-way analysis of variance (ANOVA), pvalue
\textless{} 0.05.

\textbf{Figure 2B.} Quantification of fucoxanthin content (mg
g\textsuperscript{-1}) of \emph{P. tricornutum} cultivated
in\textbf{Photo1 (P1):} Photoautotrophic control culture with additions
of 882 \selectlanguage{greek}µ\selectlanguage{english}M sodium nitrate (NaNO\textsubscript{3}); \textbf{Photo 2
(P2):}Photoautotrophic control culture with additions of 441 \selectlanguage{greek}µ\selectlanguage{english}M urea and
882 \selectlanguage{greek}µ\selectlanguage{english}M sodium nitrate (NaNO\textsubscript{3}); \textbf{Photo 3
(P3):}Photoautotrophic control culture with additions 441 \selectlanguage{greek}µ\selectlanguage{english}M of
urea;\textbf{Mixo 1 (M1):} Mixotrophic culture with additions of 0.1M
glycerol and 882 \selectlanguage{greek}µ\selectlanguage{english}M sodium nitrate (NaNO\textsubscript{3}); \textbf{Mixo
2 (M2):} Mixotrophic culture with additions of 0.1M glycerol, 441 \selectlanguage{greek}µ\selectlanguage{english}M of
urea and 882 \selectlanguage{greek}µ\selectlanguage{english}M sodium nitrate (NaNO\textsubscript{3}); \textbf{Mixo 3
(M3):} Mixotrophic culture with additions of 0.1M glycerol and 441 \selectlanguage{greek}µ\selectlanguage{english}M of
urea conditions. Values indicate mean (n = 3) with standard error; *
indicate statistical significance by one-way analysis of variance
(ANOVA), pvalue \textless{} 0.05.

\textbf{Table Legends}

\textbf{Table 1.} Performance of microalgal dry biomass concentration on
day 10, specific growth rate, carbohydrate titer, protein titer, and
lipid titer of \emph{P. tricornutum} under \textbf{Photo1
(P1):}Photoautotrophic control culture with additions of 882 \selectlanguage{greek}µ\selectlanguage{english}M sodium
nitrate (NaNO\textsubscript{3}); \textbf{Photo 2 (P2):} Photoautotrophic
control culture with additions of 441 \selectlanguage{greek}µ\selectlanguage{english}M urea and 882 \selectlanguage{greek}µ\selectlanguage{english}M sodium nitrate
(NaNO\textsubscript{3}); \textbf{Photo 3 (P3):} Photoautotrophic control
culture with additions 441 \selectlanguage{greek}µ\selectlanguage{english}M of urea; \textbf{Mixo 1 (M1):} Mixotrophic
culture with additions of 0.1M glycerol and 882 \selectlanguage{greek}µ\selectlanguage{english}M sodium nitrate
(NaNO\textsubscript{3}); \textbf{Mixo 2 (M2):} Mixotrophic culture with
additions of 0.1M glycerol, 441 \selectlanguage{greek}µ\selectlanguage{english}M of urea and 882 \selectlanguage{greek}µ\selectlanguage{english}M sodium nitrate
(NaNO\textsubscript{3}); \textbf{Mixo 3 (M3):} Mixotrophic culture with
additions of 0.1M glycerol and 441 \selectlanguage{greek}µ\selectlanguage{english}M of urea; \textbf{Mixo 4
(M4):}Mixotrophic culture with additions of 0.1M glycerol, feeded with
441 \selectlanguage{greek}µ\selectlanguage{english}M of urea post 36 hours of cultivation. Values indicate mean (n =
3) with standard error; * indicate statistical significance by one-way
analysis of variance (ANOVA), pvalue \textless{} 0.05.

\textbf{Table 2.} Total fatty acid methyl esters (FAMEs) (\% FAME
profile) using gas chromatography-mass spectrometry (GC-MS) of \emph{P.
tricornutum} sp. in the presence of glycerol, NaNO\textsubscript{3}, and
urea. Values indicate mean (n = 3) with standard error; * indicate
statistical significance by

one-way analysis of variance (ANOVA), p-value \textless{} 0.05.

\textbf{Table 3.} Eicosapentaenoic acid and fucoxanthin titer (mg
L\textsuperscript{-1}) in \emph{P. tricornutum} sp. in \textbf{Photo1
(P1):} Photoautotrophic control culture with additions of 882 \selectlanguage{greek}µ\selectlanguage{english}M sodium
nitrate (NaNO\textsubscript{3}); \textbf{Photo 2 (P2):} Photoautotrophic
control culture with additions of 441 \selectlanguage{greek}µ\selectlanguage{english}M urea and 882 \selectlanguage{greek}µ\selectlanguage{english}M sodium nitrate
(NaNO\textsubscript{3}); \textbf{Photo 3 (P3):} Photoautotrophic control
culture with additions 441 \selectlanguage{greek}µ\selectlanguage{english}M of urea; \textbf{Mixo 1 (M1):} Mixotrophic
culture with additions of 0.1M glycerol and 882 \selectlanguage{greek}µ\selectlanguage{english}M sodium nitrate
(NaNO\textsubscript{3}); \textbf{Mixo 2 (M2):} Mixotrophic culture with
additions of 0.1M glycerol, 441 \selectlanguage{greek}µ\selectlanguage{english}M of urea and 882 \selectlanguage{greek}µ\selectlanguage{english}M sodium nitrate
(NaNO\textsubscript{3}); \textbf{Mixo 3 (M3):} Mixotrophic culture with
additions of 0.1M glycerol and 441 \selectlanguage{greek}µ\selectlanguage{english}M of urea; \textbf{Mixo 4
(M4):}Mixotrophic culture with additions of 0.1M glycerol, feeded with
441 \selectlanguage{greek}µ\selectlanguage{english}M of urea post 36 hours of cultivation condition. Values indicate
mean (n = 3) with standard error; * indicate statistical significance by
one-way analysis of variance (ANOVA), p-value \textless{} 0.05.

\textbf{Table 1.}\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
\textbf{Parameters/Conditions} & \textbf{P1} & \textbf{P2} & \textbf{P3}
& \textbf{M1} & \textbf{M2} & \textbf{M3} & \textbf{M4}\tabularnewline
\textbf{Specific growth rate (day\textsuperscript{-1})} & 0.26 \selectlanguage{ngerman}± 0.01 &
0.25 \selectlanguage{ngerman}± 0.03 & 0.22 \selectlanguage{ngerman}± 0.02 & 0.37 \selectlanguage{ngerman}± 0.03* & 0.32 \selectlanguage{ngerman}± 0.04* & 0.38 \selectlanguage{ngerman}± 0.01* &
0.35 \selectlanguage{ngerman}± 0.03*\tabularnewline
\textbf{Biomass (g L\textsuperscript{-1})} & 0.35 \selectlanguage{ngerman}± 0.03 & 0.36 \selectlanguage{ngerman}± 0.03 &
0.32 \selectlanguage{ngerman}± 0.01 & 0.54 \selectlanguage{ngerman}± 0.02* & 0.53 \selectlanguage{ngerman}± 0.06* & 0.46 \selectlanguage{ngerman}± 0.02* & 0.77 \selectlanguage{ngerman}±
0.01*\tabularnewline
\textbf{Lipid ( mg L\textsuperscript{-1})} & 55.85 \selectlanguage{ngerman}± 0.89 & 61.77 \selectlanguage{ngerman}± 1.73
& 60.97 \selectlanguage{ngerman}± 1.17 & 167.66 \selectlanguage{ngerman}± 4.41* & 152.85 \selectlanguage{ngerman}± 4.09* & 208.12 \selectlanguage{ngerman}± 4.45* &
227.29 \selectlanguage{ngerman}± 11.46*\tabularnewline
\textbf{Carbohydrates (mg L\textsuperscript{-1})} & 88.83 \selectlanguage{ngerman}± 2.45 & 86.91
\selectlanguage{ngerman}± 5.37 & 97.5 \selectlanguage{ngerman}± 6.12 & 93.83 \selectlanguage{ngerman}± 3.01* & 124.5 \selectlanguage{ngerman}± 2.71* & 164.50 \selectlanguage{ngerman}± 0.23* &
231.45 \selectlanguage{ngerman}± 3.50*\tabularnewline
\textbf{Proteins (mg L\textsuperscript{-1})} & 169.49 \selectlanguage{ngerman}± 10.50 & 194.06 \selectlanguage{ngerman}±
1.20 & 132.10 \selectlanguage{ngerman}± 0.75 & 148.78 \selectlanguage{ngerman}± 1.59* & 217.52 \selectlanguage{ngerman}± 2.20* & 138.69 \selectlanguage{ngerman}± 4.46*
& 361.80 \selectlanguage{ngerman}± 3.12*\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
\textbf{Total Fatty acid (\% FAME)} & \textbf{Total Fatty acid (\%
FAME)} & \textbf{Total Fatty acid (\% FAME)} & \textbf{Total Fatty acid
(\% FAME)} & \textbf{Total Fatty acid (\% FAME)} & \textbf{Total Fatty
acid (\% FAME)} & \textbf{Total Fatty acid (\% FAME)} & \textbf{Total
Fatty acid (\% FAME)}\tabularnewline
\textbf{Fatty acid} & \textbf{P1} & \textbf{P2} & \textbf{P3} &
\textbf{M1} & \textbf{M2} & \textbf{M3} & \textbf{M4}\tabularnewline
C14 & 7.6 \selectlanguage{ngerman}± 0.01 & 7.6 \selectlanguage{ngerman}± 0.04 & 7.7 \selectlanguage{ngerman}± 0.15 & 10.6 \selectlanguage{ngerman}± 0.53 & 9.5 \selectlanguage{ngerman}± 0.17 &
9.9 \selectlanguage{ngerman}± 0.07 & 9.7 \selectlanguage{ngerman}± 0.04\tabularnewline
C16 & 15.8 \selectlanguage{ngerman}± 0.03 & 14 \selectlanguage{ngerman}± 0.01 & 17.5 \selectlanguage{ngerman}± 0.21* & 16.9 \selectlanguage{ngerman}± 0.11 & 16.3 \selectlanguage{ngerman}± 0.34
& 17.3 \selectlanguage{ngerman}± 0.27* & 8.9 \selectlanguage{ngerman}± 0.46*\tabularnewline
C16:1 & 20.2 \selectlanguage{ngerman}± 0.46 & 17.4 \selectlanguage{ngerman}± 0.39 & 23.1 \selectlanguage{ngerman}± 0.5 & 24.4 \selectlanguage{ngerman}± 0.12 & 23.4 \selectlanguage{ngerman}±
0.15 & 25.9 \selectlanguage{ngerman}± 0.52 & 15.7 \selectlanguage{ngerman}± 0.42\tabularnewline
C16:2 & 4.7 \selectlanguage{ngerman}± 0.03 & 7.5 \selectlanguage{ngerman}± 0.03* & 4.2 \selectlanguage{ngerman}± 0.14 & 2.1 \selectlanguage{ngerman}± 0.15 & 2.8 \selectlanguage{ngerman}± 0.02
& 2.4 \selectlanguage{ngerman}± 0.07 & 3.4 \selectlanguage{ngerman}± 0.08*\tabularnewline
C16:3 & 6.2 \selectlanguage{ngerman}± 0.03 & 7.8 \selectlanguage{ngerman}± 0.03 & 4.9 \selectlanguage{ngerman}± 0.11 & 2.7 \selectlanguage{ngerman}± 0.05 & 3.4 \selectlanguage{ngerman}± 0.03 &
2.6 \selectlanguage{ngerman}± 0.06* & 4.3 \selectlanguage{ngerman}± 0.07*\tabularnewline
C16:4 & NA & 1.1 \selectlanguage{ngerman}± 0.02 & NA & NA & NA & 0.5 \selectlanguage{ngerman}± 0.01 & 0.7 \selectlanguage{ngerman}±
0.07\tabularnewline
C18 & 4.8 \selectlanguage{ngerman}± 0.15* & 3.7 \selectlanguage{ngerman}± 0.02 & 2.6 \selectlanguage{ngerman}± 0.08 & 2.6 \selectlanguage{ngerman}± 0.88 & 2.7 \selectlanguage{ngerman}± 0.89 &
2.4 \selectlanguage{ngerman}± 0.05 & 1.7 \selectlanguage{ngerman}± 0.08*\tabularnewline
C18:1 & 11.8 \selectlanguage{ngerman}± 0.69 & 9.1 \selectlanguage{ngerman}± 0.09 & 11.8 \selectlanguage{ngerman}± 0.01 & 16.1 \selectlanguage{ngerman}± 0.21 & 14.9 \selectlanguage{ngerman}±
0.06 & 14 \selectlanguage{ngerman}± 1.75* & 9.7 \selectlanguage{ngerman}± 0.05*\tabularnewline
C18:2 & 1.2 \selectlanguage{ngerman}± 0.01 & 3.7 \selectlanguage{ngerman}± 0.09 & 3.6 \selectlanguage{ngerman}± 0.32 & 3.0 \selectlanguage{ngerman}± 0.13 & 3.1 \selectlanguage{ngerman}± 0.01 &
2.3 \selectlanguage{ngerman}± 0.16 & 3 \selectlanguage{ngerman}± 0.01\tabularnewline
C18:3 & 2.3 \selectlanguage{ngerman}± 0.11 & 2.5 \selectlanguage{ngerman}± 0.16 & 2.3 \selectlanguage{ngerman}± 0.12 & 3.0 \selectlanguage{ngerman}± 0.06 & 2.7 \selectlanguage{ngerman}± 0.09 &
4.0 \selectlanguage{ngerman}± 0.14* & 2.4 \selectlanguage{ngerman}± 0.04*\tabularnewline
C18:4 & 2.7 \selectlanguage{ngerman}± 0.11 & 1.7 \selectlanguage{ngerman}± 0.01 & 2.0 \selectlanguage{ngerman}± 0.14 & 4.2 \selectlanguage{ngerman}± 0.17 & 4.4 \selectlanguage{ngerman}± 0.05 &
3.6 \selectlanguage{ngerman}± 0.12 & 5 \selectlanguage{ngerman}± 0.11\tabularnewline
C20:4 & NA & NA & NA & NA & NA & 1.1 \selectlanguage{ngerman}± 0.02 & 1.6 \selectlanguage{ngerman}± 0.08\tabularnewline
C20:5 & 18.1 \selectlanguage{ngerman}± 0.04 & 18.4 \selectlanguage{ngerman}± 0.15 & 15.4 \selectlanguage{ngerman}± 0.22 & 11.3 \selectlanguage{ngerman}± 0.23 & 12.1 \selectlanguage{ngerman}±
0.15 & 12.6 \selectlanguage{ngerman}± 0.38* & 28.1 \selectlanguage{ngerman}± 0.02*\tabularnewline
C24 & 4.5 \selectlanguage{ngerman}± 0.06 & 5.7 \selectlanguage{ngerman}± 0.24 & 4.7 \selectlanguage{ngerman}± 0.11 & 3.1 \selectlanguage{ngerman}± 0.08 & 4.9 \selectlanguage{ngerman}± 0.05 &
4.0 \selectlanguage{ngerman}± 0.23 & 5.9 \selectlanguage{ngerman}± 1.68\tabularnewline
SFA & 32.8 \selectlanguage{ngerman}± 0.05 & 31.0 \selectlanguage{ngerman}± 0.24 & 32.5 \selectlanguage{ngerman}± 0.54 & 33.2 \selectlanguage{ngerman}± 0.63 & 33.3 \selectlanguage{ngerman}±
0.11 & 33.6 \selectlanguage{ngerman}± 0.62 & 26.2 \selectlanguage{ngerman}± 2.19*\tabularnewline
MUFA & 32.0 \selectlanguage{ngerman}± 0.22 & 26.4 \selectlanguage{ngerman}± 0.48 & 35.0 \selectlanguage{ngerman}± 0.51 & 40.4 \selectlanguage{ngerman}± 0.08 & 38.3 \selectlanguage{ngerman}±
0.08 & 39.9 \selectlanguage{ngerman}± 1.23 & 25.3 \selectlanguage{ngerman}± 0.47*\tabularnewline
PUFA & 35.2 \selectlanguage{ngerman}± 0.27 & 42.6 \selectlanguage{ngerman}± 0.24 & 32.5 \selectlanguage{ngerman}± 0.04 & 26.4 \selectlanguage{ngerman}± 0.71 & 28.4 \selectlanguage{ngerman}±
0.24 & 29.1 \selectlanguage{ngerman}± 0.61 & 48.5 \selectlanguage{ngerman}± 0.02*\tabularnewline
Total FAME (mg L\textsuperscript{-1}) & 54.2 \selectlanguage{ngerman}± 0.28 & 50.6 \selectlanguage{ngerman}± 0.62* &
51.7 \selectlanguage{ngerman}± 0.24 & 88.4 \selectlanguage{ngerman}± 3.04 & 88.3 \selectlanguage{ngerman}± 1.91 & 88.9 \selectlanguage{ngerman}± 1.95 & 96.54 \selectlanguage{ngerman}±
0.51*\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

\textbf{Table 2.}



\textbf{Table 3.}\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\textbf{Conditions} & \textbf{EPA concentrations (mg
L\textsuperscript{-1})} & \textbf{Fucoxanthin concentrations (mg
L\textsuperscript{-1})}\tabularnewline
P1 & 9.09 \selectlanguage{ngerman}± 0.23* & 5.44 \selectlanguage{ngerman}± 0.22*\tabularnewline
P2 & 11.28 \selectlanguage{ngerman}± 0.33 & 6.62 \selectlanguage{ngerman}± 0.81\tabularnewline
P3 & 10.44 \selectlanguage{ngerman}± 0.12 & 3.66 \selectlanguage{ngerman}± 0.44\tabularnewline
M1 & 11.00 \selectlanguage{ngerman}± 0.17 & 3.05 \selectlanguage{ngerman}± 0.17\tabularnewline
M2 & 14.29 \selectlanguage{ngerman}± 0.34 & 4.01 \selectlanguage{ngerman}± 0.87\tabularnewline
M3 & 12.6 \selectlanguage{ngerman}± 0.37* & 2.88 \selectlanguage{ngerman}± 0.93*\tabularnewline
M4 & 27.07 \selectlanguage{ngerman}± 0.38* & 11.01 \selectlanguage{ngerman}± 0.75*\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2A/Figure-2A}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2B/Figure-2B}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

