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\begin{document}

\title{Biomass and Pigment Production for Arthrospira platensis via
Semi-Continuous Cultivation in Photobioreactors: Temperature Effects}



\author[1]{Ankush Karemore}%
\author[2]{Yanhui Yuan}%
\author[3]{William Porubsky}%
\author[3]{Ronald Chance}%
\affil[1]{Georgia Institute of Technology}%
\affil[2]{Algenol Biofuels}%
\affil[3]{Algenol Biotech}%


\vspace{-1em}



  
  \date{June 14, 2024}


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\selectlanguage{english}
\begin{abstract}
Abstract This study describes the response of Arthrospira platensis to a
variety of temperature conditions as reflected in variations of
photosynthetic parameters, pigmentation, and biomass productivity in
indoor photobioreactor (PBR) cultivations. These experiments are
designed to better understand the impact of temperature, seasonal
variations, and acclimation effects on outdoor biomass production. The
irradiance level and temperature range (20 -- 39\selectlanguage{ngerman}°C) are chosen to enable
modeling of semi-continuous operation of large-scale outdoor PBR
deployments. Overall, the cultivations were quite stable with some
pigment-related instabilities after prolonged high temperature exposure.
Changes in productivity with temperature, as reflected in measured
photosynthetic parameters, are immediate and mainly attributable to the
temperature dependence of the photosaturation parameter, a secondary
factor being variation in pigment content on a longer time scale
corresponding to turnover of the culture population. Though pigment
changes have minimum impact on productivity, prolonged exposure at 35°C
and above yields a clear degradation in performance. Productivities in a
semi-continuous operation are quantitatively reproduced with a
productivity model incorporating photosynthetic parameters measured
herein. This study confirms the importance of temperature for biomass
and pigment production in Arthrospira cultivations and provides a basis
for risk assessments related to temperature mitigation for large-scale
outdoor cultivations. Keywords: Arthrospira Platensis, photosynthetic
parameters, pigment production, productivity modeling, photobioreactors%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Introduction}

The filamentous cyanobacterium \emph{Arthrospira platensis
(Spirulina)}is an oxygenic photosynthetic organism able to grow in
tropical and subtropical environments, and one of only a few microalgal
systems that has been successfully commercialized and approved by United
States Food and Drug Administration (FDA) as a food supplement (Trabelsi
et al., 2009). \emph{Arthrospira} cultivation and processing yields
valuable biochemical components including protein, carbohydrates, fatty
acids and pigments such as phycocyanin (PC), which can be used in
nutritional, pharmacological, and cosmetic products. Due to these high
value applications, as well as relatively easy harvesting and extraction
processes, \emph{Arthrospira} cultivation has been deployed commercially
at moderate scale (10 -- 100 acre open ponds) for many decades (Lu et
al., 2011). It is important to note that \emph{Arthrospira} is an
extremophile, in that it can maintain high productivity under high
alkalinity, high pH conditions; this limits predation and competition
sufficiently to allow commercial production in open pond systems. Algal
cultures are influenced by various abiotic variables such as
temperature, irradiance levels, and nutrient availability, all of which
play a significant role in regulating photosynthetic activity, biomass
composition and overall productivity. Under outdoor cultivation
conditions, temperature and light intensity are the two key external
factors that determine photosynthetic activity and biomass growth rates.
Obviously, both factors are highly variable on a daily and seasonal
basis in the natural environment, and spatially within the culture as
well (Chaiklahan et al., 2007; Vonshak and Novoplansky, 2008).
Typically, \emph{Arthrospira} is cultivated outdoors for mass production
in raceway ponds, where cells encounter fluctuating environments in
terms of irradiance, temperature, and nutrient supply. Though the PBR
environment tends to be more homogeneous, similar fluctuations are
present and temperatures are generally higher due to absorptive heating
and the absence of evaporative cooling. Outdoor algal cultures are
subjected to high light intensity as well as possible high temperature
stress that can negatively impact photosynthetic activity (Torzillo et
al., 1991b). These factors can change both the photosynthesis and
respiration rates, thereby directly influencing the growth and the
chemical composition of the biomass produced (Trabelsi et al., 2009).

Overall, the existing literature is consistent with an optimal
temperature range for stable production of roughly 20-35 \selectlanguage{ngerman}°C. Our
screening studies are consistent with that range and also consistent
with an activation energy of about 60 kJ mole\textsuperscript{-1} (Q10
\textasciitilde{}2) under saturating light conditions over that
temperature range. It is well-known that productivity is enhanced in
semi-continuous operation where the impact of photosaturation effects is
lessened. We know of no detailed studies dealing with the effect of
temperature and acclimation response on growth and pigment content
of\emph{Arthrospira} in a semi-continuous production mode for extended
time scales under tightly controlled (laboratory) conditions. The
intention here is to determine what portion of previous learnings
translate to semi-continuous operation and the dynamic
(light/temperature) conditions experienced outdoors, and examine the
responses to abrupt changes in temperature. Therefore, in the present
work we will examine temperature effects at a longer time scale, and
carry out the experiments in semi-continuous operation mode in PBRs at
20 °C, 30 °C and 35 °C. In subtropical conditions, the outdoor culture
temperature in the summer months can be very high in PBRs, reaching up
to 35-45 °C for several hours. We have only a limited understanding of
temperature impacts on photosynthetic parameters, and pigment
accumulation in that outdoor environment. Thus, the scope of this work
includes \emph{Arthrospira} growth under a variety of temperature
conditions with a work plan that includes assessment of temperature
response and recovery, and quantification of the dynamic change in
biomass and pigment content of \emph{Arthrospira} during the
experiments. There is no doubt that high irradiance levels can be a
confounding factor both at low and high temperatures. We limit the
current study to ``average'' irradiance conditions in Fort Myers,
Florida. The combination of vertical PBR arrays, which dilute the
average irradiance levels from about 800 \selectlanguage{greek}μ\selectlanguage{english}E/m2-s to about 200 \selectlanguage{greek}μ\selectlanguage{english}E/m2-s,
and the rapid mixing, which distributes the heat from light absorption
more evenly within the PBR volume, lessen the potential for extreme
effects due to high irradiance. That is born out by cultivation field
observations of \emph{Arthrospira} growth and laboratory studies of
irradiance effects with the same approach used here. Regarding lower
temperatures than the 20\selectlanguage{ngerman}°C included here, our screening studies do not
suggest any issues down to 10°C and field experience in the environment
of interest shows that the concerns lie at high temperatures.

The work was performed in three phases (Figure 1a): Phase I employs
constant temperature conditions (same for day and night cycles), Phase
II shifts the Phase 1 cultures to opposing temperature conditions (low
to high, and high to low), and Phase III continues the examination under
dynamic summer temperature profiles (hourly variations) in a
semi-continuous operation mode. The experimental setup, shown in Figure
1b, involves vertically oriented tubular photobioreactors, designed to
be predictive of outdoor performance in large PBR arrays.

\textbf{Material and Methods}

\textbf{Algal Strain and culture condition}

The algal strain used in this study was \emph{ArthrospiraPlatensis}
maintained in Zarrouk's medium with the following macro and micro
ingredients (mM): NaHCO\textsubscript{3} (200),
K\textsubscript{2}HPO\textsubscript{4} (3.7), NaNO\textsubscript{3}(30),
K\textsubscript{2}SO\textsubscript{4} (5.7), NaCl (18),
CaCl\textsubscript{2} 2H\textsubscript{2}O (0.27),
FeSO\textsubscript{4}7H\textsubscript{2}O (0.036),
Na\textsubscript{2}EDTA 2H\textsubscript{2}O (0.215), NaOH (0.1),
H\textsubscript{3}BO\textsubscript{3} (0.045),
MnCl\textsubscript{2}4H\textsubscript{2}O (0.009), ZnSO\textsubscript{4}
7H\textsubscript{2}O (0.001), NaMoO\textsubscript{4}
2H\textsubscript{2}O (0.000083), and CuSO\textsubscript{4}
5H\textsubscript{2}O (0.00032) (Zarrouk, 1966). Unless stated otherwise,
Zarrouk´s medium with 200 mM NaHCO\textsubscript{3} was used for all
culture cultivations. The seed culture was sourced from a private
collection and cultivated at 30 °C for one week to reach a biomass
concentration of 0.75 gDW L\textsuperscript{-1} (optical density at
750nm, OD\textsubscript{750}, equal to 1.5). Under most conditions the
DW to OD\textsubscript{750}ratio was about 0.5 gDW L\textsuperscript{-1}
per OD\textsubscript{750}which is regarded as normal for this organism.
Although some variation within the relationship between OD and DW can
occur with differing temperature conditions (Jahnke et al., 2011;
Torzillo et al., 1991a), such variations were generally minor in the
experiments reported here and only occurred at the latter stages of
growth at 35 °C where the cultures displayed clear instabilities
(declines) in pigmentation. Lower pigmentation, especially in the red
spectral region, can lead to lower refractive indices at 750 nm, lower
light scattering, and thus high DW/OD ratios, as observed. The DW
measurements involve collecting algal cells on pre-rinsed glass fiber
filters (1.5 \selectlanguage{greek}µ\selectlanguage{english}m pore size, 47 mm ProWeigh Filters, Cole-Parmer) by
filtration, washing three times in deionized water, and then drying to a
constant weight at 60 \selectlanguage{ngerman}°C for 48 h. The specification for the drying
method calls for a water content of no more than 3\% after 24 h. The
remaining salt content after this procedure was generally less than 2\%,
and to that extent our DW is equivalent to ash-free DW (AFDW).

\emph{Arthrospira platensis} , the most common commercial strain, is
interchangeably referred to as ``\emph{Spirulina} '' in the literature.
We have used \emph{Arthrospira} throughout this article to refer to the
organism under study here and literature results cited
for\emph{Arthrospira platensis} .

\textbf{0.35-liter PBR cultivation conditions}

For 0.35 L PBR operation, the reactors were inoculated with
the\emph{Arthrospira} strain grown at 30 °C to a concentration of 0.5
gDW L\textsuperscript{-1}. The pH value was maintained between 9.2-9.8
with an aeration rate of 80 mL min\textsuperscript{-1} supplying 0.75 \%
CO\textsubscript{2} during the light phase and 0.20 \%
CO\textsubscript{2} in the dark phase for cultures grown at 30 °C and 35
°C. The cultures grown at 20 ° C were supplied with 0.38 \%
CO\textsubscript{2} during the light phase, and 0.2 \%
CO\textsubscript{2} in the night phase. The aeration rate chosen for
these experiments is higher than normal due to the filamentous nature
of\emph{Arthrospira} that leads to settling under less stringent
conditions. Thus the CO\textsubscript{2} concentration, required to
supply the cultures and replenish the bicarbonate consumed by the
organism, appears low but is confirmed to be sufficient by the pH
monitoring, as well as extensive experience with outdoor cultivations.
The light regime for the cultures was a 12 h light:12 h dark cycle with
light intensity of 230 \selectlanguage{greek}μ\selectlanguage{english}mol m\textsuperscript{-2}
s\textsuperscript{-1}from one side of the PBRs using fluorescent lamps
(Plusrite). This irradiance level was chosen to model annual average
conditions for typical vertical PBR arrays deployed in Fort Myers,
Florida. For CO\textsubscript{2} aeration and mixing of the cultures, a
custom designed porous air diffuser (0.5 mm diameter) was used in order
to generate mm-size gas bubbles for aeration at a constant flow rate of
80 mL min\textsuperscript{-1}.

\textbf{Semi-continuous cultivation and experimental set-up in PBR}

For semi-continuous cultivation, culture dilution with Zarrouk\selectlanguage{ngerman}´s medium
was carried out on alternate days maintaining OD\textsubscript{750} =
2.0 (\textasciitilde{}1 gDW L\textsuperscript{-1}) as the starting point
for the next production cycle. The experiment was continued over a time
course of 43 days in this semi-continuous operation mode. The
experimental program was divided into three phases with eight PBRs
designated in alphabetical order from A to H. These eight PBRs were
divided in four sets of duplicate PBRs indicated as `AB' for A and B,
`CD' for C and D, etc. (Figure 1). OD750nm was monitored as a surrogate
for biomass concentration, with the OD-concentration relationship
periodically monitored. That relationship (typically DW in g
L\textsuperscript{-1} = 0.5*OD750nm) varied only slightly during the
course of these experiments as discussed above.

In Phase I, four reactors each were cultivated at constant 20 °C (AB,
CD) and 35 °C (EF, GH) for 15 days. In Phase II, the four reactors at
20°C were shifted to 30 °C (CD) and 35 °C (AB) whereas the other set of
four reactors at 35°C were shifted to 20 °C (EF) and 30°C (GH). The
Phase II shift was started on the 15\textsuperscript{th} day and kept in
place until the 33\textsuperscript{rd} day. To acclimate the cultures to
the newer temperature conditions, cultures were grown in batch mode for
four days (15 -- 19) without performing any dilution. In the final Phase
III, the culture conditions were shifted from constant temperature to
dynamic summer temperature profiles (derived from historical climate
data for Fort Myers, Florida) with hourly changes in temperature over
the course of 24 hours. This phase lasted from day 33 to day 43. In this
phase, the cultures grown at constant 20 °C (EF) and 30 °C (CD) in Phase
II were shifted to average summer profile with 35/21 °C as
maximum/minimum temperatures, the culture at constant 35 °C (AB) was
moved to extreme summer profile with 39/26 °C as maximum/minimum
temperatures, and final set of two reactors cultured at constant 30°C
(GH) were shifted to constant day (31 °C) and night (22 °C) temperature
based on summer average of day and night temperatures. Summer is defined
as June 1 through August 31 for creating these profiles from historical
data.

\textbf{Determination of chlorophyll and phycocyanin}

Chlorophyll-a (Chl-a) was measured using a standard methanol-based
methodology (Marsac and Houmard, 1988).

PC extraction and quantification was carried out using repeated
freeze-thaw cycles based on Yoshikawa and Belay (2008). Briefly, this
method extracts PC from fresh biomass using a repeated freeze-thaw and
soaking regime and then quantifies PC spectro-photometrically based on
absorbance at three wavelengths: 620 nm, 650 nm and 680 nm. PC content
is calculated following the Yoshikawa equation, where cPC is
C-phycocyanin and aPC is allophycocyanin:

cPC, mg/mL = 0.162 × OD\textsubscript{620} -- 0.098 ×
OD\textsubscript{650} (1)

aPC, mg/mL = 0.180 × OD\textsubscript{650} -- 0.042 ×
OD\textsubscript{620} (2)

Photosynthetic parameters

The oxygen PE (O\textsubscript{2} production vs irradiance) curves were
determined in Algenol's custom-designed system. The system is comprised
of a white light LED source, photosynthetically active radiation (PAR)
light sensor, and an O\textsubscript{2} sensor, all in a
temperature-controlled cassette (Legere, 2017). The optical path length,
d, is 1 cm. The cell contains 4 ml of fluid stirred at 400rpm with no
head space. For measurement of O\textsubscript{2}, needle-type oxygen
sensors (OXR50, Pyroscience) composed of fiber-optical cable connected
to FireSting O\textsubscript{2} sensors were used. Response times to
changing conditions were rapid with no indication of mass transfer
limitations. The temperature is controlled to ±1 °C over the range from
10 °C to 50 °C. The culture samples were taken from the PBR and
incubated at 30 °C for 1.5 hr for dark acclimation, and then diluted to
an absorptivity of exp(-kd)=0.1 (concentration\textasciitilde{} 1 mg
Chl.a L\textsuperscript{-1}) using fresh Zarrrouk's media, where k is
the absorption coefficient for the whole cell \emph{Arthrospira}averaged
over the PAR range (400-750 nm). In determining k, spectra are first
corrected for scattering (approximately) by subtracting at all
wavelengths the absorbance at 750 nm (OD\textsubscript{750}). The first
10 min of oxygen uptake data in the dark is used to calculate the dark
respiration rate. The light is then ramped up to 1000 \selectlanguage{greek}µ\selectlanguage{english}E
m\textsuperscript{-2} s\textsuperscript{-1} stepwise with 3 min at each
step (typically 15 steps). The oxygen evolution rate is fitted with a
Monod model (Bechet 2013) form to report photosynthetic parameters: \selectlanguage{greek}α,
\selectlanguage{english}E\textsubscript{k}, and P\textsubscript{max} (limited quantum yield in
low light limit, photosaturation parameter, and max photosynthetic rate
in high light limit). The Monod mathematical model is used in order to
be consistent with the approach taken for the Algenol Productivity Model
(see Supplementary Material). All measurements are carried out in
duplicate.

\textbf{Productivity Modeling}

Ethanol productivity from genetically modified cyanobacteria has been
successfully modeled by Algenol using a Monod modeling approach (Legere
2017). The Algenol Productivity Model has been adapted for biomass-only
production and used in conjunction with PE-derived photosynthetic
parameters to estimate expected outdoor productivities for PBR
deployments in Fort Myers (and elsewhere around the world). The daily
biomass volumetric productivity can be described as:

\(P_{\text{Biomass}}=\alpha E_{k}\selectlanguage{greek}\text{γln}\selectlanguage{english}\left[\frac{E_{k}+E_{0}}{E_{k}+E_{0}e^{-kD}}\right]\frac{t_{1}}{D}-R_{0}C_{c}\gamma t_{2}\)(3)

where \(\alpha\) is the quantum yield in the low light limit
(mol C /mol photons), E\textsubscript{k} is the photosaturation
parameter (\selectlanguage{greek}µ\selectlanguage{english}E m\textsuperscript{-2} s\textsuperscript{-1}),
\(\gamma\) is the conversion between fixed C to dry weight
biomass (gDW mol C\textsuperscript{-1}), E\textsubscript{0} is the
incident light intensity (\selectlanguage{greek}µ\selectlanguage{english}E m\textsuperscript{-2}
s\textsuperscript{-1}) at the culture surface (corrected approximately
for reflection losses), k is the absorptivity coefficient of biomass
(m\textsuperscript{-1}), D is the effective light path (m),
R\textsubscript{0} is the specific respiration rate (\selectlanguage{greek}µ\selectlanguage{english}mol C
mgChl.a\textsuperscript{-1}min\textsuperscript{-1}), C\textsubscript{c}
is the Chl.a concentration (mgChl.a m\textsuperscript{-3}),
t\textsubscript{1} is the time for light-on (sec), and
t\textsubscript{2} is the time for respiration load (min). Light-on time
(t\textsubscript{1}) is about half of the respiration load time
(t\textsubscript{2}) for outdoor cultivation. For the indoor PBR
experiment, 12 hr light/12 hr dark cycle, E\textsubscript{0} is constant
at 230 \selectlanguage{greek}µ\selectlanguage{english}E m\textsuperscript{-2}s\textsuperscript{-1}, and the average
light path (D) is approximately the radius of the reactor tubes with
illumination from one side. The productivity data are quoted as the mean
values \selectlanguage{ngerman}± SD (\emph{n} = 2) for the two independent replicate cultures.
The derivation of Equation (3) is included in Supplementary Material
along with application of model to a large scale outdoor PBR cultivation
of \emph{Arthrospira} (Chance and Roessler 2019). The model applies to a
static system, in that none of the mixing rates involved in these
cultivations involve significant movement of culture components on the
time scale of the photosynthetic reactions. (See Supplemental Material.)

\textbf{Results and Discussion}

Cell growth characteristics

Cell growth was evaluated based on the determination of optical cell
density (OD\textsubscript{750}), converted to dry weight (DW). Pigment
content was determined by extraction and quantification as described
above. All results reflect duplicate measurements, plus at least two
biological replicates.

Startup: Phase I

The biomass growth profiles of \emph{Arthrospira} under photoautotrophic
conditions in three phases are shown in Figure 2. In Phase I, the growth
response to constant low temperature (LT) (20 °C±1) (AB \& CD) and high
temperature (HT) (35°C±1) (EF \& GH) was assessed. PBRs were cultivated
in batch mode for initial 5 days to reach OD\textsubscript{750} 2.0 and
beyond, and then operated in semi-continuous mode maintaining the
OD\textsubscript{750} = 2.0 starting point from day 5 to 15 with
harvest/dilution every two days. For the chosen temperatures of 20°C and
35°C, the average pre-dilution concentrations of the cultures were 1.30
gDW L\textsuperscript{-1} (OD\textsubscript{750} = 2.6) and 1.62 gDW
L\textsuperscript{-1} (OD\textsubscript{750} = 3.2) respectively (Figure
2) yielding biomass growth rates 0.17 gDW
L\textsuperscript{-1}d\textsuperscript{-1} and 0.20 gDW
L\textsuperscript{-1}d\textsuperscript{-1} at 20 °C and 35 °C,
respectively. These results are summarized in Table 1. As can be seen in
the table, good reproducibility is found for all results (including
pigment contents to be discussed later).

Transition: Phase II

As noted earlier, in Phase II (day 15 to 33) the four cultures (ABCD)
grown previously at 20 °C were shifted to 35 °C (AB) and 30 °C (CD), and
the four cultures at 35 °C (EFGH) were shifted to 20 °C (EF) and 30 °C
(GH). As a recovery phase and to acclimate the cultures after transition
from Phase I to Phase II, the cultures in the newer temperature
conditions were grown in batch mode without dilution for four days from
day 15-19. The responses to temperature change were assessed at the end
of the fourth day. As expected, the culture shifted from lower
temperature (LT) (20 °C) to higher temperatures (30 °C and 35 °C) showed
higher growth to 1.90 gDW L\textsuperscript{-1} (from 0.93 to 1.90 gDW
L\textsuperscript{-1} over 4 days). The cultures shifted from higher
temperature (35 °C) to low temperature (20 °C and 30 °C) grew more
slowly, with biomass concentration reaching around 1.71 gDW
L\textsuperscript{-1} (from 1.17 to 1.71 gDW L\textsuperscript{-1}) on
day 19 after four days of batch cultivation (Figure 2 a and b). The time
scale of temperature response for \emph{Arthrospira} , as judged from
these OD750nm measurements, is essentially instantaneous within the
noise of these measurements.

During semi-continuous operation in Phase II, different algal growth
patterns are found under the chosen temperature conditions. The average
pre-dilution DW biomass concentrations for the different temperature
treatments are shown in Table 1. The 20 °C average is the only one that
is clearly distinguishable. The most favorable temperature appears to be
30 °C, which is close to the optimum temperature for
\emph{Arthrospira}for achieving maximum productivity under our growth
conditions. The results are consistent with that of Colla et al.
(2007)\emph{,} where higher temperatures had a clear negative effect on
\emph{Arthrospira}biomass production. An optimization study carried out
by Sánchez\selectlanguage{english}-Luna et al. (2007) in batch cultivations reported 29 degC as
best growth temperature. For the two cultures in our Phase II study
maintained at 30 degC (one originating from the 20 degC Phase I experiment
and the other from the 35 degC Phase I experiment), the results are
essentially the same. Thus, the extreme of temperatures and prolonged
exposure to high temperatures at 35 degC in Phase I is thought to have
caused stress to the cells, and that has been observed by others to
result in decline in biomass production and protein content, with
simultaneous accumulation of carbohydrate and EPS (Panyakampol et al.,
2015; Trabelsi et al., 2009). There was some decline in growth rate at
35 degC, though a stress response is clearer in the pigment content, as
discussed below. Noticeable decline in growth and a lower cell density
were observed in the cultures that were shifted from 35 degC\selectlanguage{english}-20 degC. This
is normal temperature dependence (Kumar et al., 2011). The relative
dilution rates were 0.12 day\textsuperscript{-1} at 20 degC, 0.16
day\textsuperscript{-1}at 30 degC, and 0.13 day\textsuperscript{-1} at 35
degC. The highest dilution rate, and therefore productivity, was seen at
30 degC. This agrees with the results cited above and also with Trabelsi
et al. (2009) where maximum growth rate for \emph{Arthrospira platensis}
was found at 30 degC.

\textbf{Outdoor Simulation: Phase III}

Phase III involved exposing the cultures to dynamic temperature profiles
with hourly changes in temperature that are representative of outdoor
summer culture temperature profiles (Supplementary Material). This final
phase of cultivation was carried out from day 33-43 with scheduled
alternate day dilutions and with no adaptation period. The cultures
grown at constant 20 degC (EF) and one set of the constant 30 degC (CD)
cultures were shifted to average summer profile (AvSP) with 35 degC/21 degC
as maximum/minimum temperatures during the course of day/night
temperature ramping. The cultures at constant 35 degC (AB) were shifted to
an extreme summer profile (ExSP) with 39 degC/26 degC as maximum/minimum
temperatures, and the final set of two photobioreactors at constant 30
degC (GH) were shifted to constant summer profile (CtSP) where day/night
temperature were maintained at constant 31 degC/21 degC, selected based on
Fort Myers summer profile and averaging the day and night temperatures
separately.

The average pre-dilution cell concentration in gDW
L\textsuperscript{-1}for the temperature profile treatments are shown in
Table 1. The relative dilution rates were about 0.16
day\textsuperscript{-1} in AvSP (CD), AvSP (EF) and CtSP (GH), and about
20\% lower (0.135 day\textsuperscript{-1}) for ExSP (AB).

From visual observations, it is worth noting that during the processing
of biomass samples for the various analyses, agglomeration or clumping
of the algal cultures occurred for cultures grown at higher temperature
(constant 35 degC and ExSP). This is attributed to a stress response. In
addition, the dried samples from these temperature exposures showed a
flaky texture on the dry weight plate membrane surface. In
cyanobacteria, high temperature stress can result in a rise in fluidity
of membranes which can cause disintegration of the lipid bilayer and
many other alterations in the physical properties of the cells that
result in the loss of functionality of photosynthetic machinery
(Panyakampol et al., 2015; Panyakampol et al., 2016).

Biomass Productivity

Table 1 summarizes productivity results obtained in different phases of
temperature treatment. These productivities are averaged over the entire
phase and indicate an apparent peak at 30 degC. Figure 3 summarizes the
productivity results found towards the end of the various phases with
average values plotted against average daytime temperatures. This plot
is intended to explore the final results of the extended periods at the
various temperature conditions. The variation from 20 degC to 30 degC is
attributed to the temperature dependence of P\textsubscript{max} (and
thus E\textsubscript{k}) and is less than the commonly observed Q10 = 2
behavior due to the irradiance level (230 \selectlanguage{greek}µ\selectlanguage{english}E
m\textsuperscript{-2}s\textsuperscript{-1}) being substantially below
E\textsubscript{k} as shown below. This is the expected behavior when
the system is stable and unstressed. No acclimation, other than the
normal E\textsubscript{k}dependence on temperature, is indicated. The
slight downturn at 35 \selectlanguage{ngerman}°C and higher in Figure 3 is attributed to a
stress response that is more apparent in the pigment results discussed
below. The consequences of the stress response are continuing throughout
the time period of the phases, consistent with a degradation as opposed
to an acclimation process. The downturn in productivity and pigmentation
was enhanced for batch experiments at 35 °C to 45 °C that are not
discussed here.

The choice of 230 \selectlanguage{greek}µ\selectlanguage{english}E m\textsuperscript{-2} s\textsuperscript{-1} was
based on the average annual irradiance at the exposed culture surfaces
of PBR arrays in Fort Myers, Florida with a height-to-spacing ratio
chosen to maximize productivity (Legere, 2017). To convert the quoted
biomass productivities from g L\textsuperscript{-1}d\textsuperscript{-1}
to g m-\textsuperscript{2} d\textsuperscript{-1}, multiply by a
geometric factor of 95 L m\textsuperscript{-2}. Thus, taking 30 \selectlanguage{ngerman}°C as a
reasonable estimate of the annual average daytime temperature in Fort
Myers, a biomass productivity of 21 g m\textsuperscript{-2}
day\textsuperscript{-1} is obtained. This is very close to the observed
annual average of 23 g m\textsuperscript{-2}day\textsuperscript{-1}
found experimentally for large PBR arrays (24,000 L culture) tested for
over 1 year at the Algenol site in Fort Myers (Chance and Roessler,
2019; see also Supplementary Material).

Chlorophyll and Phycocyanin Production

During the cultivations, chlorophyll (Chl-a) content and phycocyanin
(PC) content for \emph{Arthrospira} were monitored. The two main
components of the PC content were determined separately: allophycocyanin
(aPC) and C-phycocyanin (cPC) via spectroscopic determination (Yoshikawa
and Belay, 2008). In addition, whole cell (WC) absorption spectra were
measured (Figure 4) for qualitative pigment analysis. Results for
pigment content are summarized in Table 1 and displayed in detail in
Figure 5. All quoted contents are expressed as a percentage of whole
cell dry weight.

First, from the Phase I results in Table 1, good reproducibility between
the biological duplicates (AB-CD and EF-GH) is found for all the
measurements. It can also be seen from Figure 5 that the AB and CD
experiments at 20 °C are very stable in their pigment content, both PC
and Chl-a. That is not the case for the 35 °C results where both PC and
Chl-a are decreasing steadily throughout the Phase I residence time in
Figure 5c. The decline in PC is about 20\% and the decline in Chl-a is
about 40\% over the course of the Phase I experiments at 35 °C. It is
unlikely that the degradation in pigment content in either case is due
to thermal damage to the pigments, as these pigments are known to be
stable to much high temperatures. It is more likely due to a slow
alteration of the photosynthetic apparatus, which we regard as
biologically irreversible (as distinguished from recovery from culture
turnover in the semi-continuous mode). The decrease in PC content could
in fact be a consequence of the decrease in Chl-a content as the light
harvesting machinery re-balances the optimal ratio for these pigments.

In Phase II, the AB culture goes from 20 °C to 35 °C with an initial
sharp increase in PC content followed by a slow decline (Figure 5a). The
Chl-a content stays constant initially and then declines slowly over the
Phase II residence time, the overall decrease being similar to that for
EF in Phase I (Figure 5c). The CD culture, which transitioned from 20 °C
to 30 °C (Figure 5b), shows much more stable behavior, with a slower
increase in PC content before reaching an apparent steady concentration
of about 12\%. The Chl-a concentration is stable at about 1.7\%, similar
to Phase I at 20 °C. There is no indication of instability at 30 °C. The
CD culture, transitioned from 35 °C to 20 °C (Figure 5c), shows a steady
decline in PC to the expected level for 20 °C production
(\textasciitilde{}8 \%). The Chl-a content increases slowly, eventually
reaching the level expected for 20 °C production
(\textasciitilde{}1.5\%). The time scale for these changes are
consistent with the expected time scale for culture turnover (roughly
20-30\% per 2 day cycle). At the end of Phase II, the EF culture is
almost exactly at the expected pigment contents found in the AB and CD
Phase I experiments. The GH culture, transitioned from 35 °C to 30 °C
(Figure 5d), achieves an overall increase in PC content and Chl-a
content, with a slightly enhanced time scale for reaching stable levels
for both pigments.

Phase III observations from Figure 5 are consistent with the above
observations. AB (35 °C to ExSP) shows an initial decline in pigment
contents and then some recovery over time. CD shows essentially no
change in going from 30 °C to AvSP. EF shows expected changes in going
from 20 °C to AvSP. GH (30 °C to CtSP) shows little or no change in
pigment content.

According to the literature for shorter duration experiments, there is a
narrow temperature range between 35 °C and 37 °C for optimal growth with
40 °C being definitely detrimental for \emph{Arthrospira} (Kumar et al.,
2011; Torzillo et al., 1991b). Our results suggest extended periods at
35 °C are also not favorable for sustained growth, though the effects
are largely reversible on a culture basis and most of the variation is
in pigment production. A similar trend was seen in whole cell spectrum
in Figure 4, where relatively higher peak at \textasciitilde{}680nm
(Chl-a) and lower peak at 620 nm (cPC) was found at lower temperatures,
and thus indicates a higher Chl-a to PC ratio for low temperature
cultures compared to those after prolonged high temperature exposure.
The culture at 35 °C turned bluish green with Chl-a reduction (by
\textgreater{}50 \%) after prolonged exposure to this modestly elevated
temperature. The spectra in Figure 4 are consistent with this visual
observation. These results are generally consistent with Watras et al.,
(2017) where a progressive decrease in chlorophyll and phycocyanin
fluorescence with increasing temperature was reported in most of the
cultures of green and blue-green algae (e.g., \emph{Scenedesmus
dimorphus, Selenastrum minutum, and Synechococcus leopoliensis} ).

Photosynthetic parameters

Photosynthesis-irradiance (PI) curves have been extensively used to
evaluate the photosynthetic response to various abiotic stresses
experienced by algae (Falkowski and Raven, 2007). Photosynthetic
parameters, including P\textsubscript{max} (\selectlanguage{greek}µ\selectlanguage{english}mol
O\textsubscript{2}L\textsuperscript{-1} hr\textsuperscript{-1}), \selectlanguage{greek}α\selectlanguage{english}' (mol
O\textsubscript{2} mol photon\textsuperscript{-1}),
R\textsubscript{0}\textsuperscript{'} (\selectlanguage{greek}µ\selectlanguage{english}mol
O\textsubscript{2}L\textsuperscript{-1} min\textsuperscript{-1}) and
E\textsubscript{k}(\selectlanguage{greek}µ\selectlanguage{english}E m\textsuperscript{-2} s\textsuperscript{-1}), were
evaluated at different temperature treatments during Phases I and II
using PE curves (Figure 6 and Table 2) to test consistency with the
above observations for changing conditions and provide parameters for
productivity modeling. The culture samples from different temperature
treatments were first incubated at 30 \selectlanguage{ngerman}°C under dark conditions for 1 h.
Testing for the different treatments was conducted at a single
temperature (30 °C) to avoid the normal temperature dependence wherein
P\textsubscript{max} and E\textsubscript{k} display a
Q\textsubscript{10} = 2 dependence (about 60 kJ
mol\textsuperscript{-1}). The PE curves were measured (in duplicate) for
all treatments, with average values reported. The photosynthetic
response patterns from cultures grown at 20 °C, 30 °C and 35 °C Phase I
are shown in Figure 6, with results summarized in Table 2. It is clear
that with this experimental protocol none of the samples in Figure 6
shows a significant difference from the others, the only possible
exceptions being the AB-ExSP sample exposed to the most severe summer
profile conditions and the A sample from Phase II (constant 35 °C). PE
curves measured at 20 °C for culture samples from Phase I (20 °C
treatment) yield a P\textsubscript{max} of 240 \selectlanguage{greek}µ\selectlanguage{english}mol
O\textsubscript{2}L\textsuperscript{-1} hr\textsuperscript{-1} and
E\textsubscript{k} as 85 \selectlanguage{greek}µ\selectlanguage{english}E m\textsuperscript{-2} s\textsuperscript{-1},
which is roughly Q\textsubscript{10} of 2 when compared to results from
PE curves measured at 30 \selectlanguage{ngerman}°C. In fact a more extensive testing (not
presented here) of PE curves measured over the temperature range 15-35
°C yields an activation energy for P\textsubscript{max} of 60 kJ
mol\textsuperscript{-1}. This activated process can be attributed
entirely to E\textsubscript{k}, as the limiting quantum yield (\selectlanguage{greek}α\selectlanguage{english}) has
been shown to be independent of temperature over the range studied. As
noted earlier, these observations are typical of temperature response in
photosynthetic organisms (Falkowski and Raven, 2007). The constant
exposure to 35 \selectlanguage{ngerman}°C, also measured at 35 °C, (Table 2) yields
photosynthetic parameters similar to the other tests at 30 °C. In Phase
I there is some indication of a stress response at sustained high
temperatures in these results, though this is not as clear as the
pigment variation. There is no indication in Phase III of dynamic high
temperature exposure having an adverse effect. These observations are
consistent those made in conjunction with biomass and pigment
production.

There was no significant difference in values of \selectlanguage{greek}α\selectlanguage{english}' (limited quantum
yield for O\textsubscript{2} production) which were close to
\textasciitilde{} 0.070 mol O\textsubscript{2}/mol photon for all the
treatments. The lowest R\textsubscript{0}\textsuperscript{'}(respiration
rate) of 0.12 \selectlanguage{greek}µ\selectlanguage{english}mol O\textsubscript{2}/L-min was observed at 20\selectlanguage{ngerman}°C while
the maximum of 0.55 \selectlanguage{greek}µ\selectlanguage{english}mol O\textsubscript{2}/L-min was found at 35\selectlanguage{ngerman}°C.
R\textsubscript{0}\textsuperscript{'} determinations have effects due to
the light exposure history (Falkowski and Raven, 2007). Little
temperature dependence is expected for \selectlanguage{greek}α\selectlanguage{english}', consistent with the results
from this study. The ratio \selectlanguage{greek}α\selectlanguage{english}'/\selectlanguage{greek}α \selectlanguage{english}is the photosynthetic quotient
(O\textsubscript{2} per fixed carbon) which is expected to be in the
range 1.1-1.3 (Falkowski and Raven, 2007). We use 1.2 for the modeling
analysis to follow. The same value applies to R\textsubscript{0}, the
respiration rate on a carbon basis required for application in the
Algenol Productivity Model, Equation 3.

It is noteworthy that large changes in pigment content and light
absorption level are seen with very little change in biomass
productivity, whether measured directly or inferred from the PE curves.
This is consistent with the relatively minor impacts of low pigment
mutants on productivity in other organisms (Kirst et al., 2014;
Lea-Smith et al., 2014).

Productivity Modeling Analysis

The Algenol Productivity Model (Legere, 2017; Chance and Roessler, 2019)
is used to analyze these indoor PBR experiment results, and determine if
a set of photosynthetic parameters can be developed to adequately
represent all experimental results. A representative model parameter set
for the productivity model is derived from the PE data sets with
{[}\(\alpha\), E\textsubscript{k}, R\textsubscript{0}{]} =
{[}0.061 fixed C/photon, 240 \selectlanguage{greek}µ\selectlanguage{english}E m\textsuperscript{-2}
s\textsuperscript{-1}, 0.1 \selectlanguage{greek}µ\selectlanguage{english}mol C mgChl.a\textsuperscript{-1}
min\textsuperscript{-1}{]} at 30 \selectlanguage{ngerman}°C providing a reasonable
representation of the entire data base. The R\textsubscript{0} value at
the reference 30 °C temperature was taken as 0.1 \selectlanguage{greek}µ\selectlanguage{english}mol C
mgChl.a\textsuperscript{-1} min\textsuperscript{-1}consistent with
conclusions from outdoor experiments on a carbon basis (Legere 2017;
Chance and Roessler, 2019) and recognizing that R\textsubscript{0}'
determinations from PE curves will show an irradiance-related
enhancement (Falkowski and Raven, 2007). To model temperature effects,
E\textsubscript{k} is set as a function of temperature (activation
energy 60 kJ mol\textsuperscript{-1} ), and the respiration rate
(R\textsubscript{0}) was modeled as a function of temperature
(activation energy as 27 kJ mol\textsuperscript{-1}), with the
activation energy estimates being consistent with previous studies
(Legere, 2017). Table 4 gives a summary of the model parameter values.
Comparison between the modeled and experimental productivities are shown
in Figure 7. The model results are in good in agreement with experiment
results for all cases considered here. Even at 35 \selectlanguage{ngerman}°C, where clear
changes in pigmentation are seen, the agreement is satisfactory. For
example, with temperature increased from 20 °C to 30 °C, the biomass
productivity increases by 28\% (experimental) and 26\% (productivity
model). At higher light intensities
\textgreater{}\textgreater{}E\textsubscript{k}, an increase of 100\%, or
Q\textsubscript{10} = 2, would be expected. Good agreement between
biomass productivities for the small, L scale, experiments reported here
and the large, 24000 L scale, outdoor experiments (Chance and Roessler
2019) was noted earlier. This consistency can be extended to the PE
experiments (mL scale) where the derived photosynthetic parameters are
in good agreement with those employed for model representations of large
scale outdoor experiments (Chance and Roessler, 2019; Supplementary
Material).

\textbf{Conclusions}

We have provided here a detailed study of temperature impacts
on\emph{Arthrospira} \emph{platensis} biomass production in
semi-continuous operation. This temperature study of \emph{Arthrospira
platensis} in photobioreactor cultivations demonstrates that
temperatures in the 20 - 35 °C range are favorable for achieving
consistent productivities, though long term exposure to 35 °C caused
some modest changes in productivity and more obvious changes in
pigmentation. Exposure to simulated conditions for summer temperature
profiles for Southwest Florida shows some issues for the most extreme
conditions but a general tolerance for the short term, mid-day exposures
to higher temperatures. The response of the cultures to abrupt changes
in temperature is immediate for biomass production and quantitatively
consistent with the temperature dependence observed for
P\textsubscript{max} in smaller scale photosynthetic response
experiments. Pigment variations with abrupt changes in temperature
occurrs on a time scale that was essentially the same as that expected
for turnover of the cell population under semi-continuous operation. No
other acclimation effects were identified. These results all involve
annual average irradiance conditions. Extension of this study to higher
irradiance conditions in the summer may cause additional issues in
combination with extreme temperature exposures, high or low.
Productivity modeling based on photosynthetic parameters derived from
periodic sampling of the cultures provides excellent agreement with
experiment and consistency with the performance of large scale outdoor
PBR cultivations.

\textbf{Acknowledgements}

We thank Professor John Coleman and Dr. Paul Roessler for many helpful
discussions and suggestions. We thank Professors Valerie Thomas and
Matthew Realff for their support and helpful comments. We acknowledge
Matthew Anderson, Vedanta Malhoe, and Lucas Eastham for technical
support. This material is based upon work supported by the U.S.
Department of Energy's Office of Energy Efficiency and Renewable Energy
(EERE) under the Bioenergy Technologies Office Award Number
DE-EE0007690.

\textbf{Disclaimer:}

This report was prepared as an account of work sponsored by an agency of
the United States Government. Neither the United States Government nor
any agency thereof, nor any of their employees, makes any warranty,
express or implied, or assumes any legal liability or responsibility for
the accuracy, completeness, or usefulness of any information, apparatus,
product, or process disclosed, or represents that its use would not
infringe privately owned rights. Reference herein to any specific
commercial product, process, or service by trade name, trademark,
manufacturer, or otherwise does not necessarily constitute or imply its
endorsement, recommendation, or favoring by the United States Government
or any agency thereof. The views and opinions of authors expressed
herein do not necessarily state or reflect those of the United States
Government or any agency thereof.

\textbf{Authorship}

All authors made substantive contributions to the experimental design,
data analysis, and interpretation of results.

All authors actively participated in the drafting and revision of the
manuscript.

All authors have approved the final version of the manuscript and agree
to be accountable for all aspects of the work

All authors have agreed on the order in which their names are listed on
the manuscript

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\textbf{Tables}\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\begin{minipage}[b]{0.17\columnwidth}\raggedright\strut
\textbf{Table 1}. Summarized results indicating various growth and
pigment parameters in response to temperature conditions in Phase I, II
and III. Phase I starts with 20 \selectlanguage{ngerman}°C for both AB and CD, shifted to 35 °C
(AB) and 30 °C (CD) in Phase II, then to ExSP (AB) and AvSP (CD) in
Phase III, whereas culture EF and GH start with 35 °C in Phase I,
shifted to 20 °C (EF) and 30 °C (GH) in Phase II, then to AvSP (EF) and
CtSP (GH) in Phase III, respectively. Biomass Concentration is the
concentration prior to dilution, averaged over the relevant Phase period
(semi-continuous operation regions only). Error bars are +/- one
standard deviation.\strut
\end{minipage} & \begin{minipage}[b]{0.17\columnwidth}\raggedright\strut
\textbf{Table 1}. Summarized results indicating various growth and
pigment parameters in response to temperature conditions in Phase I, II
and III. Phase I starts with 20 \selectlanguage{ngerman}°C for both AB and CD, shifted to 35 °C
(AB) and 30 °C (CD) in Phase II, then to ExSP (AB) and AvSP (CD) in
Phase III, whereas culture EF and GH start with 35 °C in Phase I,
shifted to 20 °C (EF) and 30 °C (GH) in Phase II, then to AvSP (EF) and
CtSP (GH) in Phase III, respectively. Biomass Concentration is the
concentration prior to dilution, averaged over the relevant Phase period
(semi-continuous operation regions only). Error bars are +/- one
standard deviation.\strut
\end{minipage} & \begin{minipage}[b]{0.17\columnwidth}\raggedright\strut
\textbf{Table 1}. Summarized results indicating various growth and
pigment parameters in response to temperature conditions in Phase I, II
and III. Phase I starts with 20 \selectlanguage{ngerman}°C for both AB and CD, shifted to 35 °C
(AB) and 30 °C (CD) in Phase II, then to ExSP (AB) and AvSP (CD) in
Phase III, whereas culture EF and GH start with 35 °C in Phase I,
shifted to 20 °C (EF) and 30 °C (GH) in Phase II, then to AvSP (EF) and
CtSP (GH) in Phase III, respectively. Biomass Concentration is the
concentration prior to dilution, averaged over the relevant Phase period
(semi-continuous operation regions only). Error bars are +/- one
standard deviation.\strut
\end{minipage} & \begin{minipage}[b]{0.17\columnwidth}\raggedright\strut
\textbf{Table 1}. Summarized results indicating various growth and
pigment parameters in response to temperature conditions in Phase I, II
and III. Phase I starts with 20 \selectlanguage{ngerman}°C for both AB and CD, shifted to 35 °C
(AB) and 30 °C (CD) in Phase II, then to ExSP (AB) and AvSP (CD) in
Phase III, whereas culture EF and GH start with 35 °C in Phase I,
shifted to 20 °C (EF) and 30 °C (GH) in Phase II, then to AvSP (EF) and
CtSP (GH) in Phase III, respectively. Biomass Concentration is the
concentration prior to dilution, averaged over the relevant Phase period
(semi-continuous operation regions only). Error bars are +/- one
standard deviation.\strut
\end{minipage} & \begin{minipage}[b]{0.17\columnwidth}\raggedright\strut
\textbf{Table 1}. Summarized results indicating various growth and
pigment parameters in response to temperature conditions in Phase I, II
and III. Phase I starts with 20 \selectlanguage{ngerman}°C for both AB and CD, shifted to 35 °C
(AB) and 30 °C (CD) in Phase II, then to ExSP (AB) and AvSP (CD) in
Phase III, whereas culture EF and GH start with 35 °C in Phase I,
shifted to 20 °C (EF) and 30 °C (GH) in Phase II, then to AvSP (EF) and
CtSP (GH) in Phase III, respectively. Biomass Concentration is the
concentration prior to dilution, averaged over the relevant Phase period
(semi-continuous operation regions only). Error bars are +/- one
standard deviation.\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{Parameters/Temperature treatments}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{AB}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{CD}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{EF}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{GH}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{Phase I: 0-15 Days (n=4)}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\selectlanguage{ngerman}\textbf{20°C}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\selectlanguage{ngerman}\textbf{20°C}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\selectlanguage{ngerman}\textbf{35°C}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\selectlanguage{ngerman}\textbf{35°C}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{Biomass Concentration (g L\textsuperscript{-1})}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.29\selectlanguage{ngerman}±0.07\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.30\selectlanguage{ngerman}±0.05\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.62\selectlanguage{ngerman}±0.05\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.61\selectlanguage{ngerman}±0.08\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{Biomass Productivity (g L\textsuperscript{-1}
day\textsuperscript{-1})}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.17\selectlanguage{ngerman}±0.03\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.17\selectlanguage{ngerman}±0.02\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.20\selectlanguage{ngerman}±0.04\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.21\selectlanguage{ngerman}±0.03\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{PC \% (\%DW)}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
8.08\selectlanguage{ngerman}±0.3\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
8.09\selectlanguage{ngerman}±0.4\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
9.95\selectlanguage{ngerman}±1.1\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
9.73\selectlanguage{ngerman}±0.8\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{aPC \% (\%DW)} \textbf{cPC \% (\%DW)}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
2.85\selectlanguage{ngerman}±0.09\% 5.22±0.22\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
2.83\selectlanguage{ngerman}±0.10\% 5.26±0.35\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
2.68\selectlanguage{ngerman}±0.21\% 7.27±0.57\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
2.61\selectlanguage{ngerman}±0.15\% 7.11±0.42\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{Chl-a \%}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.61\selectlanguage{ngerman}±0.10\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.61\selectlanguage{ngerman}±0.13\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.98\selectlanguage{ngerman}±0.17\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.0\selectlanguage{ngerman}±0.12\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{Phase II: 15-33 Days (n=4)}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\selectlanguage{ngerman}\textbf{35°C}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\selectlanguage{ngerman}\textbf{30°C}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\selectlanguage{ngerman}\textbf{20°C}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\selectlanguage{ngerman}\textbf{30°C}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{Biomass Concentration (g L\textsuperscript{-1})}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.57\selectlanguage{ngerman}±0.17\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.48\selectlanguage{ngerman}±0.18\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.40\selectlanguage{ngerman}±0.09\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.58\selectlanguage{ngerman}±0.14\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{Biomass Productivity (g L\textsuperscript{-1}
day\textsuperscript{-1})}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.18\selectlanguage{ngerman}±0.04\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.21\selectlanguage{ngerman}±0.02\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.15\selectlanguage{ngerman}±0.03\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.23\selectlanguage{ngerman}±0.06\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{PC \% (\%DW)}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
11.50\selectlanguage{ngerman}±0.8\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
11.31\selectlanguage{ngerman}±0.6\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
7.45\selectlanguage{ngerman}±0.3\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
10.35\selectlanguage{ngerman}±1.0\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{aPC\% (\%DW)} \textbf{cPC\% (\%DW)}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
3.20\selectlanguage{ngerman}±0.13\% 8.30±0.36\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
3.49\selectlanguage{ngerman}±0.15\% 7.82±0.39\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
2.22\selectlanguage{ngerman}±0.08\% 5.23±0.23\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
2.91\selectlanguage{ngerman}±0.23\% 7.44±0.45\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{Chl-a \%}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.32\selectlanguage{ngerman}±0.05\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.70\selectlanguage{ngerman}±0.11\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.14\selectlanguage{ngerman}±0.23\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.42\selectlanguage{ngerman}±0.18\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\textbf{Phase III: 33- 43 Days (n=4)}\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\textbf{Ext. Summer} \selectlanguage{ngerman}\textbf{(39/26°C)}\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\textbf{Ave. Summer} \selectlanguage{ngerman}\textbf{(35/21°C)}\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\textbf{Ave. Summer} \selectlanguage{ngerman}\textbf{(35/21°C)}\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\textbf{Ct. Summer} \selectlanguage{ngerman}\textbf{(31/21°C)}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{Biomass Concentration (g L\textsuperscript{-1})}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.59\selectlanguage{ngerman}±0.03\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.52\selectlanguage{ngerman}±0.02\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.45\selectlanguage{ngerman}±0.05\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.58\selectlanguage{ngerman}±0.04\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{Biomass Productivity (g L\textsuperscript{-1}
day\textsuperscript{-1})}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.22\selectlanguage{ngerman}±0.02\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.25\selectlanguage{ngerman}±0.02\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.24\selectlanguage{ngerman}±0.02\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.25\selectlanguage{ngerman}±0.02\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{PC \% (\%DW)}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
10.85\selectlanguage{ngerman}±0.4\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
12.42\selectlanguage{ngerman}±0.5\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
11.85\selectlanguage{ngerman}±0.6\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
11.49\selectlanguage{ngerman}±0.4\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{aPC\% (\%DW)} \textbf{cPC\% (\%DW)}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
2.81\selectlanguage{ngerman}±0.07\% 8.04±0.40\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
3.61\selectlanguage{ngerman}±0.18\% 8.81±0.39\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
3.57\selectlanguage{ngerman}±0.14\% 8.28±0.54\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
3.34\selectlanguage{ngerman}±0.12\% 8.15±0.33\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{Chl-a \%}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.08\selectlanguage{ngerman}±0.12\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.83\selectlanguage{ngerman}±0.16\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.93\selectlanguage{ngerman}±0.09\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.86\selectlanguage{ngerman}±0.04\%\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}\selectlanguage{english}
\begin{longtable}[]{@{}lllllllll@{}}
\toprule
\textbf{Table 2}. Summary of photosynthetic parameters at different
temperature treatments in the three phases obtained from Monod fits to
the PI response curve & \textbf{Table 2}. Summary of photosynthetic
parameters at different temperature treatments in the three phases
obtained from Monod fits to the PI response curve & \textbf{Table 2}.
Summary of photosynthetic parameters at different temperature treatments
in the three phases obtained from Monod fits to the PI response curve &
\textbf{Table 2}. Summary of photosynthetic parameters at different
temperature treatments in the three phases obtained from Monod fits to
the PI response curve & \textbf{Table 2}. Summary of photosynthetic
parameters at different temperature treatments in the three phases
obtained from Monod fits to the PI response curve & \textbf{Table 2}.
Summary of photosynthetic parameters at different temperature treatments
in the three phases obtained from Monod fits to the PI response curve &
\textbf{Table 2}. Summary of photosynthetic parameters at different
temperature treatments in the three phases obtained from Monod fits to
the PI response curve & \textbf{Table 2}. Summary of photosynthetic
parameters at different temperature treatments in the three phases
obtained from Monod fits to the PI response curve & \textbf{Table 2}.
Summary of photosynthetic parameters at different temperature treatments
in the three phases obtained from Monod fits to the PI response
curve\tabularnewline
\midrule
\endhead
& \textbf{Phase I} & \textbf{Phase I} & \textbf{Phase II} &
\textbf{Phase II} & \textbf{Phase II} & \textbf{Phase III} &
\textbf{Phase III} & \textbf{Phase III}\tabularnewline
\textbf{Temperature treatments} & 20 \selectlanguage{ngerman}°C & 35 \selectlanguage{ngerman}°C & 20 \selectlanguage{ngerman}°C & 30 \selectlanguage{ngerman}°C & 35 \selectlanguage{ngerman}°C
& 31/21 \selectlanguage{ngerman}°C & 35/21 \selectlanguage{ngerman}°C & 39/26 \selectlanguage{ngerman}°C\tabularnewline
\textbf{PI Temperature} & 20 \selectlanguage{ngerman}°C & 35 \selectlanguage{ngerman}°C & 30 \selectlanguage{ngerman}°C & 30 \selectlanguage{ngerman}°C & 30 \selectlanguage{ngerman}°C & 30 \selectlanguage{ngerman}°C
& 30 \selectlanguage{ngerman}°C & 30 \selectlanguage{ngerman}°C\tabularnewline
\textbf{sOD750} & 2.6 & 2.7 & 2.5 & 2.75 & 2.5 & 3.0 & 2.95 &
2.8\tabularnewline
\textbf{Chl.a-Extract/sOD (mg/L)} & 7.5 & 8.15 & 6.7 & 8.0 & 7.7 & 10.3
& 10.1 & 7.3\tabularnewline
\selectlanguage{greek}\textbf{P\textsubscript{max} (µmolO\textsubscript{2}/L-hr)}\selectlanguage{english} & 240 & 620
& 583 & 650 & 510 & 730 & 710 & 580\tabularnewline
\selectlanguage{greek}\textbf{α' (molO\textsubscript{2}/mol photon)}\selectlanguage{english} & 0.075 & 0.075 & 0.060 &
0.075 & 0.070 & 0.090 & 0.080 & 0.060\tabularnewline
\selectlanguage{greek}\textbf{E\textsubscript{k} (µE/m\textsuperscript{2}-s)}\selectlanguage{english} & 85 & 230 & 270
& 240 & 200 & 220 & 240 & 270\tabularnewline
\selectlanguage{greek}\textbf{R\textsubscript{0}\textsuperscript{'}
(µmolO\textsubscript{2}/L-min)}\selectlanguage{english} & 0.12 & 0.48 & 0.47 & 0.37 & 0.55 &
0.49 & 0.25 & 0.35\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 3} : Productivity Model Parameters\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\textbf{Model Parameters} & \textbf{Unit} & \textbf{Culture Density 1
gDW/L}\tabularnewline
\midrule
\endhead
\selectlanguage{greek}α & \emph{mol C/mol photon} & 0.061\tabularnewline
E\textsubscript{k} & \selectlanguage{greek}\emph{µE/m\textsuperscript{2}-s}\selectlanguage{english} & \emph{240 @
30C}\tabularnewline
R\textsubscript{0} & \selectlanguage{greek}\emph{µmolC/mgChl.a-min}\selectlanguage{english} & \emph{0.1
@30C}\tabularnewline
F & \emph{-} & \emph{1}\tabularnewline
E\textsubscript{0} & \selectlanguage{greek}\emph{µE/m\textsuperscript{2}-s}\selectlanguage{english} &
\emph{230}\tabularnewline
D & \emph{m} & \emph{0.0254}\tabularnewline
k & \emph{1/m at 1 gDW/L} & \emph{175}\tabularnewline
kD & \emph{-} & \emph{4.45}\tabularnewline
C\textsubscript{c} & \emph{mgChl.a/m\textsuperscript{3} at 1 gDW/L} &
\emph{18,000}\tabularnewline
g & \emph{gDW/molC} & \emph{22.68}\tabularnewline
t\textsubscript{1} & \emph{sec} & \emph{43,200}\tabularnewline
t\textsubscript{2} & \emph{sec} & \emph{86,400}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Figure legends}

\textbf{Figure 1.} a) Experimental program illustrating timing for the
three experimental phases and the sequencing of the eight reactors. The
temperature profiles for Phase III are based on historical climate data
in Fort Myers Florida. The Extreme Profile is based on summer
temperatures only; the average profile is based on annual average. Both
involve hourly temperature variations in the reactors. The Constant
Profile has constant values on a 12-12 cycle based on annual day and
night averages. b) Photobioreactor setup for cultures in Phase I where
Reactors A, B and C, D were cultivated at 20 \selectlanguage{ngerman}°C and (b) Reactors E, F
and G, H were cultivated at 35 °C. Dye-1 and Dye-2 were the dummy
reactors used for monitoring temperature, and reactors W-1 and W-2 were
connected to the reactor exhaust and used as waste collectors due to
minor foaming and evaporation loss. Reactors are brought outside of the
incubator for sampling and photography.

\textbf{Figure 2.} OD (750 nm) results for cultures in three different
phases (a) AB and CD and (b) EF and GH at various temperature
treatments. Results shown are the average of two determinations with
error bars showing the range of values. Temperature conditions for the
various phases are shown.

\textbf{Figure 3.} Late-phase average biomass productivities
of\emph{Arthrospira} \emph{platensis} at various temperature treatments
in three phases: Phase I temperature were 20 and 35 °C; in Phase II
temperature were 20, 30 and 35 °C and in Phase III 31 °C (CtSP), 32 °C
(AvSP) and 36 °C (ExSP), where temperature designations are the average
daytime values. Error bars are +/- one standard deviation for the
averages over multiple days of semi-continuous operation.

\textbf{Figure 4} . Representative whole cell (WC) spectra of cultures
at different temperature treatments in the three phases of the
experiment (a) Phase I at 20 °C (A and C) and 35 °C (E and G), (b) Phase
II at 35 °C (A), 30 °C (C) 20 °C (E) and 30 °C (G), and (c) Phase III
with Extreme Summer, ExSP (A), Average Summer, AvSP (C) and Constant
Summer, CtSP (G). The absorbance values are normalized to 1 gDW
L\textsuperscript{-1}.

\textbf{Figure 5.} PC and Chl-a content (\%, DW) in the three phases of
the experimental plan at different temperature conditions. Experiments
were performed in duplicate.

\textbf{Figure 6} . Photosynthesis irradiance (PI) response curves
(measured at 30 °C) for the algal cultures at different temperature
treatments in (a) Phase II on day 27, A-35 °C, C-30 °C, F-20 °C, and
H-35 °C, and (b) Phase III on day 43, AB-ExSP, CD and EF-AvSP and
GH-CtSP.

\textbf{Figure 7} . Experiment results of biomass productivity in
comparison to the productivity model simulation. The productivity model
parameter set, representative of the PI database at 30 °C, is {[}\selectlanguage{greek}α,
\selectlanguage{english}E\textsubscript{k}, R\textsubscript{0}{]} = {[}0.061 fixed C/photon, 240
\selectlanguage{greek}µ\selectlanguage{english}E/m\textsuperscript{2}-s, 0.1 \selectlanguage{greek}µ\selectlanguage{english}molC/mgChl.a-min{]}.

Figure 1\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Figure 2\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

Figure 3\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

Figure 4\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

Figure 5\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

Figure 6\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

Figure 7\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure1/Figure1}

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

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-3/Figure-3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-4/Figure-4}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-5/Figure-5}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-6/Figure-6}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-7/Figure-7}

\end{center}
\end{figure}

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

