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Algal Research 71 (2023) 102996
Available online 3 February 2023
2211-9264/© 2023 Battelle Memorial Institute and The Author(s). Published by Elsevier B. V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
DISCOVR strain pipeline screening – Part I: Maximum specic growth rate
as a function of temperature and salinity for 38 candidate microalgae for
biofuels production
Michael Huesemann
a
,
*
, Scott Edmundson
a
, Song Gao
a
, Sangeeta Negi
b
, Taraka Dale
b
,
Andrew Gutknecht
a
, Hajnalka E. Daligault
b
, Carol K. Carr
b
, Jacob Freeman
a
, Theresa Kern
b
,
Shawn R. Starkenburg
b
, Cheryl D. Gleasner
b
, William Louie
a
, Robert Kruk
a
, Sean McGuire
a
a
Pacic Northwest National Laboratory, Marine and Coastal Research Laboratory, Sequim, WA, USA
b
Los Alamos National Laboratory, Los Alamos, NM, USA
ARTICLE INFO
Keywords:
Microalgal biofuels production strains
Salinity tolerance
Temperature tolerance
18S and 16S rDNA sequencing
Activation energies
Growing season
ABSTRACT
To identify high productivity strains for microalgal biofuels generation, the maximum specic growth rate of 38
strains was measured as a function of salinity (i.e., 5, 15, and 35 PSU) and temperature (i.e., at 8 temperatures
along a linear gradient from ca. 5 to 45 ◦C) to determine the most suitable growth medium salinity and best
growing season, respectively, for outdoor raceway pond cultivation. The following strains were evaluated:
Agmenellum quadruplicatum UTEX 2268, Anabaena sp. ATCC 33081, Arthrospira fusiformis UTEX 2721, Arthrospira
platensis UTEX 3086, Chlorella vulgaris NREL 4-C12, Chlorella autotrophica CCMP 243, Chlorella sorokiniana
DOE1044, Chlorella sorokiniana DOE 1116, Chlorella sorokiniana DOE 1412 (UTEXB3016), Chlorella vulgaris LRB
AZ-1201, Chlorococcum littorale UTEX 117, Chlorococcum sp. UTEX-B P7, Chloromonas reticulata CCALA 870,
Coelastrella sp. DOE 0202, Cyanobacterium sp. AB1, Micractinium reisseri NREL 14-F2, Microchloropsis gaditana
CCMP1894, Microchloropsis salina CCMP 1776, Monoraphidium sp. MONOR1, Monoraphidium minutum 26B-AM,
Nannochloropsis oceanica CCAP 849/10, Oscillatoria cf. priestleyi CCMEE 5020.1-1, Picochlorum celeri TG2-WT-
CSM/EMRE, Picochlorum oklahomensis CCMP 2329, Picochlorum renovo NREL 39-A8, Picochlorum soloecismus
DOE 101, Porphyridium cruentum CCMP 675, Scenedesmus acutus LRB-AP-0401, Scenedesmus obliquus DOE 0152.z,
Scenedesmus obliquus UTEX393,Scenedesmus rubescens NREL 46B-D3, Scenedesmus sp. IITRIND2, Stichococcus
minor CCMP 819, Stichococcus minutus CCALA 727, Synechococcus elongatus UTEX2973.1, Tetraselmis striata LANL
1001, Tisochrysis lutea CCMP 1324, and Tribonema minus UTEXB3156. For each strain, the identity and the
presence of bacterial cohorts was determined using 18S and 16S rDNA sequencing, respectively. The maximum
specic growth rate versus temperature data were also used to determine the activation energies (Arrhenius
equation) for most strains. For all strains, the measured salinity and temperature tolerance data were compared
to those reported in the literature. The fastest growing strains were down-selected for subsequent biomass
productivity measurements in climate-simulation photobioreactors, as reported in the next paper in the issue.
1. Introduction
In our overview of the DISCOVR consortium project in the intro-
ductory paper of this Special Issue ([1] this issue), we described how
candidate algal cultures move through the DISCOVR strain pipeline and
were down-selected based on testing at three different scales, i.e., in
50–100 mL ask cultures (Tier I), 1 L indoor climate simulation pho-
tobiorectors (Tier II), and 100 L outdoor raceway ponds (Tier III).
During the Tier I screening, a diverse set of strains from different habi-
tats and phylogenies were obtained from culture collections and in-
dustrial partners and were evaluated in ask cultures in terms of their
respective temperature and salinity tolerance. The rationale was that if a
strain cannot grow fast and reliably under optimized ask culture con-
ditions (i.e., nutrient-replete medium, sparged continuously with CO
2
-
enriched air, saturation light intensity, constant temperature, and pH), it
is unlikely to be competitive and stable under more harsh outdoor pond
* Corresponding author.
E-mail address: michael.huesemann@pnnl.gov (M. Huesemann).
Contents lists available at ScienceDirect
Algal Research
journal homepage: www.elsevier.com/locate/algal
https://doi.org/10.1016/j.algal.2023.102996
Received 20 April 2022; Received in revised form 22 December 2022; Accepted 26 January 2023
Algal Research 71 (2023) 102996
2
conditions, such as varying light and water temperatures, and biotic
disturbances such as competing phototrophs, predators, and infectious
agents. Therefore, testing at the ask scale removes non-promising
strains at a faster rate and minimizes the overall screening effort,
compared to the more time- and resource-consuming work at larger
scales.
Here we report on the temperature and salinity tolerance experi-
ments that were conducted at the ask culture scale (Tier I). Specically,
we measured the maximum specic growth rate as a function of tem-
perature and salinity, to determine for each respective DISCOVR strain
(a) the likely best growing season (winter, summer, spring/fall) in out-
door ponds, and (b) the most suitable medium salinity (brackish, ma-
rine, etc.) for cultivation. Results from DISCOVR strain testing at the
LEAPS photobioreactor culture scale (Tier II) and outdoor pond culture
scale (Tier III) are reported in subsequent papers in this Special Issue ([2]
this issue, [3] this issue).
2. Material and methods
2.1. Microorganisms and media
In casting the net for candidate algal strains to test, we looked to
available industry strains and previous phyco-prospecting and screening
efforts, while simultaneously emphasizing both taxonomic and habitat
diversity (Table 1). We included strains from industry efforts, previous
phyco-prospecting efforts such as the Department of Energy (DOE)
Aquatic Species Program [4], a broad phyco-prospecting effort in the
South Western USA [5], the City University of New York (CUNY)-led
effort under the National Alliance for Advanced Biofuels and Bio-
products (NAABB) phyco-prospecting efforts [6,7], the DOE Regional
Algae Feedstock Testbed (RAFT) project [8], the Arizona State Univer-
sity (ASU)-led Algae Testbed Public-Private Partnership (ATP3) project
[9], and the Culture Collection of Algae and Protozoa's (CCAP) screening
effort [10]. Details on the original isolation location and subsequent
acquisition of each strain are given in the respective results section.
In order to have a direct comparison of strains and reduce the
number of variables in our screening approach, we chose to cultivate
strains in a common medium that was industrially-relevant for large-
scale cultivation efforts. This medium, termed the DISCOVR medium,
was designed to provide nitrogen in a reduced form (ammonium) as
would be expected from plant nutrient recycling operations after fuel
conversion (anaerobic digestion of residuals or direct recycle from hy-
drothermal liquefaction) as assumed in NREL's Algae Farm Model [11].
The sulfate salt was chosen based on previous experimental work on
recycling nitrogen and phosphorus after actual biocrude conversion
processes [12]. Concentrations of elements in many maintenance media
(e.g., BG-11) are excessively high (i.e., 1.5 g/ L or 17.6 mM sodium
nitrate) and economically inhibitory for large scale biomass production
operations. Therefore, we chose reasonable nutrient levels (3.5 mM N)
to allow for robust growth to at least 500 mg/ L ash-free dry weight
(AFDW), the likely approximate harvest concentration for outdoor open
ponds operated under continuous cultivation conditions.
All strains were grown in this DISCOVR medium, which per liter
contained 300 mg sodium bicarbonate, 200 mg ammonium sulfate, 90
mg diammonium phosphate, 4.36 mg disodium ethylenediaminetetra-
acetate dihydrate, 3.16 mg ferric (III) chloride hexahydrate, 180
μ
g
manganese (II) chloride tetrahydrate, 22
μ
g zinc sulfate heptahydrate,
10
μ
g cobalt (II) chloride hexahydrate, 9.18
μ
g copper (II) sulfate pen-
tahydrate, 6.29
μ
g sodium molybdate dihydrate, and 0.5
μ
g cyanoco-
balamin (Table S1). Vitamin B
12
, cyanocobalamin, was added due to the
general physiological requirement observed in many phytoplankton
[13]. It was also assumed that in the envisioned industrial open pond
cultivation scenarios, naturally occuring bacterial consortia would
provide this vitamin to the cultivated algae culture [14]. Media of
different salinities (i.e., 5, 15, and 35 PSU, Practical Salinity Units) were
made by adding articial sea salts (Crystal Sea Marinemix, Bioassay
Table 1
Algal strains included in the screening efforts and sources from which the algae
were obtained.
# Alga strain Source
1 Agmenellum quadruplicatum UTEX2268 UTEX
2 Anabaena sp. ATCC33081 ATCC
3 Arthrospira fusiformis UTEX2721 UTEX
4 Arthrospira platensis UTEX3086 UTEX
5 Chlorella autotrophica CCMP243 CCMP
6 Chlorella sorokiniana DOE1044 CUNY/UTEX
7 Chlorella sorokiniana DOE1116/UTEXBP15 CUNY/UTEX
8 Chlorella sorokiniana DOE1412 CUNY
9 Chlorella vulgaris NREL4-C12 NREL
10 Chlorella vulgaris AZ-1201 AzCATI
11 Chlorococcum littorale UTEX117 UTEX
12 Chlorococcum sp. DOE1426/UTEX BP7 UTEX
13 Chloromonas reticulata CCALA870 CCALA
14 Coelastrella sp. DOE0202 CUNY
15 Cyanobacterium sp. AB1 Industrial
16 Micractinium sp. NREL14-F2 NREL
17 Microchloropsis gaditana CCMP1894 CCMP
18 Microchloropsis salina CCMP1776 CCMP
19 Monoraphidium minutum 26B-AM TAMU
20 Monoraphidium sp. MONOR1 ASP
21 Nannochloropsis oceanica CCAP849/10 CCAP
22 Oscillatoria cf. priestleyi CCMEE5020.1-1 CCMEE
23 Picochlorum celeri TG2-CSM/EMRE Industrial
24 Picochlorum oklahomensis CCMP2329 CCMP
25 Picochlorum renovo NREL39-A8 NREL
26 Picochlorum soleocismus DOE101 LANL
27 Porphyridium cruentum CCMP675 CCMP
28 Scenedesmus acutus LRB-AZ-0401 AzCATI
29 Scenedesmus obliquus DOE0152.z CUNY
30 Scenedesmus obliquus UTEX393 UTEX
31 Scenedesmus rubescens NREL46B-D3 NREL
32 Scenedesmus sp. IITRIND2 IIT
33 Stichococcus minor CCMP819 CCMP
34 Stichococcus minutus CCALA727 CCALA
35 Synechococcus elongatus UTEX2973.1 UTEX
36 Tetraselmis striata LANL1001 LANL
37 Tisochrysis lutea CCMP1324 CCMP
38 Tribonema minus UTEXB3156 UTEX
39
a
Chaetoceros muelleri CCMP194 CCMP
40
a
Surirella sp. CCMP3162 CCMP
41
a
Rhodomonas salina CCMP1319 CCMP
42
a
Thalassiosira weissogii CCMP1051 CCMP
43
a
Tisochrysis lutea CCMP463 CCMP
44
a
Desmodesmus sp. DOE1357 CUNY/UTEX
45
a
Stichogloea doederleinii CCMP823 CCMP
46
a
Pleurochrysis carterae CCMP647 CCMP
47
a
Synechococcus elongatus CCMP1630 CCMP
48
a
Leptolyngbya sp. CCMEE5010.3-1 CCMEE
49
a
Phormidium cf. autumnale CCMEE5034.1-3 CCMEE
50
a
Oscillatoria sp. CCMEE5011.3-1 CCMEE
51
a
Phormidium cf. autumnale CCMEE5019.2-2 CCMEE
52
a
Nitzschia sp. CCMP580 CCMP
53
a
Oocystis alpina UTEX2541 UTEX
54
a
Cyclotella cryptica CCMP333 CCMP
55
a
Cyclotella cryptica CCMP332 CCMP
ASP – Aquatic Species Program.
ATTC – American Type Culture Collection.
AzCATI- Arizona Center for Algal Technology and Innovation.
CCALA - Culture Collection of Autotrophic Organisms.
CCAP- Culture Collection of Algae and Protozoa.
CCMEE - Culture Collection of Microbes from Extreme Environments.
CCMP - National Center for Marine Algae and Microbiota (formerly the Culture
Collection of Marine Phytoplankton).
CUNY- City University of New York.
IIT - Indian Institute for Technology.
LANL- Los Alamos National Laboratory.
NREL – National Renewable Energy Laboratory.
TAMU- Texas A&M University.
UTEX – University of Texas Culture Collection of Algae.
a
Strains exhibiting poor or unstable growth were not fully characterized in
this study in terms of salinity and temperature tolerance (Section 3).
M. Huesemann et al.
Algal Research 71 (2023) 102996
3
Laboratory Formula, Marine Enterprises International, LLC) to deion-
ized water at approximately 1.12 g per liter per 1 unit of measured
salinity. The exception being the lowest salinity medium (0.4 PSU),
which was used in limited experiments. This medium did not use arti-
cial sea salts, but instead used the same base recipe described above
with the addition of 4 mg calcium chloride dihydrate, 8 mg magnesium
sulfate heptahydrate, and 10 mg potassium chloride on a per liter basis.
Salinity was measured via conductance (YSI model 30, YSI Inc., Yellow
Springs, Ohio) and for simplicity is reported in this effort as PSU. All
media were sterile ltered via 0.2
μ
m polyethersulfone (PES)
membranes.
2.2. Microscopy
As cellular morphology can change signicantly due to culture
environment and growth cycle, microscopy for the generation of
representative images was performed during active culture growth (i.e.,
linear growth phase) at 25 ◦C at the determined optimal salinity. An
Olympus BX51 microscope with either an Olympus DP73 or an OMAX
A35180U3 camera was used to visualize and photograph cells. Cells
were photographed with 100×objective lens using immersion oil, un-
less otherwise stated.
2.3. DNA extraction, 16S/18S sequencing and analysis
2.3.1. DNA extraction
Liquid culture samples were prepped for DNA extraction by spinning
them down in a centrifuge (Beckman GS-6R) at 4.5 ×1000g for 5–10
min. The supernatant was discarded, and samples were stored at −80 ◦C.
DNA extraction consisted of using the Quick-DNA Fungal/Bacterial Kit
(ZYMO Cat. #D6006) following the manufacturer's protocol. A Nano-
Drop (NanoDrop™ One/OneC Microvolume UV–Vis Spectrophotom-
eter) was used to assess DNA quantity and quality before sequencing.
2.3.2. Amplicon sequencing - 16S, ITS
16S PCR amplication of the V3-V4 region of bacterial rDNA, and
addition of the Illumina over-hang sequences, was performed using
degenerate primers (341F-806R) as previously described [15]. In the
case of the Nannochloropsis samples, amplication of the hypervariable
ITS region of rDNA was undertaken using different degenerate primers
(ITS3-ITS4 pair). In all cases 10 ng of DNA was used as the template. The
KAPA HiFi HotStart Ready Mix was used with the following PCR pro-
tocol: a denaturation temperature of 95 ◦C for 3 min, 20 cycles of 95 ◦C
for 30 s, 55 ◦C for 30 s and 72 ◦C for 30 s and followed by an extension of
72 ◦C for 5 min before holding at 4 ◦C. A second round of PCR was
performed to add unique 8 base Nextera XT v2 indexes (Illumina) and
adapters complementary to the ow cell. The second round of PCR used
a denaturation temperature of 95 ◦C for 3 min, 8 cycles of 95 ◦C for 30 s,
55 ◦C for 30 s and 72 ◦C for 30 s, followed by an extension of 72 ◦C for 5
min before holding at 4 ◦C. AMPure XP beads (Beckman Coulter, Cat.
#A63881) were used to clean up the amplicons. A control was also
included which contained no template. However, because no ampli-
cation band was seen in the V4 region, the sample was not processed
further. Amplicons were pooled for multiplexing. The concentration of
the combined sample was quantied using a Qubit dsDNA HS Assay
(ThermoFisher Scientic, Cat. #Q32854). An Agilent High Sensitivity
DNA Kit (Agilent, Cat. #5067-4626) was used to determine the average
library size. A complete library quantication was performed using a
Library Quantication Kit – Illumina/Universal Kit (KAPA Biosystems,
Cat. #KK4824). Illumina MiSeq and MiSeq Reagent Kits v3 (600-cycle)
(Illumina, Cat. #MS-102-3003) were used to sequence the amplicon
pool, generating paired-end 301 bp reads, with the expectation that each
amplicon would produce approximately 100,000 reads.
2.3.3. Amplicon sequencing - 18S
In order to amplify the 18S small subunit of eukaryotic and algal rDNA,
primers from the earth microbiome project (http://earthmicrobiome.org/
protocols-and-standards/18s/; F1391-5′-GTACACACCGCCCGTC-3′; R-
EukBr-5′-TGATCCTTCTGCAGGTTCACCTAC-3′) were amended with
Nextera XT v2 indexes and adapters complimentary to the Ilumina Miseq
owcell as described above in 5.a. The F1391-EukBr primers (0.2 uM nal
concentration) plus 10 ng of DNA from each sample were combined with
KAPA HiFi HotStart Ready Mix. 18s ssu DNA was amplied via the
following PCR protocol: a 94 ◦C denaturation step for 3 min, 20 cycles of
94 ◦C for 45 s, 57 ◦C for 60 s, and 72 ◦C for 60 s and followed by an extension
of 72 ◦C for 10 min before holding at 4 ◦C. Amplicon clean-up, sample
pooling, concentration and quality measurements, and MiSeq kits were all
performed and used as described in Section 2.3.2.
2.3.4. Taxonomic assessment
Post sequencing, the Miseq amplicon reads were trimmed for quality
(q >29 across 50 % of the read length with no ambiguous N base calls)
and analyzed using QIIME2 software (versions 2017.5 and .11) [16].
Taxonomy for the 16s and 18s operating taxonomic units (OTUs) were
assigned and/or veried using the Greengenes and SILVA reference
databases, respectively. In cases of assignment ambiguity, PCR amplicon
sequences were queried in NCBI BLAST, in order to increase condence
in OTU assignments.
2.4. Measurement of salinity and temperature tolerance proles
For salinity prole determination, cultures were grown in 125 mL
Erlenmeyer asks at selected salinities (i.e., generally 5, 15, and 35 PSU)
on PNNL's Salinity Gradient Incubator (SGI) at 25.5 ±0.3 ◦C. For tem-
perature prole determination, cultures were grown in the highest
tolerated salinity medium with no statistically signicant reduction in
growth as measured by a simple t-test (alpha =0.05). Higher salinity
media were favored as it is expected to both reduce the freshwater
footprint for large scale outdoor cultivation as well as reduce the salt
management costs, which can contribute signicantly to the cost of
cultivation [11]. After determination of the maximum salinity without a
reduction in growth rate (as dened above), each culture was then
grown in eight separate asks on PNNL's Thermal Gradient Incubator
(TGI) [17,18], to determine the temperature tolerance of the alga. The
temperature ranges tested were from ca. 4 to 46 ◦C, each ask at a
different temperature approximately 5 ◦C cooler or warmer than the
subsequent ask. Illumination for both the SGI and TGI was provided by
neutral white (4000 K) LED panels at ~450
μ
mol m
−1
s
−1
, set to a 12:12
light:dark photoperiod. The actual light intensity within each ask at the
surface of the culture medium was ca. 450
μ
mol m
−1
s
−1
, exceeding
saturating light intensities for all strains. Each ask was topped with a
foam stopper and sparged with sterile-ltered, CO
2
-enriched (0.5 % v/v)
humidied air owing from one 24-way gas distribution manifold,
resulting in CO
2
replete conditions with a culture of pH of ca. 7.0–7.5.
All cultures were allowed 48 h to acclimate to salinity or temperature
conditions prior to measuring growth rates. Growth rates were obtained
by diluting cultures to below an optical density at 750 nm (OD
750
) of 0.1
via a dual-beam spectrophotometer (Genesys 10S UV–Vis, Thermo Sci-
entic). Measurements were recorded over the course of the light period
only in optically thin, exponentially growing cultures, with at least three
time points per photoperiod. Maximum specic growth rates (
μ
max
)
were calculated by taking the slope of the natural log-transformed OD
750
measurements along a minimum of 3 time points. Data sets with linear
regression slopes having R
2
<0.95 were disregarded. Growth trials were
repeated 3–7 times for each cultivation condition, and the average
μ
max
values and respective standard deviations were calculated.
2.5. Determination of activation energies (E
a
)
Arrhenius-type kinetics is widely adopted to describe the tempera-
ture dependence of microbial growth [19–21]. The activation energy,
E
a
, an important parameter that indicates the sensitivity of growth rate
M. Huesemann et al.
Algal Research 71 (2023) 102996
4
to temperature, varies from species to species. Depending on the specic
growth rate and E
a
, the relative competitiveness among strains may vary
as temperature shifts [22]. In the current study, both the maximum
specic growth rates and E
a
were measured under the same condition,
which allows direct comparison of the strains for different seasons. The
Arrhenius equation is:
μ
=Ae−Ea/RT
where,
μ
is the maximum specic growth rate, A is Arrhenius constant,
E
a
is the activation energy, R is the ideal gas constant and T is temper-
ature (Kelvin). The activation energy (E
a
) was determined from the
linearized Arrhenius equation by multiplying R and the slope of the
logarithm of the growth rate versus the reciprocal of temperature:
ln
μ
=lnA −Ea
R
1
T→Ea=R*(slope of ln
μ
vs.1
T)
Due to the denaturation of proteins at high temperature, the Arrhe-
nius equation doesn't account for the steep decline in growth rate once
the temperature surpasses the optimal temperature, or the breakpoint
temperature, of a microorganism [23,24]. Therefore, in the current
study, the activation energy is only calculated for the range where
growth rate is positively related to temperature, including at least 3 data
points with a linear regression t of R
2
>0.95. Since in this study
activation energy (E
a
) values were not used for down-selecting strains
(from Tier I to Tier II), we include the E
a
data for each respective strain
for reference only, in case there is interest in this parameter for modeling
or other purposes.
2.6. Determination of seasonal water temperature ranges in AzCATI
outdoor ponds
In order to identify the optimum growing season for a specic DIS-
COVR strain whose temperature tolerance prole has been experimen-
tally determined (see Section 3), it is necessary to know the minimum
and maximum pond water temperatures for each season. The change in
temperature in 20 cm deep outdoor ponds at the AzCATI testbed site
(Mesa, Arizona) was predicted in hourly intervals for each day of the
year by carrying out energy balance calculations via the PNNL Biomass
assessment Tool, using 30 year averaged meteorological data for Mesa,
Arizona [25]. Fig. 1 shows the average minimum and maximum pond
water temperature for the rst day of each month. These data were then
used to determine the pond water temperature ranges for the four sea-
sons: 6.9–29 ◦C for Spring (Mar, Apr, May), 14.3–34 ◦C for Summer
(Jun, Jul, Aug), 10.4–33 ◦C for Fall (Sep, Oct, Nov), and 4.5–16 ◦C for
Winter (Dec, Jan, Feb).
2.7. Statistics
Average values, standard deviations, standard error of the mean, and
the statistical signicance between given means assessed by the Stu-
dent's t-test (two-tailed, unequal variance, alpha =0.05) were all
calculated in Microsoft Excel (Redmond, WA USA).
3. Results and discussion
The DISCOVR strain screening effort included 12 distinct taxonomic
groups at the Class level, and 12 distinct habitat types (Figs. 2 and 3). Of
the 55 strains tested, 17 failed to grow reliably after receipt from culture
collections, despite multiple attempts at revival. These strains, which are
marked with an asterisk in Table 1, were Chaetoceros muelleri CCMP194,
Surirella sp. CCMP3162, Rhodomonas salina CCMP1319, Thalassiosira
weissogii CCMP1051, Tisochrysis lutea CCMP463, Stichogloea doederleinii
CCMP823, Pleurochrysis carterae CCMP647, Synechococcus elongatus
CCMP1630, Leptolyngbya sp. CCMEE5010.3-1, Phormidium cf. autum-
nale CCMEE5034.1-3, Oscillatoria sp. CCMEE5011.3-1, Phormidium cf.
autumnale CCMEE5019.2-2, Nitzschia sp. CCMP580, Oocystis alpina
UTEX2541, Cyclotella cryptica CCMP333, and Cyclotella cryptica
CCMP332. These strains were eliminated from further testing in terms of
their salinity and temperature tolerance. One strain was found to
contain two algae via 18S analysis (Desmodesmus sp. DOE1357) and was
eliminated from further testing.
The results from the screening of the remaining 38 strains are indi-
vidually summarized and discussed in detail below. The strains are listed
in alphabetical order so that the related characterization data is easy to
nd. The limitation of using a single medium is the elimination of some
strains due to medium incompatibility. We observed approximately 30
% of the attempted strains had poor or unstable growth in the revival
and prescreening efforts and were therefore not fully characterized in
this effort. The observed incompatibility is likely due to ammonia
sensitivity in tested strains, as has been observed in the literature [26].
In this screening effort, we considered ammonia tolerance as a prereq-
uisite for strains to integrate successfully with waste nutrient (nitrogen
and phosphorus) recycle for sustainable large-scale production of algal
biomass [12,27].
3.1. Agmenellum quadruplicatum UTEX2268
The marine cyanobacterium Agmenellum quadruplicatum UTEX2268
was obtained from the University of Texas (UTEX) Culture Collection of
Algae at the recommendation of Dr. Jerry Brand (former Curator of the
Culture Collection of Algae at the University of Texas-Austin).
14.5 15.5
19.7
24.5
28.9
31.8
34.0 34.0 33.1
28.1
22.0
15.6
4.5 4.8
6.9
9.5
11.4
14.3
17.2
19.8 19.4
15.1
10.4
5.4
0
5
10
15
20
25
30
35
40
1-Jan 1-Feb 1-Mar 1-Apr 1-May 1-Jun 1-Jul 1-Aug 1-Sep 1-Oct 1-Nov 1-Dec
Water Temperature (°C)
Month
Fig. 1. The range of water temperature variation for the rst day of each month in 20 cm outdoor ponds at the DOE State of Technology testbed at AzCATI, located in
Mesa, Arizona, U.S.A.
M. Huesemann et al.
Algal Research 71 (2023) 102996
5
A. quadruplicatum UTEX2268 was collected from the mud of a sh pen
on Magueyes Island, Puerto Rico and isolated by Van Baalen [28]. We
conrmed the genus as Agmenellum via 16S rDNA identication. The
strain was axenic with no bacterial cohort, which was determined by
16S identication. Robust growth was observed in the DISCOVR me-
dium (Table S1), over a wide range of salinities (5 to 35 PSU) (Fig. 4A).
At 25 ◦C, the highest maximum specic growth rate (3.2 day
−1
) was
measured at 15 PSU salinity, but this was not signicantly (statistically)
greater than the 3.0 day
−1
observed at 35 PSU. At this salinity (35 PSU),
vigorous growth occurred within a temperature tolerance range of 24 to
41 ◦C, and the optimum maximum specic growth rate (7.25 day
−1
) was
measured at 41 ◦C (Fig. 4B), indicating that this strain should be suitable
for summer season outdoor pond cultivation (Table 2 in Section 4). The
activation energy (E
a
), determined over a temperature range of 24–35
◦C, was 1092 J mol
−1
(Fig. S2).
Although relatively thermotolerant, A. quadruplicatum UEX2268 was
not able to tolerate a constant temperature of 46 ◦C and failed to grow
consistently at this temperature. The strain was markedly sensitive to
temperatures below 20 ◦C, completely failing to grow at these lower
temperatures under the tested conditions. The lack of observed growth
at mesophilic conditions indicates a generally limited applicability to
unheated open pond cultivation conditions in the temperate United
States. Cultivation in enclosed photobioreactors may be suitable for this
strain, though properties such as sensitivity to dissolved oxygen were
not determined.
Initial isolation and characterization of Agmenellum quadruplicatum
UTEX2268, also known as A. quadruplicatum PR-6, indicated a require-
ment for vitamin B
12
[28]. Van Baalen [28] also observed a wide salinity
tolerance from 0 to 50 ppt with an optimum around 20 ppt, similar to the
results obtained in this screening where optimum growth was found at
15 ppt. Van Baalen [28] further determined growth rates at 29.5 and 37
◦C as 2.90 and 4.95 day
−1
. These rates are similar to the rates deter-
mined in the current study, i.e., 3.18 and 4.98 day
−1
at temperatures of
29.2 and 35.0 ◦C, respectively. Agmenellum belongs to the order
Bacillariophyceae
Chlorodendro
p
h
y
ceae
Chlorophyceae
Coccolithophyceae
Cryptophyceae
Cyanophyceae
Eusgmatophyceae
Mediophyceae
Phaeothamnio
p
h
y
ceae
Porphyridiophyceae
Trebouxiophyceae
Xanthophyceae
Fig. 2. DISCOVR strain taxonomic diversity at the Class level. Note: The taxonomic diversity of DISCOVR strains at the Phylum level is shown in Fig. S1.
Marine
Estuary
Terrestrial
Saline
Lake Brine
PoolAlpine
Sub-
Glacial
Freshwater
Marine Mudflat
Alkaline Lake
Polar
Opportunisc
Contaminant
Waste water Unknown
Fig. 3. DISCOVR strain sourced-habitat diversity.
M. Huesemann et al.
Algal Research 71 (2023) 102996
6
Synechococcales, and therefore is related to Synechococcus elongatus
UTEX2973.1, which was demonstrated to be also heat tolerant and
exhibit comparatively high maximum specic growth rates (5.1 day
−1
)
(see below).
3.2. Anabaena sp. ATCC 33081
Anabaena sp. ATCC 33081, also known as Anabaena sp. 2C, is a
cyanobacterium originally isolated by Gotto et al. [29] from an
enrichment culture inoculated with sediment collected from the shallow
bays and mud ats near the University of Texas Port Aransas Laboratory
on the Gulf of Mexico. We conrmed that this organism is in the Nos-
tocaceae but were unable to conrm the genus as Anabaena via 16S
identication. The strain was non-axenic with an unidentied Alphap-
roteobacterial cohort, which was determined by 16S identication. To
prevent clumping due to the lamentous nature of this strain, we used
ca. 0.5 g of 3 mm sterile glass beads placed within the 125 mL Erlen-
meyer cultivation asks to gently homogenize the culture and facilitate
uniform absorbance readings. The maximum specic growth rate,
determined at 25 ◦C in N-free DISCOVR medium (Table S1), declined
with increasing salinity, from 2.5 day
−1
at 5 PSU to 1.3 day
−1
at 35 PSU
(Fig. 5A). At 5 PSU salinity, vigorous growth occurred within a tem-
perature tolerance range of 18 to 40 ◦C, and the optimum maximum
specic growth rate (3.6 day
−1
) was measured at 40 ◦C (Fig. 5B), indi-
cating that this strain should be suitable for summer season outdoor
pond cultivation (Table 2 in Section 4). The activation energy (E
a
),
determined over a temperature range of 18.5–35 ◦C, was 1269 J mol
−1
(Fig. S3).
After the original isolation of Strain 2C (Anabaena sp. ATCC 33081),
Gotto et al. [29] observed vigorous growth between 30 and 45 ◦C, with
optimum growth occurring at 42 ◦C, which is comparable to our results.
Also similar to our ndings, these authors reported declining growth
rates with increasing salinity, testing concentrations ranging from 5 to
40 g/L NaCl (ca. 5 to 40 PSU), with optimum growth occurring at 5 g/L
NaCl (ca. 5 PSU). No growth was observed in their freshwater medium
(0 g/L NaCl).
However, Patel et al. [30] reported a specic growth rate at 25 ◦C of
only 0.76 day
−1
for Anabaena cylindrica, which is about 50 % of what
was measured in our study for Anabaena sp. ATCC 33081 at the same
temperature (1.3 day
−1
). The most likely explanation, other than the
difference in species, is that Patel et al. [30] measured maximum specic
growth rates using an incident light intensity of only 92.5
μ
mol m
−2
s
−1
,
which in conjunction with light attenuation in the culture, results in cells
being exposed, on average, to signicantly below saturating light
intensities.
AB
C
Agmenellum quadruplicatum UTEX2268
Fig. 4. Maximum specic growth rate of Agmenellum quadruplicatum UTEX2268 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at
salinity of 35 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3).
M. Huesemann et al.
Algal Research 71 (2023) 102996
7
3.3. Arthrospira fusiformis UTEX2721
Arthrospira fusiformis UTEX 2721, is a cyanobacterium originally
isolated from Lake Chitu in Ethiopia, was obtained from the University
of Texas (UTEX) Culture Collection of Algae. This strain was found to be
identical to Arthrospira maxima [31]. We conrmed the genus as
Arthrospira via 16S rDNA identication. The strain was non-axenic with
a multi-bacterial cohort including the genera Rhodobacas and Alka-
limonas, which were determined by 16S rDNA identication. To prevent
clumping due to the lamentous nature of this strain, we used ca. 0.5 g
of 3 mm sterile glass beads placed within the 125 mL Erlenmeyer
cultivation asks to gently homogenize the culture and facilitate uni-
form absorbance readings. The maximum specic growth rate, deter-
mined at 25 ◦C in DISCOVR medium (Table S1), was highest (1.5 day
−1
)
at 15 PSU salinity and slightly lower (1.3 and 1.2 day
−1
) at 5 PSU and 35
PSU salinity, respectively (Fig. 6A). At 5 PSU salinity, healthy growth
occurred within a temperature tolerance range of 18 to 38 ◦C, and the
optimum maximum specic growth rate (2.9 day
−1
) was measured at
32 ◦C (Fig. 6B), indicating that this strain should be suitable for spring,
summer, and fall season outdoor pond cultivation (Table 2 in Section 4).
There is no published temperature or salinity tolerance data for
Arthrospira fusiformis UTEX2721. This strain likely would have a
different growth response if a higher alkalinity medium had been used
for cultivation, more similar to that found in Lake Chitu (>500 meq/L,
Kebede 1994). Nevertheless, it is remarkable that this strain was able to
survive in the standard DISCOVR screening medium with only ~4 meq/
L, <1 % of the alkalinity found in the source location. Future tests should
explore the growth response of this strain as a function of alkalinity in
conjunction with salinity and temperature.
3.4. Arthrospira platensis UTEX3086
Arthrospira platensis UTEX3086, a cyanobacterium originally isolated
from Lake Chad in Africa, was obtained from the University of Texas
(UTEX) Culture Collection of Algae. Synonyms for this species are
Spirulina platensis, Limnospira platensis, and Ocillatoria platensis. A closely
related strain is Arthrospira platensis NIES-39. Arthrospira platensis
UTEX3086 was genetically characterized by Fujisawa et al. [32], and a
working draft genomic sequence is available for this strain (https:
//www.ncbi.nlm.nih.gov/nuccore/AP011615). We conrmed the
genus as Arthrospira via 16S identication. The strain was non-axenic
with a multi-bacterial cohort dominated by the genus Rhodobacas and
other unidentied bacteria, which was determined by 16S identica-
tion. To prevent clumping due to the lamentous nature of this strain,
we used ca. 0.5 g of 3 mm sterile glass beads placed within the 125 mL
Erlenmeyer cultivation asks to gently homogenize the culture and
facilitate uniform absorbance readings. The maximum specic growth
rate, determined at 25 ◦C in DISCOVR medium (Table S1), declined with
increasing salinity, from 1.6 day
−1
at 5 PSU to 0.9 day
−1
at 35 PSU
(Fig. 7A). At 5 PSU salinity, healthy growth occurred within a temper-
ature tolerance range of 12 to 38 ◦C, and the optimum maximum specic
growth rate (1.8 day
−1
) was measured at 32 ◦C (Fig. 7B), indicating that
this strain should be suitable for spring, summer, and fall season outdoor
pond cultivation (Table 2 in Section 4).
This strain likely would have a different growth response if a higher
alkalinity medium had been used for cultivation. However, previous
studies have noted the ability of this alkali-tolerant species to grow on
the freshwater medium BG-11 [33]. Future tests should explore the
growth response of this strain as a function of alkalinity in conjunction
with salinity and temperature.
There is no published temperature or salinity tolerance data for
Arthrospira platensis UTEX3086. However, Olaizola and Duerr [34] grew
Spirulina (Arthrospira) platensis UTEX1928 at 36 ◦C in culture tubes with
465
μ
mol m
−2
s
−1
incident light intensity and measured a specic
growth rate of 1.8 day
−1
(2.7 doublings/day), about three times higher
than was observed at this temperature in our study, but very similar to
growth rates observed at 32 ◦C, the optimum temperature of A. platensis
UTEX3086 (Fig. 7B). Kebede [34] reported a maximum specic growth
rate of 2.14 day
−1
for Spirulina platensis under turbidostat cultivation at
ca. 300
μ
mol m
−2
s
−1
incident light intensity and 30 ◦C. Trabelsi et al.
[35] cultured Arthrospira platensis sp. Comp´
ere 1968/3786 at 30, 35, and
40 ◦C and observed a linear decrease in specic growth rate with
increasing temperature, as was also the case in our study, with the
highest value of 0.41 day
−1
being measured at 30 ◦C and 180
μ
mol m
−2
s
−1
light intensity. Finally, Mohite and Wakte [36] measured a specic
growth rate of 0.45–0.5 day
−1
at 25 ◦C in Spirulina platensis cultures
isolated from Lonar Crater Lake in India, which is about 50 % less than
was observed in our study. Strain specic differences and the potential
use of sub-saturating light intensities most likely explain this
discrepancy.
3.5. Chlorella autotrophica CCMP243
Chlorella autotrophica CCMP243 (Chlorella sp. UTEX580) was ob-
tained from the National Center for Marine Algae and Microbiota
(NCMA). This strain was isolated from a seawater tank in Milford, CT by
R. Lewin in 1952. We conrmed the genus as Chlorella via 18S identi-
cation. The strain was axenic with no detected bacterial cohort, as
determined by 16S identication. The maximum specic growth rate,
determined at 25 ◦C in DISCOVR medium (Table S1), was highest at 35
PSU salinity (1.0 day
−1
), followed by 0.6 and 0.5 day
−1
at 5 and 15 PSU
salinity, respectively, indicating that this strain is most suitable for
cultivation in seawater medium (Fig. 8A). At 35 PSU salinity, growth
occurred within a temperature tolerance range of 18 to 40 ◦C, and the
optimum maximum specic growth rate (3.2 day
−1
) was measured at
35 ◦C (Fig. 8B), indicating that this strain should be suitable for spring,
summer, and fall season outdoor pond cultivation (Table 2 in Section 4).
The activation energy (E
a
), determined over a temperature range of
17.8–35.2 ◦C, was 1087 J mol
−1
(Fig. S4).
There are no published temperature tolerance data and limited
salinity tolerance data for Chlorella autotrophica CCMP243. Ahmad and
Hellebust [37] reported C. autotrophica clone 580 as an “extreme eury-
haline” strain maintaining photosynthetic activity in salinities up to 600
% articial seawater (presumably 210 ppt) and surviving complete
desiccation. Growth rates at standard salinity (presumably 35 ppt) were
approximately 0.8 day
−1
, similar to the growth rates measured in this
study. By contrast, Ahmad and Hellebust [37] measured increased
growth rates up to (ca. 1 day
−1
) at lower salinities (as low as 1 % of
seawater), whereas growth rates measured in our experimentation fol-
lowed a reverse trend, decreasing with decreasing salinity. Growth and
rates at lower salinities were still stable, agreeing with the broad salinity
tolerance of this strain, but unfortunately the temperature for the
growth experiments reported in Ahmad and Hellebust [37] were not
given and so it is difcult to further assess why results between the two
efforts differ.
3.6. Chlorella sorokiniana DOE1044
Chlorella sorokiniana DOE1044 (UTEXB3021, P13, NAABB2044) was
isolated by Dr. Juergen Polle (Brooklyn College, New York) from an
undisclosed habitat in Texas as part of the U.S. Department of Energy
National Consortium of Advanced Biofuels and Bioproducts (NAABB)
[6], and subsequently deposited as “unknown species” UTEXB3021 in the
University of Texas (UTEX) Culture Collection of Algae. We identied
the species as Chlorella sorokiniana via 18S. The strain was non-axenic
with low level contamination of Alphaproteobacteria (specically
Rhizobium), which was determined by 16S identication. The maximum
specic growth rate, determined at 25 ◦C in DISCOVR medium (Table
S1), was highest at 5 PSU salinity (4.3 day
−1
), followed by 4.2 day
−1
at
15 PSU (not statistically different from 4.3 day
−1
at 5 PSU), 3.5 day
−1
at
35 PSU, and 3.1 day
−1
at 0.4 PSU (Fig. 9A), indicating that this strain
can be cultivated in brackish and seawater growth medium. At 5 PSU
M. Huesemann et al.
Algal Research 71 (2023) 102996
8
salinity, robust growth occurred within a temperature tolerance range of
12 to 41 ◦C, and the optimum maximum specic growth rate (5.6 day
−1
)
was measured at 35 ◦C (Fig. 9B), indicating that this strain should be
suitable for spring, summer, and fall season outdoor pond cultivation
(Table 2 in Section 4). The activation energy (E
a
), determined over a
temperature range of 11.9–29.3 ◦C, was 920 J mol
−1
(Fig. S5).
There is no published temperature or salinity tolerance data for
Chlorella sorokiniana DOE1044. However, published results on the
temperature and salinity tolerance of other Chlorella sorokiniana strains
are given in Section 3.8 for Chlorella sorokiniana DOE1412.
3.7. Chlorella sorokiniana DOE1116
Chlorella sorokiniana DOE1116 (P15, NAABB2116) was isolated by
Dr. Juergen Polle (Brooklyn College, New York) from an estuary in Texas
as part of the U.S. Department of Energy National Consortium of
Advanced Biofuels and Bioproducts (NAABB) [6], and subsequently
deposited as “green alga, not designated” UTEX-P15 in the University of
Texas (UTEX) Culture Collection of Algae. We identied this organism as
a strain of Chlorella sorokiniana via 18S. The strain was non-axenic with
the presence of Alphaproteobacteria (Erythromicrobium, Agrobacterium,
Sphingomonas) and Gammaproteobacteria (Stenotrophomonas) detected
by 16S sequencing. The maximum specic growth rate, determined at
25 ◦C in DISCOVR medium (Table S1), was highest at 5 PSU salinity (4.0
day
−1
), followed by 3.9 day
−1
, 3.2 day
−1
, and 3.0 day
−1
at 15 PSU, 0.4
PSU and 35 PSU, respectively (Fig. 10A), indicating that this strain can
be cultivated in freshwater, brackish-water, and seawater growth me-
dium. At 5 PSU salinity, robust growth occurred within a temperature
tolerance range of 17 to 41 ◦C, and the optimum maximum specic
growth rate (4.7 day
−1
) was measured at 29 ◦C (Fig. 10B), indicating
that this strain should be suitable for spring, summer, and fall season
outdoor pond cultivation (Table 2 in Section 4). The activation energy
(E
a
), determined over a temperature range of 17.3–29.2 ◦C, was 799 J
mol
−1
(Fig. S6).
There is no published temperature or salinity tolerance data for
Chlorella sorokiniana DOE1116. However, published results on the
temperature and salinity tolerance of other Chlorella sorokiniana strains
are given in Section 3.8 for Chlorella sorokiniana DOE1412.
3.8. Chlorella sorokiniana DOE1412 (UTEXB3016)
Chlorella sorokiniana DOE1412 (UTEXB3016, NAABB2412) was iso-
lated by Dr. Juergen Polle (Brooklyn College, New York) as part of the U.
S. Department of Energy National Consortium of Advanced Biofuels and
Bioproducts (NAABB), where it was ranked as one of the top performing
strains [6]. This strain was subsequently deposited as Chlorella sor-
okiniana UTEX B3016 in the University of Texas (UTEX) Culture
Collection of Algae. We conrmed the species as Chlorella sorokiniana via
18S identication. The strain was axenic with no detected bacterial
cohort, as determined by 16S identication. The maximum specic
growth rate, determined at 25 ◦C in DISCOVR medium (Table S1), was
highest at 5 PSU salinity (4.6 day
−1
), followed by 3.9 day
−1
at 15 PSU,
Anabaena sp. ATCC 33081
AB
C
Fig. 5. Maximum specic growth rate of Anabaena sp. ATCC 33081 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at the optimum
salinity of 5 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3).
M. Huesemann et al.
Algal Research 71 (2023) 102996
9
AB
C
Arthrospira fusiformis UTEX2721
Fig. 6. Maximum specic growth rate of Arthrospira fusiformis UTEX 2721 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at the optimum
salinity of 5 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥2).
M. Huesemann et al.
Algal Research 71 (2023) 102996
10
3.1 day
−1
at 0.4 PSU, and 2.8 day
−1
at 35 PSU salinity (Fig. 11A),
indicating that this strain can be cultivated in brackish-water growth
medium. At 5 PSU salinity, robust growth occurred within a temperature
tolerance range of 10 to 46 ◦C, and the optimum maximum specic
growth rate (5.2 day
−1
) was measured at 36 ◦C (Fig. 11B), indicating
that this strain should be suitable for spring, summer, and fall season
outdoor pond cultivation (Table 2 in Section 4). The activation energy
(E
a
), determined over a temperature range of 10–22.8 ◦C, was 2303 J
mol
−1
(Fig. S7).
Temperature tolerance data for this strain were published earlier by
Huesemann et al. [18], who measured the optimum maximum specic
growth rate of 6 day
−1
at 37 ◦C in 1.2 ppt (freshwater) BG-11 medium.
Vona et al. [38] cultured Chlorella sorokiniana 211/8K at 20, 25, 30, and
35 ◦C with a photon ux density of 130
μ
mol m
−2
s
−1
and reported the
optimum specic growth rate of 3.2 day
−1
at 35 ◦C. Li et al. [39]
measured specic growth rates of Chlorella sorokiniana UTEX1602 at
temperatures ranging from 21 to 45 ◦C and observed the fastest growth
(1.6 day
−1
) at 37 ◦C. Numerous studies, given here in chronological
order, conrm the high specic growth rates of different Chlorella sor-
okiniana strains: 3.1 day
−1
and 5.8 day
−1
at 40 ◦C for Chlorella sor-
okiniana UTEX1230 and Chlorella sorokiniana H-84, respectively (Sakai
et al., 1995); 6.5 day
−1
at 35.5–37.5 ◦C for Chlorella sorokiniana
CCAP211/8K [40]; 5.8 day
−1
at 40 ◦C for Chlorella sorokiniana H-84
[41]; 5.8 day
−1
at 37 ◦C for Chlorella sorokiniana CCAP211/8K [42]; and
4.3 day
−1
at 38 ◦C for Chlorella sorokiniana CCAP211/8K [43]. It should
be noted that Chlorella sorokiniana UTEX1230 and CCAP211/8k are
closely related strains maintained at different culture collections, both
derived from the initial isolation and characterization by Sorokin and
Myers and originally referred to as “Tx 71105” [44].
Salinity tolerance data over a much wider range than reported in this
study were published by Huesemann et al. [45], who observed a linear
decrease in maximum specic growth rate withincreasing NaCl con-
centration, i.e.,
μ
(day
−1
) =4.09–0.1055⋅NaCl (g/L) ~
4.09–0.1055⋅PSU. Kim et al. [46] cultured Chlorella sorokiniana
AG20740 and Chlorella sorokiniana HS1 in BG-11 medium with NaCl
concentrations varying from 0 to 50 g/L, and reported the highest spe-
cic growth rates of ca. 0.33 and 0.4 day
−1
, respectively, at 0 to 10 g/L
NaCl, and declining growth rates with increasing salinity. In conclusion,
Chlorella sorokiniana grows well in brackish waters (5 to 15 ppt) while
tolerating both fresh and marine waters.
3.9. Chlorella vulgaris NREL4-C12
Chlorella vulgaris NREL4-C12, among hundreds of other strains, was
originally isolated from a bioprospecting effort in the Southwest United
States [5]. The strains were then down-selected using a screen of NREL's
AB
C
Arthrospira platensis UTEX3086
Fig. 7. Maximum specic growth rate of Arthrospira platensis UTEX3086 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at the optimum
salinity of 5 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3, with the exception of
μ
max
measurements between 18.5 ◦C and 34.7 ◦C where n
<3).
M. Huesemann et al.
Algal Research 71 (2023) 102996
11
resultant algal culture collection. The species was recently determined
to be Chlorella vulgaris by NREL via 18S identication [47]. The strain
was assumed to be non-axenic, and the bacterial cohort was not deter-
mined. The maximum specic growth rate, determined at 25 ◦C in
DISCOVR medium (Table S1), declined with increasing salinity, from
4.5 day
−1
at 5 PSU to 2.7 day
−1
at 35 PSU (Fig. 12A). At 5 PSU salinity,
healthy growth occurred within a temperature tolerance range of 3 to
40 ◦C, and the optimum maximum specic growth rate (4.1 day
−1
) was
measured at 23 ◦C (Fig. 12B), indicating that this strain should be
suitable for spring, summer, fall, and winter season outdoor pond
cultivation (Table 2 in Section 4). The activation energy (E
a
), deter-
mined over a temperature range of 3.3–22.9 ◦C, was 1070 J mol
−1
(Fig.
S8).
Temperature and salinity tolerance data for Chlorella vulgaris NREL4-
C12 were examined in [48] in modied f/2 seawater medium, who re-
ported a temperature tolerance from 10 to 30 ◦C and a maximum growth
rate of ca. 1.1 day
−1
at 25 ◦C, which agrees with the temperature opti-
mum determined in this study. The growth rates of Dahlin [48] are likely
much lower than observed here due to the sampling method used, which
measured optical density only once per day and thus does not reect a
maximum specic growth rate. Salinity tolerance was assessed by Dahlin
[48] using endpoint biomass concentrations (not growth rates). Both
Dahlin [48] and the current study show that this strain has a moderate
salinity tolerance, capable of robust growth at seawater salinities (ca. 35
ppt) and below. Published results on the temperature and salinity
tolerance of other Chlorella vulgaris strains are given in the Section 3.10
for Chlorella vulgaris LRB-AZ-1201. Furthermore, Chlorella vulgaris
NREL4-C12, together with other candidate strains, was tested in outdoor
ponds in Arizona during the winter season (February) and was found to
exhibit a maximum areal biomass productivity of ca. 6 g/m
2
-day, with
pond water temperatures ranging from ca. 0 to 20 ◦C, conrming its
suitability as a winter season strain [47].
3.10. Chlorella vulgaris LRB AZ-1201
Chlorella vulgaris LRB-AZ-1201, a benchmark strain from the unied
eld studies conducted under the DOE Algae Testbed Public-Private
Partnership (ATP
3
) [49], was obtained from Arizona State University.
We conrmed the species as Chlorella vulgaris via 18S identication. The
strain was non-axenic with an unidentied Alphaproteobacterial cohort,
which was determined by 16S identication. The maximum specic
growth rate, determined at 25 ◦C in DISCOVR medium (Table S1), was
highest at 15 PSU salinity (3.6 day
−1
), followed by 3.3 day
−1
at 5 PSU
(not statistically different from 3.6 day
−1
at 15 PSU), and 2.7 day
−1
at 35
AB
C
Chlorella autotrophica CCMP243
Fig. 8. Maximum specic growth rate of Chlorella autotrophica CCMP243 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at the optimum
salinity of 35 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3).
M. Huesemann et al.
Algal Research 71 (2023) 102996
12
PSU (Fig. 13A), indicating that this strain can be successfully cultivated
in brackish waters. At 15 PSU salinity, robust growth occurred within a
temperature tolerance range of 10 to 42 ◦C, and the optimum maximum
specic growth rate (4.4 day
−1
) was measured at 30 ◦C (Fig. 13B),
indicating that this strain should be suitable for spring, summer, and fall
season outdoor pond cultivation (Table 2 in Section 4). The activation
energy (E
a
), determined over a temperature range of 4.6–22.9 ◦C, was
796 J mol
−1
(Fig. S8).
There is no published temperature or salinity tolerance data for
Chlorella vulgaris LRB-AZ-1201. However, there are a number of studies
related to other Chlorella vulgaris strains. Dauta et al. [50] measured the
specic growth rates of Chlorella vulgaris at 10, 15, 20, 25, 30, and 35 ◦C
using incident light intensities ranging from 5 to 800
μ
mol m
−2
s
−1
, and
reported the highest specic growth rate (1.3 day
−1
) at 30 ◦C, using a
photon ux density of 140
μ
mol m
−2
s
−1
. Goncalves et al. [51] cultured
Chlorella vulgaris CCAP211/11B at 15, 25, and 35 ◦C with incident light
intensities ranging from 15 to 180
μ
mol m
−2
s
−1
and observed the
highest specic growth rate (1.2 day
−1
) at 25 ◦C and 180
μ
mol m
−2
s
−1
.
Our results conrm the previously reported temperature optimum range
of 25 to 30 ◦C, but the maximum specic growth rates in our study were
signicantly higher (up to 4.4 day
−1
) than those reported by Dauta et al.
[50] and Goncalves et al. [51], which is most likely due to sparging our
cultures with CO
2
enriched air (0.5 % v/v) instead of just with air (as in
the published studies), and the much higher light intensities used in our
experiments. Regarding salinity tolerance, Pandit et al. [52] cultured
Chlorella vulgaris KY436509.1 in media containing 0, 0.06, 0.08, 0.1, 0.3,
and 0.4 M NaCl (equivalent to ca. 0, 3.5, 4.7, 5.8, 17.5, and 23 ppt
salinity, respectively) and reported specic growth rates of 0.105, 0.127,
0.123, 0.119, 0.109, and 0.093 day
−1
, respectively. Our results conrm
the large salinity tolerance range observed by Pandit et al. [52].
3.11. Chlorococcum littorale UTEX117
Chlorococcum littorale UTEX117 (Chlorococcum minutum CCAP 213/
7, CCALA290, SAG 213-7) was obtained from the University of Texas
Culture Collection of Algae (UTEX). This strain was isolated by H.C. Bold
from soil in Bombay, India. The culture we received was visibly
contaminated and was difcult to cultivate in liquid media. Therefore,
we treated the culture with 50
μ
g/mL ampicillin to reduce the bacterial
load and enrich for algal cells. Under these conditions, we conrmed the
genus as Chlorococcum via 18S identication. Even after antibiotic
treatment, the strain was non-axenic and contaminated with many
different bacteria. Alphaproteobacteria (Aminobacter, Agrobacterium,
Sphingopyxis, Brevundimonas, Sphingomonas), Flavobacteriia (Fla-
vobacterium, Chryseobacterium), Actinobacteria (Gordonia), and Beta-
proteobacteria (Achromobacter), which was determined by 16S
identication. No attempts were made to generate an axenic culture.
The maximum specic growth rate, determined at 25 ◦C in DISCOVR
AB
C
Chlorella sorokiniana DOE1044
Fig. 9. Maximum specic growth rate of Chlorella sorokiniana DOE1044 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at the optimum
salinity of 15 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3).
M. Huesemann et al.
Algal Research 71 (2023) 102996
13
medium (Table S1), was highest (4.3 day
−1
) at 15 PSU salinity, followed
by 3.2 day
−1
at 5 PSU, and 1.9 day
−1
at 35 PSU (Fig. 14A), indicating
that this strain can be cultivated in brackish-water growth media. It
should be noted that the error bars in Fig. 14A are relatively large which
was caused by higher-than-average variability in optical density mea-
surements due to cell clumping problems. At 15 PSU salinity, robust
growth occurred within a temperature tolerance range of 16 to 36 ◦C,
and the optimum maximum specic growth rate (2.8 day
−1
) was
measured at 36 ◦C (Fig. 12B), indicating that this strain should be
suitable for spring, summer, and fall season outdoor pond cultivation
(Table 2 in Section 4).
There is no published temperature or salinity tolerance data for
Chlorococcum littorale UTEX117. Hu et al. [53] extensively studied the
growth of a Clorococcum littorale strain of unknown origin in a half
seawater medium (ca. 15 ppt) under high light intensity in at plate
reactors and found a maximum specic growth rate of 1.68 day
−1
. This
growth rate corresponded to a biomass productivity of 380 mg/ L h
−1
,
one of the highest reported biomass productivities in the literature. The
extreme conditions of high cell density (20 g/ L) and a short light path
(1 cm) and 5 % CO
2
likely facilitated this high productivity. Zhang et al.
[54] measured the maximum specic growth rate, using an incident
light intensity of 200
μ
mol m
−2
s
−1
, of a locally isolated Chlorococcum sp.
as a function of temperature (25, 30, 35, 40, 45 ◦C) and observed the
optimum specic growth rate of 1.6 day
−1
at 30 ◦C. Leya et al. [55]
measured specic growth rates of 0.2 and 0.4 day◦at 8 ◦C for Chlor-
ococcum sp. CCCryo 006-99 and 039-99, respectively, two strains that
were isolated from Spitsbergen (Norway) permafrost. Feng et al. [56]
reported a specic growth rate of 1.5 day
−1
for another locally isolated
Chlorococcum sp. grown at 25 ◦C with a photon ux density of 200
μ
mol
m
−2
s
−1
. Aravantinou and Manariotis [57] measured a specic growth
rate of 0.6 day
−1
for Chlorococcum sp. SAG 22.83 grown at 21 ◦C in ask
cultures receiving 100
μ
mol m
−2
s
−1
incident light intensity. Overall, the
growth rates reported in the literature are lower than in this study,
which could be caused by strain-specic differences but also by the sub-
saturating light intensities employed by these investigators.
3.12. Chlorococcum sp. UTEX-BP7
Chlorococcum sp. UTEXBP7 (DOE1426, NAABB2426) was obtained
from the University of Texas Culture Collection of Algae (UTEX). This
strain was a high biomass producer in bubble column tests conducted in
the National Alliance for Advanced Biofuels and Bioproducts (NAABB)
consortium project [6]. The 18S sequence analysis aligned with se-
quences from multiple other algae strains and therefore we were not
able to conrm this culture as a species of Chlorococcum. A more in-
depth sequence analysis, such as genome sequencing, would have to
be undertaken to identify this strain, which was beyond the scope of this
work. The strain was non-axenic with Alphaproteobacteria
AB
C
Chlorella sorokiniana DOE1116
Fig. 10. Maximum specic growth rate of Chlorella sorokiniana DOE1116 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at salinity of 0.4
PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3).
M. Huesemann et al.
Algal Research 71 (2023) 102996
14
AB
C
Chlorella sorokiniana DOE1412
Fig. 11. Maximum specic growth rate of Chlorella sorokiniana DOE1412 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at the optimum
salinity of 5 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3).
M. Huesemann et al.
Algal Research 71 (2023) 102996
15
Chlorella vulgaris NREL4-C12
AB
C
Fig. 12. Maximum specic growth rate of Chlorella vulgaris NREL4-C12 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at the optimum
salinity of 5 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3, with the exception of
μ
max
measurements at 3.3 ◦C, 10.82 ◦C, and ≥35.5 ◦C where
n <3).
M. Huesemann et al.
Algal Research 71 (2023) 102996
16
(Erythromicrobium, Rhizobium) bacterial cohort, which was determined
by 16S identication. The maximum specic growth rate, determined at
25 ◦C in DISCOVR medium (Table S1), decreased with increasing
salinity, from 3 day
−1
at 0.4 PSU, and 2.4 day
−1
at 5 PSU to 1 day
−1
at 35
PSU (Fig. 15A), indicating that this strain is most suitable for cultivation
in lower salinity media. At 0.4 PSU, robust growth occurred within a
temperature tolerance range of 11 to 40 ◦C, and the optimum maximum
specic growth rate (4 day
−1
) was measured at 29 ◦C (Fig. 15B), indi-
cating that this strain should be suitable for spring, summer, and fall
season outdoor pond cultivation (Table 2 in Section 4).
There is no published temperature or salinity tolerance data for
Chlorococcum sp. UTEXBP7.
However, published results on the temperature tolerance of other
Chlorococcum species are given in Section 3.11 for Chlorococcum littorale
UTEX117.
3.13. Chloromonas reticulata CCALA870
Chloromonas reticulata CCALA870 (Chloromonas clathrata) was ob-
tained from the Culture Collection of Autotrophic Organisms (CCALA) of
the Institute of Botany, Trebon, Czech Republic. This culture was iso-
lated from snow on Ben Navis, Scotland. We conrmed the genus as
Chloromonas via 18S identication. The strain was non-axenic with a
multi-bacterial cohort, including Rhodobacter, Aquimonas, Mycoplana,
Erythromicrobium, Phyllobacterium, along with unidentied members of
the Sphingobacteriales and Rhizobaceae, which were determined by 16S
identication. The maximum specic growth rate, determined at 25 ◦C
in DISCOVR medium (Table S1), was highest (2.5 day
−1
) at 5 PSU
salinity, followed by 1.2 day
−1
at 15 PSU, and 1.1 day
−1
at 35 PSU
(Fig. 16A), indicating that this strain can be cultivated in brackish-water
growth media. At 5 PSU salinity, robust growth occurred within a
temperature tolerance range of 6 to 24 ◦C, and the optimum maximum
specic growth rate (2.6 day
−1
) was measured at 19 ◦C (Fig. 16B),
indicating that this strain should be suitable for late fall, winter, and
early spring season outdoor pond cultivation (Table 2 in Section 4).
There is no published temperature or salinity tolerance data for
Chloromonas reticulata CCALA870. However, Leya et al. [55] cultured
Chloromonas nivalis and measured a specic growth rate of 0.6 day
−1
at
13 ◦C, which is lower than reported in this study (2.2 day
−1
at 13 ◦C).
Hoham [58], evaluating the temperature tolerance range of different
snow algae, reported that Chloromonas pichinchae grew best between 1
and 5 ◦C. In a later study, the same author reported an optimum tem-
perature range of 4 to 15 ◦C for four strains of Chloromonas rosae v.
psychrophila (UTEX SNO 11, 56, 50, 51), and an optimum range of 2.5 to
5 ◦C for ve strains of Chloromonas tughillensis (UTEX SNO 88, 89, 90, 91,
92) and Chloromonas chenangoensis (UTEX SNO 147) [59].
3.14. Coelastrella sp. DOE 0202
Coelastrella sp. DOE 0202 (UTEX B 3026, NAABB1202) was isolated
by Dr. Juergen Polle (Brooklyn College, New York) from water in garden
pot in San Pablo, CA, as part of the U.S. Department of Energy National
Alliance for Advanced Biofuels and Bioproducts (NAABB). Under
bubble-column testing, this strain had one of the highest recorded
Chlorella vulgaris LRB-AZ-1201
AB
C
Fig. 13. Maximum specic growth rate of Chlorella vulgaris LRB-AZ-1201 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at the optimum
salinity of 15 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3, with the exception of the
μ
max
measurement at 41.8 ◦C where n =1).
M. Huesemann et al.
Algal Research 71 (2023) 102996
17
biomass productivities [7]. Coelastrella sp. was also noted as unique in its
ability to grow at high densities with relatively high lipid content [6].
Indoor pond climate simulated cultivation of this strain showed pro-
ductivities similar to the benchmark strain Acutodesmus obliquus
DOE0152.Z [60]. This strain is also carotenogenic, producing high
concentrations of carotenoids in stationary phase [61]. We conrmed
the strain as Coelastrella via 18S identication. The strain was non-
axenic with an unidentied multi-bacterial cohort, which was deter-
mined by 16S identication. The maximum specic growth rate,
determined at 25 ◦C in DISCOVR medium (Table S1), was 3.8 day
−1
at
both 5 PSU and 15 PSU, but then declined to 0.9 day
−1
at 35 PSU
(Fig. 17A). At 15 PSU salinity, healthy growth occurred within a tem-
perature tolerance range of 14 to 37 ◦C, and the optimum maximum
specic growth rate (3.5 day
−1
) was measured at 28 ◦C (Fig. 17B),
indicating that this strain should be suitable for spring, summer, and fall
season outdoor pond cultivation (Table 2 in Section 4).
There is no published temperature or salinity tolerance data for
Coelastrella sp. DOE 0202. However, Minyuk et al. [62] measured a
maximum specic growth rate of 1.11 day
−1
at 25 ◦C for Coelastrella
rubescens, which is about three times lower than observed for Coelastrella
sp. DOE 0202. This discrepancy could be due to strain-specic differ-
ences or Minyuk et al. [62] using sub-saturating light intensities in their
growth rate measurements.
3.15. Cyanobacterium sp. AB1
Cyanobacterium sp. AB1 is a proprietary unicellular cyanobacterium
that was isolated by Algenol, Inc. from a non-disclosed location [63].
The strain was selected by Algenol because of its insensitivity to high
dissolved oxygen concentration and high thermal tolerance, enabling
the culturing of AB1 in closed photobioreactors. The genome of AB1 was
sequenced and genetic tools were developed to generate ethanolgenic
strains derived from AB1 [63]. The strain was tested as received with no
16S or 18S analyses or attempt to make it axenic. Robust growth was
observed in the DISCOVR medium (Table S1), over a wide range of sa-
linities (5 to 35 PSU) (Fig. 18A). At 25 ◦C, the highest maximum specic
growth rate (3.8 day
−1
) was measured at 5 and 15 PSU salinity, but this
was not signicantly (statistically) greater than the 3.2 day
−1
observed
at 35 PSU. At this salinity (35 PSU), vigorous growth occurred within a
temperature tolerance range of 11 to 48 ◦C, and the optimum maximum
specic growth rate (7 day
−1
) was measured at 48 ◦C (Fig. 18B), indi-
cating that this strain has exceptional thermal tolerance and thus is
suitable for enclosed photobioreactor cultivation. However, given its
relatively low maximum specic growth rate below temperatures of 25
◦C, the average daytime summer season water temperature in 20 cm
deep outdoor ponds in Arizona (Fig. 1), this strain is unlikely to exhibit
exceptional biomass productivity in open raceways. However, it should
still be suitable for summer season outdoor pond cultivation. In repeated
AB
C
Chlorococcum liorale UTEX117
Fig. 14. Maximum specic growth rate of Chlorococcum littorale UTEX117 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at the optimum
salinity of 15 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3).
M. Huesemann et al.
Algal Research 71 (2023) 102996
18
experiments, growth rates observed in the characterization of this strain
were temporally variable for unknown reasons. It remains unclear why
the growth rates observed in the salinity gradient at 25 ◦C were higher
than the expected growth rate at 25 ◦C based on the thermal growth
curve. The activation energy (E
a
), determined over a temperature range
of 4–41.8 ◦C, was 960 J mol
−1
(Fig. S10).
3.16. Micractinium reisseri NREL14-F2
Micractinium reisseri NREL14-F2 (C2B2-14-f2) was originally isolated
from Broad Lake Bog in Arvada, CO in a bioprospecting effort in the
Southwest United States [5]. M. reisseri NREL14-F2 was down-selected
as a promising winter strain using a screen of NREL's resultant algal
culture collection and the strain was identied as Micractinium reisseri
via 18S identication [47]. The strain is assumed to be non-axenic,
although the bacterial cohort for this strain was not determined. The
maximum specic growth rate, determined at 25 ◦C in DISCOVR me-
dium (Table S1), declined with increasing salinity, from 4.4 day
−1
at 5
PSU to 2.8 day
−1
at 35 PSU (Fig. 19A). At 5 PSU salinity, vigorous
growth occurred within a temperature tolerance range of 4 to 33 ◦C, and
the optimum maximum specic growth rate (3.9 day
−1
) was measured
at 28 ◦C (Fig. 19B), indicating that this strain should be suitable for fall,
winter, and spring season outdoor pond cultivation (Table 2 in Section
4). The activation energy (E
a
), determined over a temperature range of
3.6–22.2 ◦C, was 776 J mol
−1
(Fig. S11).
Temperature and salinity tolerance for Micractinium reisseri NREL14-
F2 was examined in [48], who reported a temperature tolerance from 10
to 30 ◦C and a maximum growth rate of ca. 1.8 day
−1
at 25 and 30 ◦C,
which agrees with the temperature optimum determined in this study.
Growth rates given in Dahlin [48] are likely much lower than observed
here due to the sampling method used, which measured optical density
only once per day and is not reective of maximum specic growth rates.
Salinity tolerance was also assessed by Dahlin [48] using endpoint
biomass concentrations. Both Dahlin [48] and the current study show
that M. reisseri NREL14-F2 has a wide salinity tolerance, capable of
robust growth at salinities above and below typical seawater (ca. 35
ppt). This strain, together with other candidates, was tested in outdoor
ponds in Arizona during the winter season (February) and was found to
exhibit the highest productivity (ca. 6.5 g/m
2
-day), with pond water
temperatures ranging from ca. 0 to 20 ◦C, conrming its suitability as a
winter season strain [47]. The specic growth rate of other Micractinium
strains, i.e., Micractinium reisseri and Micractinium inermum, were
respectively reported to be 1.15 day
−1
at 27 ◦C [64] and 1.61 day
−1
at
20 ◦C [65]. These specic growth rate values are signicantly lower than
those for Micractinium reisseri 14-F2 in our study (>3 day
−1
) since these
authors used incident light intensities that were likely signicantly
below saturation, i.e., 40
μ
mol m
−2
s
−1
[64] and 50
μ
mol m
−2
s
−1
[65].
3.17. Microchloropsis gaditana CCMP1894
Microchloropsis gaditana CCMP1894 (formerly Nannochloropsis gadi-
tana) was obtained from the National Center for Marine Algae and
C
Chlorococcum sp. DOE1426/UTEXBP7
AB
Fig. 15. Maximum specic growth rate of Chlorococcum sp. UTEXBP7 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at the optimum
salinity of 0.4 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3).
M. Huesemann et al.
Algal Research 71 (2023) 102996
19
Microbiota. The strain was isolated from Comacchio Lagoons, near
Ferrara, Italy. We conrmed the genus as Microchloropsis via 18S iden-
tication. The strain was axenic with no detectable bacterial cohort, as
determined by 16S identication. The strain exhibited growth over a
wide range of salinities (from 5 to 75 PSU), with the largest maximum
specic growth rates (ca. 1.8 day
−1
) being observed at 35 and 50 PSU
salinity (Fig. 20A). At 35 PSU salinity, growth occurred within a tem-
perature tolerance range of 12 to 30 ◦C, and the optimum maximum
specic growth rate (2.5 day
−1
) was measured at 30 ◦C (Fig. 20B),
indicating that this strain should be suitable for spring and fall season
outdoor pond cultivation (Table 2 in Section 4).
There are no published data on specic growth rates as a function of
temperature or salinity for Nannochloropsis gaditana CCMP1894. How-
ever, Ma et al. [66] compared the specic growth rates of 9 different
Nannochloropsis strains in 400 mL bubble column bioreactors at 25 ◦C
under continuous illumination (100
μ
mol m
−2
s
−1
) and reported a value
of 0.18 day
−1
for Nannochloropsis gaditana CCMP527. Their specic
growth rate value is about <1/10 of the maximum specic growth rate
measured in our study at the same temperature for N. gaditana
CCMP1894. The observed discrepancy could be strain-specic but is
most likely the result of Ma et al. [66] using sub-saturating incident light
intensities. This specic strain was included in the screening effort due
to the remarkable increase in lipid productivity through the application
of CRISPR–Cas9 as described by Ajjawi et al. [67].
3.18. Microchloropsis salina CCMP1776
Microchloropsis (formerly known as Nannochloropsis) salina
CCMP1776 (CCAP 849/2, Millport 201; CSIRO CS-190; SMBA 201) was
obtained from the National Center for Marine Algae and Microbiota. The
strain was originally isolated by M. Droop from Skate Point, Isle of
Cumbrae, Scotland in 1965. We conrmed the genus as Microchloropsis
via 18S identication. The strain was non-axenic with an unidentied
bacterial cohort within the Alphaproteobacteria, which was determined
by 16S identication. No growth was observed at 5 PSU salinity
(Fig. 21A). The maximum specic growth rate, determined at 25 ◦C in
DISCOVR medium (Table S1), increased with increasing salinity, from
1.3 day
−1
at 15 ppt to 1.7 day
−1
at 35 PSU (Fig. 21A). At 35 PSU salinity,
vigorous growth occurred within a temperature tolerance range of 11 to
35 ◦C, and the optimum maximum specic growth rate (1.8 day
−1
) was
measured at 24 ◦C (Fig. 21B), indicating that this strain should be most
suitable for spring and fall season outdoor pond cultivation (Table 2 in
Section 4).
Bartley et al. [68] cultured Nannochloropsis salina CCMP1776 in
media with salinities ranging from 10 to 58 ppt and observed the highest
specic growth rates (ca. 0.13 day
−1
) between 22 and 34 ppt, but no
AB
C
Chloromonas reculata CCALA870
Fig. 16. Maximum specic growth rate of Chloromonas reticulata CCALA870 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at the
optimum salinity of 5 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3).
M. Huesemann et al.
Algal Research 71 (2023) 102996
20
signicant growth at 10 ppt. Our study conrms these ndings. The
temperature tolerance for this strain had been determined in our lab in
earlier projects, as reported in Van Wagenen et al. [17] and Huesemann
et al. [18]. While the overall temperature tolerance range was similar in
these earlier experiments, the maximum specic growth rate at the
optimum temperature was only ca. 1 day
−1
. It is possible that the longer
and more rigorous maximum specic growth rate experiments carried
out for the current study allowed for better adaptation and thus higher
μ
max
values. Conducting steady-state chemostat experiments with Nan-
nochloropsis salina CCMP1776 at 25 ◦C in 15 g/L sea salt medium (i.e.,
ca. 15 ppt salinity), Kang et al. [69] reported a maximum specic growth
rate of 0.43 day
−1
, which is signicantly lower than observed in our
study (1.3 day
−1
). Most likely, the sub-saturating incident light intensity
(100
μ
mol m
−2
s
−1
) used by Kang et al. [69] is the causative factor.
3.19. Monoraphidium minutum 26B-AM
Monoraphidium minutum 26B-AM (Rhaphidium minutum, Rhaphi-
dium convolutum var. minutum, Selenastrum minutum, Ankistrodesmus
minutissimus, Ankistrodesmus lunulatus, Chorycystis minuta) was
collected by Dr. Louis Brown as a persistent invading species of winter
season outdoor pond cultures at the Texas A&M AgriLife Research algae
testbed in Pecos, Texas, as part of research conducted by the U.S.
Department of Energy National Consortium of Advanced Biofuels and
Bioproducts (NAABB) [70]. The strain was isolated and obtained from
Dr. Judy Brown at the University of Arizona. We conrmed the genus as
Monoraphidium via 18S identication. The strain was non-axenic with
the presence of Gammaproteobacteria (especially Pseudomonas) within
the bacterial cohort, which was determined by 16S identication. The
maximum specic growth rate, determined at 25 ◦C in DISCOVR me-
dium (Table S1), declined with increasing salinity, from 3.8 day
−1
at 5
PSU to 0.9 day
−1
at 35 PSU (Fig. 22A), indicating a severe salt-sensitivity
at seawater salinities. At 5 PSU salinity, robust growth occurred within a
temperature tolerance range of 4 to 40 ◦C, and the optimum maximum
specic growth rate (3.4 day
−1
) was measured at 28 ◦C (Fig. 22B),
indicating that this strain should be suitable for spring, summer, fall, and
winter season outdoor pond cultivation (Table 2 in Section 4). It should
be noted, however, that during summer season cultivation, this strain is
more susceptible to pond crashes. The activation energy (E
a
), deter-
mined over a temperature range of 3.6–28 ◦C, was 853 J mol
−1
(Fig.
S12).
The temperature tolerance of Monoraphidium minutum 26B-AM was
initially determined in the U.S. Department of Energy Regional Algal
Feedstock Testbed (RAFT) consortium project [8]. In this project, the
strain repeatedly demonstrated stable growth and exceptional produc-
tivity in outdoor raceways in Arizona, New Mexico, and Texas. Bouterfas
et al. [71], found maximum specic growth rates of 1.73 day
−1
at a
temperature of 35 ◦C for a strain of Selenastrum (Monoraphidium) min-
utum isolated from a eutrophic lake in Morocco. Although this strain
appears more heat tolerant than the one tested in our screening effort,
these specic growth rates are about 50 % lower than the rates we
observed. A likely factor in observing the lower growth rates is that the
AB
C
Coelastrella sp. DOE0202
Fig. 17. Maximum specic growth rate of Coelastrella sp. DOE 0202 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at salinity of 15 PSU
(B). Micrograph (C). Error bars are one standard deviation (n ≥3, with the exception of the
μ
max
measurement at 35 PSU where n =1).
M. Huesemann et al.
Algal Research 71 (2023) 102996
21
authors did not add carbon dioxide to the cultures when measuring
growth rates, sparging cultures with air only, resulting in carbon-limited
conditions. Yu et al. [72], however, reported a specic growth rate of
only 0.15 day
−1
at 25 ◦C for their Monoraphidium sp. FXY-10. Similarly,
Dhup and Dhawan [73] measured a specic growth rate of 0.089 day
−1
at 25 ◦C for their Monoraphidium sp. T4X. In addition to strain-specic
differences, these rather low reported specic growth rates, which
were at least 20 times less than observed than the maximum specic
growth rates measured in our experiments, were most likely caused by
using large culture volumes (up to 1 L) with sub-saturating incident light
intensities, resulting in very low average light intensities within the
cultures.
3.20. Monoraphidium sp. MONOR1
Monoraphidium sp. MONOR1 was isolated by W. Barclay from an
ephemeral pond in southwestern Colorado, USA, and deposited into the
SERI (Solar Energy Research Institute, the precursor of the National
Renewable Energy Laboratory, NREL) culture collection as part of
research conducted by the U.S. Department of Energy's Aquatic Species
Program from 1978 to 1996 [4,74]. The strain was obtained from NREL.
We conrmed the genus as Monoraphidium via 18S identication. The
strain was non-axenic with a multi-bacterial cohort with members of the
Alphaproteobacteria, which was determined by 16S identication. The
maximum specic growth rate, determined at 25 ◦C in DISCOVR me-
dium (Table S1), was highest between 5 and 15 PSU salinity (4.1 and 3.9
day
−1
, respectively) and slightly lower at 0.4 and 35 PSU salinity (3.2
and 2.5 day
−1
, respectively) (Fig. 23A). At 15 PSU salinity, robust
growth occurred within a temperature tolerance range of 10 to 30 ◦C,
with a maximum specic growth rate of 3.4 day
−1
at 29 ◦C. Although the
culture was able to survive, growth was unstable at 35 ◦C (Fig. 23B).
Therefore, this strain should be suitable for spring, summer, and fall
season outdoor pond cultivation (Table 2 in Section 4). The activation
energy (E
a
), determined over a temperature range of 10–23.6 ◦C, was
690 J mol
−1
(Fig. S13).
Monoraphidium sp. MONOR1 was extensively characterized by Bar-
clay et al., 1988 for salinity and temperature tolerances in two articial
island seawater media (Type I and II). The strain prefers the Type II
medium which is NaCl dominated with high bicarbonate content versus
the Type I medium, which is MgSO
4
dominated. In both media, Barclay
et al. [74] found the strain is relatively sensitive to increases in ion
concentration, with optimum growth observed at ca. 12 ppt, which
agrees well with our salinity characterization data. The maximum sus-
tained temperature tolerance of this strain was ca. 30 ◦C, and it was able
to sustain growth at 10 ◦C, which agrees with our temperature charac-
terization data. Additional information on specic growth rates of
Monoraphidium minutum 26B-AM and related species is shown below
(see next section).
C
AB
Fig. 18. Maximum specic growth rate of Cyanobacterium sp. AB1 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at salinity of 35 PSU
(B). Micrograph (C). Error bars are one standard deviation (n ≥3).
M. Huesemann et al.
Algal Research 71 (2023) 102996
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3.21. Nannochloropsis oceanica CCAP 849/10
Nannochloropsis oceanica CCAP 849/10, originally isolated from a
marine hatchery in western Norway [75], was obtained from the Culture
Collection of Algae and Protozoa. We conrmed the genus as Nanno-
chloropsis via 18S identication. The strain was axenic with no detected
bacterial cohort, as determined by 16S identication. The maximum
specic growth rate, determined at 25 ◦C in DISCOVR medium (Table
S1), was highest (2.8 day
−1
) at 15 PSU salinity, and slightly lower (2.2
day
−1
and 2.6 day
−1
) at 5 and 35 PSU salinity, respectively (Fig. 24A).
At 35 PSU salinity, vigorous growth occurred within a temperature
tolerance range of 4 to 36 ◦C, and the optimum maximum specic
growth rate (2.9 day
−1
) was measured at 30 ◦C (Fig. 24B), indicating
that this strain should be suitable for fall, winter, and spring season
outdoor pond cultivation (Table 2 in Section 4).
There are no published salinity tolerance data for Nannochloropis
oceanica CCAP849/10. Regarding temperature tolerance, Sandnes et al.
[75] measured specic growth rates of Nannochloropsis oceanica CCAP
849/10 along a temperature gradient (14.5 to 35.7 ◦C) at 6 different
incident light intensities (34 to 80
μ
mol m
−2
s
−1
) and reported the
highest specic growth (1.6 day
−1
) at 29 ◦C. No growth was observed at
35 ◦C. While our study conrmed a similar temperature optimum
around 30 ◦C, we observed higher specic growth rates (i.e., 2.9 day
−1
at 30 ◦C) since above saturation light intensities (>250
μ
mol m
−2
s
−1
)
were used. Savvidou et al. [76] cultured Nannochloropsis oceanica
CCMP1779 at 20 ◦C with a photon ux density of 100
μ
mol m
−2
s
−1
, and
reported a specic growth rate of 0.28 day
−1
, about 6 times lower than
observed in our study. Finally, Ma et al. [66] evaluated the growth of
three different Nannochloropsis oceanica strains (IMET1, 805, CCMP531)
at 25 ◦C with incident light intensity of 100
μ
mol m
−2
s
−1
, and reported
specic growth rates of 0.11 to 0.21 day
−1
, again much lower than in
our study, most likely due to the sub-saturating photon ux densities
used.
3.21.1. Oscillatoria cf. priestleyi CCMEE5020.1-1
Oscillatoria cf. priestleyi CCMEE5020.1-1 was obtained from the
Culture Collection of Microorganisms from Extreme Environments
(CCMEE). The strain was isolated from Pancreas Pond on the McMurdo
ice shelf, in Antarctica. This strain was not conrmed by genetic iden-
tication. The strain was assumed non-axenic with an unknown bacte-
rial cohort. Due to the lamentous nature of this culture, ca. 0.5 g of 3
mm, autoclaved glass beads were added to each 125 mL Erlenmeyer
cultivation ask. The glass beads in combination with the orbital mixing
of the incubator (ca. 110 rpm) were sufcient to gently fragment larger
C
Micracnium reisseri NREL14-F2
AB
Fig. 19. Maximum specic growth rate of Micractinium reisseri NREL14-F2 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at the
optimum salinity of 5 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3).
M. Huesemann et al.
Algal Research 71 (2023) 102996
23
laments and provide a more homogenous solution when measuring
changes in cell density by absorbance. However, the variability in op-
tical density measurements was higher than in non-lamentous cultures,
resulting in relatively large and unavoidable error bars. The maximum
specic growth rate, determined at 25 ◦C in DISCOVR medium (Table
S1), declined with increasing salinity, from 1.5 day
−1
at 5 PSU to 1
day
−1
at 15 PSU (Fig. 25A). No growth was observed at 35 PSU salinity.
At 5 PSU salinity, healthy growth occurred within a temperature toler-
ance range of 18 to 30 ◦C, and the optimum maximum specic growth
rate (1.4 day
−1
) was measured at 24 ◦C (Fig. 25B), indicating that this
strain should be suitable for spring and fall season outdoor pond culti-
vation (Table 2 in Section 4). Given that this strain was isolated from
Antarctica, it is surprising that no growth was observed below 18 ◦C. It
may be that light stress, which is aggravated at low temperatures, was
the causative factor.
There is no published temperature or salinity tolerance data for
Oscillatoria cf. priestleyi CCMEE5020.1-1 or any other Oscillatoria priest-
leyi strains.
3.21.2. Picochlorum celeri TG2-WT-CSM/EMRE
Picochlorum celeri TG2-WT-CSM/EMRE was originally isolated from
water samples taken at the Texas coast of the Gulf of Mexico, as
described in detail by Weissman et al. [77]. The strain was obtained
from Dr. Matthew Posewitz of Colorado School of Mines and named
Picochorlum celeri by genome sequence analysis [77]. The maximum
specic growth rate, determined at 25 ◦C in DISCOVR medium (Table
S1), was 5.1 day
−1
at 15 PSU salinity, and slightly lower at 5 ppt and 35
PSU salinities, i.e., 3.5 day
−1
and 4.0 day
−1
, respectively (Fig. 26A). At
the recommendation of Dr. Weissman, we tested the temperature
tolerance of this strain a 35 PSU salinity, in which vigorous growth
occurred within a temperature tolerance range of 13 to 46 ◦C, and the
optimum maximum specic growth rate (6.8 day
−1
) was measured at
35 ◦C (Fig. 26B), indicating that this strain should be suitable for late
spring, summer, and early fall season outdoor pond cultivation (Table 2
in Section 4). The activation energy (E
a
), determined over a temperature
range of 3.7–29.6 ◦C, was 1553 J mol
−1
(Fig. S13).
Weissman et al. [77] measured the maximum specic growth rate of
this strain (TG2 axenic), at 30 ◦C and in seawater salinity medium, and
reported a value of 0.34 h
−1
or 8.16 day
−1
[77], which is higher than in
this study, likely due to different methods (optical versus particulate
organic carbon) in determining maximum specic growth rate as well as
different media compositions and light intensities. Furthermore,
Weissman et al., [77] also reported that this strain can grow in medium
with double seawater salinity.
AB
C
Microchloropsis gaditana CCMP1894
Fig. 20. Maximum specic growth rate of Microchloropsis gaditana CCMP1894 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at the
optimum salinity of 35 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3).
M. Huesemann et al.
Algal Research 71 (2023) 102996
24
3.22. Picochlorum oklahomensis CCMP2329
Picochlorum oklahomensis CCMP2329 (SPNWR980625-4A, UTEX
2795) was obtained from the National Center for Marine Algae and
Microbiota. This alga was originally isolated from shallow evaporitic
pools at the Salt Plains National Wildlife Refuge in northwestern Okla-
homa [78]. We conrmed the genus as Picochlorum via 18S identica-
tion. The strain was axenic with no detected bacterial cohort, which was
determined by 16S identication. The maximum specic growth rate,
determined at 25 ◦C in DISCOVR medium (Table S1), was 4.2 day
−1
at
15 PSU salinity, and slightly lower (not statistically signicant) at 5 PSU
and 35 PSU salinities, i.e., 4.1 day
−1
(Fig. 27A), indicating that this is a
euryhaline strain, at the salinity ranges tested. At 35 PSU salinity,
vigorous growth occurred within a temperature tolerance range of 11 to
40 ◦C, and the optimum maximum specic growth rate (6.3 day
−1
) was
measured at 34 ◦C (Fig. 27B), indicating that this strain should be
suitable for spring, summer, and fall season outdoor pond cultivation
(Table 2 in Section 4). The activation energy (E
a
), determined over a
temperature range of 11.1–28.4 ◦C, was 1212 J mol
−1
(Fig. S15).
There are no published temperature tolerance data for Picochlorum
oklahomensis CCMP2329 (UTEX 2795). However, Annan [79] measured
specic growth rates in high/low concentration carbonate, iron, and
phosphate media as a function of salinity and found that growth rates
were similar at 10 and 50 ppt salinity, and then declined signicantly at
100 ppt salinity. Zhu and Dunford [80] reported a specic growth rate of
only 0.5 day
−1
at ca. 23 ◦C, which is signicantly lower than in our
study, most likely due to the fact that the incident light intensity was
below saturating levels (i.e., ca. 56
μ
mol m
−2
s
−1
).
3.23. Picochlorum renovo NREL 39-A8
Picochlorum renovo NREL 39-A8 (C2B2-39-A8) was originally iso-
lated from an unknown location during a bioprospecting effort in the
Southwest United States [5]. The strains were then downselected using a
screen of NREL's resultant algal culture collection, as described previ-
ously [47,48]. The genus and species were determined via 18S
sequencing by Dahlin et al. [47]. The strain was assumed to be non-
axenic, however the bacterial cohort was not determined. The
maximum specic growth rate, determined at 25 ◦C in DISCOVR me-
dium (Table S1), increased with increasing salinity, from 3.0 day
−1
at 5
PSU to 3.7 day
−1
at 35 PSU (Fig. 28A). At 35 PSU salinity, vigorous
growth occurred within a temperature tolerance range of 11 to 45 ◦C,
and the optimum maximum specic growth rate (5.1 day
−1
) was
measured at 40 ◦C (Fig. 28B), indicating that this strain should be
AB
C
Microchloropsis salina CCMP1776
Fig. 21. Maximum specic growth rate of Microchloropsis salina CCMP1776 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at the
optimum salinity of 35 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3).
M. Huesemann et al.
Algal Research 71 (2023) 102996
25
AB
C
Monoraphidium minutum 26B-AM
Fig. 22. Maximum specic growth rate of Monoraphidium minutum 26B-AM as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at the
optimum salinity of 5 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3, with the exception of
μ
max
measurements at 3.6 ◦C and 39.6 ◦C where n
=2).
M. Huesemann et al.
Algal Research 71 (2023) 102996
26
suitable for spring, summer, and fall season outdoor pond cultivation
(Table 2 in Section 4). The activation energy (E
a
), determined over a
temperature range of 11.2–28.3 ◦C, was 986 J mol
−1
(Fig. S16).
In previous detailed characterizations of Picochlorum renovo, Dahlin
et al. [48] observed this strain to have exceptional salinity tolerance,
demonstrating robust growth up to at least 107.5 g/L salinity, con-
rming our data of the euryhaline character of this strain. By contrast,
these authors reported a much narrower temperature tolerance range,
from only ca. 25 to 40 ◦C, compared to 11 to 45 ◦C in our study. The most
likely reasons for the discrepancy in observed temperature tolerance
between the two studies are that growth rates reported by Dahlin et al.
[48] were executed under continuous (24 h per day) lighting with no
dark period and no repeated acclimation of cultures over multiple
dilution cycles. Lack of a dark period and repeated dilution cycles could
likely have imparted additional stress due to lack of culture acclimation
and lack of recovery during the dark periods, impeding growth at tem-
perature extremes.
3.24. Picochlorum soloecismus DOE101
Picochlorum soloecismus DOE101 was originally isolated at Los Ala-
mos National Laboratory from a contaminated Nannochloropsis
(Microchloropsis) salina CCMP1776 culture [81]. We conrmed the
genus as Picochlorum via 18S identication. The strain was non-axenic
with low levels of Alphaproteobacteria (Rhodobacter, Mesorhizobium,
Sultobacter, Henriciella, Nitratireductor) and Flavobacteria (Muricauda),
which was determined by 16S identication. The maximum specic
growth rate, determined at 25 ◦C in DISCOVR medium (Table S1), was
4.7 day
−1
at 5 PSU and 15 PSU salinity, and slightly lower, 4.2 day
−1
, at
35 PSU salinity, generally indicating that this strain is euryhaline
(Fig. 29A). At 15 PSU salinity, vigorous growth occurred within a tem-
perature tolerance range of 10 to 35 ◦C, and the optimum maximum
specic growth rate (5.3 day
−1
) was measured at 28 ◦C (Fig. 29B),
indicating that this strain should be suitable for spring, summer, and fall
season outdoor pond cultivation (Table 2 in Section 4). The activation
energy (E
a
), determined over a temperature range of 10.5–28 ◦C, was
1277 J mol
−1
(Fig. S17).
In earlier work related to parameterizing this strain for biomass
growth modeling [18], the maximum specic growth rate was measured
as a function of temperature in f/2 medium at 35 ppt salinity. While the
temperature optimum was similarly around 30 ◦C, the maximum spe-
cic growth rates were signicantly lower compared to this study,
possibly as a result of using a different medium (f/2 vs. DISCOVR) or due
to the use of continuous light with no dark recovery cycle. Gonzalez-
AB
C
Monoraphidium sp. MONOR1
Fig. 23. Maximum specic growth rate of Monoraphidium sp. MONOR1 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at the optimum
salinity of 15 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3).
M. Huesemann et al.
Algal Research 71 (2023) 102996
27
Esquer et al. [81] measured the specic growth rate as a function of
salinity at 25 ◦C, and observed poor growth (0.3 day
−1
) in freshwater
medium (0.35 ppt) but vigorous growth (0.7 to 0.9 day
−1
) in salinities
ranging from 3.5 to 70 ppt. No sustainable growth was observed at 140
ppt.
3.25. Porphyridium cruentum CCMP675
Porphyridium cruentum CCMP675 (Porphyridium cruentum WTP, Por-
phyridium purpureum) was obtained from the National Center for Marine
Algae and Microbiota. The strain was originally collected from a pier of
the Scripps Institute of Oceanography in La Jolla, California. We
conrmed the genus as Porphyridium via 18S identication. The strain
was non-axenic with low levels of an Alphaproteobacterial cohort
(Rhodobacter), which was determined by 16S identication. The
maximum specic growth rate, determined at 25 ◦C in DISCOVR me-
dium (Table S1), was 1.6, 2.0, and 1.8 day
−1
at 5, 15, and 35 PSU
salinity, respectively (Fig. 30A). The difference in maximum specic
growth rates at these three salinities was not statistically signicant. At
35 PSU salinity, good growth occurred within a temperature tolerance
range of 17 to 36 ◦C, and the optimum maximum specic growth rate
(2.8 day
−1
) was measured at 24 ◦C (Fig. 30B), indicating that this strain
should be suitable for spring, summer, and fall season outdoor pond
cultivation (Table 2 in Section 4).
There is no published temperature or salinity tolerance data for
Porphyridium cruentum CCMP675. However, Cohen et al. [82] measured
the growth rate of P. cruentum 1380-1F, also collected from La Jolla,
California, and maintained in the Catalogue of strains at the Culture
Collection of Algae at the University of G¨
ottingen, Germany (SAG).
Cohen et al. [82] determined growth rates at 20, 25, and 30 ◦C and found
optimal growth rates at 25 ◦C of 1.2 day
−1
, close to the optimal tem-
perature observed for the CCMP675 strain of P. cruentum assessed in this
study. Further, Dermoun et al. [83] also measured maximum specic
growth rates of P. cruentum 1380-1F at temperatures ranging from 5 to
35 ◦C, with optimum values (ca. 1.37 day
−1
) occurring between 20 and
25 ◦C, which is similar to the temperature optimum observed in our
study. Oh et al. [84] measured a specic growth rate of 0.98 day
−1
at 30
◦C for Porphyridium cruentum from a Korean culture collection, which is
signicantly lower than the maximum specic growth rate (ca. 2.45
day
−1
at 29.1 ◦C) measured in our study, most likely because their
incident light intensity (10–25
μ
mol m
−2
s
−1
) was signicantly below
saturation.
3.26. Scenedesmus acutus LRB-AZ-0401
Scenedesmus acutus LRB-AZ-0401 was used extensively in efforts to
understand biochemical shifts and biomass conversions to fuels and
currently serves as the compositional benchmark strain for the DOE's
Algae State of Technology [85,86]. We conrmed the genus as Scene-
desmus via 18S identication. The strain was non-axenic with a multi-
bacterial cohort with unidentied members of the Alphaproteobac-
teria, which was determined by 16S identication. The maximum
AB
C
Nannochloropsis oceanica CCAP 849/10
Fig. 24. Maximum specic growth rate of Nannochloropsis oceanica CCAP 849/10 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at the
salinity of 35 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3).
M. Huesemann et al.
Algal Research 71 (2023) 102996
28
AB
C
Oscillatoria cf. priestleyi CCMEE5020.1-1
Fig. 25. Maximum specic growth rate of Oscillatoria cf. priestleyi CCMEE5020.1-1 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at the
optimum salinity of 5 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3, with the exception of
μ
max
measurements at 5 PSU and 18.5 ◦C where n
=2. Growth at 18.5 ◦C was unstable).
M. Huesemann et al.
Algal Research 71 (2023) 102996
29
specic growth rate, determined at 25 ◦C in DISCOVR medium (Table
S1), was highest at 5 PSU salinity (4.0 day
−1
), followed by 2.6 day
−1
at
15 PSU, and 0.8 day
−1
at 35 PSU (Fig. 31A), indicating that this strain
can be cultivated in brackish-water growth medium. At 5 PSU salinity,
vigorous growth occurred within a temperature tolerance range of 11 to
41 ◦C, and the optimum maximum specic growth rate (4.4 day
−1
) was
measured at 35 ◦C (Fig. 31B), indicating that this strain should be
suitable for spring, summer, and fall season outdoor pond cultivation
(Table 2 in Section 4). The activation energy (E
a
), determined over a
temperature range of 4.3–29.7 ◦C, was 872 J mol
−1
(Fig. S18).
There is no published temperature or salinity tolerance data for
Scenedesmus acutus LRB-AZ-0401. However, an extensive literature re-
view of published temperature and salinity tolerance data of other
Acutodesmus (Scenedesmus) obliquus strains is given in Section 3.28
below for Scenedesmus obliquus UTEX393.
3.27. Scenedesmus obliquus DOE 0152.z
Scenedesmus obliquus DOE 0152.z (Acutodesmus obliquus DOE 0152.z,
Scenedesmus obliquus UTEX3031) was isolated by Dr. Juergen Polle
(Brooklyn College, New York) from water in a turtle tank (NY) as part of
the U.S. Department of Energy National Consortium of Advanced
Biofuels and Bioproducts (NAABB) [6]. This strain was deposited in the
University of Texas Culture Collection of Algae as Scenedesmus obliquus
UTEX3031. We conrmed the genus as Scenedesmus via 18S rDNA
identication. The strain was non-axenic with an unidentied Alphap-
roteobacterial cohort, which was determined by 16S rDNA identica-
tion. The maximum specic growth rate, determined at 25 ◦C in
DISCOVR medium (Table S1), declined with increasing salinity, from
3.4 day
−1
at 5 PSU to 0.7 day
−1
at 35 PSU (Fig. 32A). This strain is highly
salt sensitive and carotogenic, generally turning an orange-brown color
under salt stress. At 5 PSU salinity, healthy growth occurred within a
temperature tolerance range of 4 to 35 ◦C, and the optimum maximum
specic growth rate (4.4 day
−1
) was measured at 29 ◦C (Fig. 32B),
indicating that this strain should be suitable for spring, summer, and fall
season outdoor pond cultivation (Table 2 in Section 4). The activation
energy (E
a
), determined over a temperature range of 3.5–35.5 ◦C, was
782 J mol
−1
(Fig. S19).
Ogden et al. [8], when culturing Scenedesmus obliquus DOE 0152.z in
standard BG-11 medium, observed similar temperature and salinity
tolerance proles as in this study. An extensive literature review of
published temperature and salinity tolerance data of other Scenedesmus
obliquus strains is given below in Section 3.28 for Scenedesmus obliquus
UTEX393.
AB
C
Picochlorum celeri TG2-WT-CSM/EMRE
Fig. 26. Maximum specic growth rate of Picochlorum celeri TG2-WT-CSM/EMRE as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at
salinity of 35 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3). Some of these data were also published by Krishnan et al. [118].
M. Huesemann et al.
Algal Research 71 (2023) 102996
30
3.28. Scenedesmus obliquus UTEX393
Scenedesmus obliquus UTEX393 (Acutodesmus obliquus UTEX393,
Tetradesmus obliquus UTEX393, Acutodesmus obliquus CCAP 276/6A,
Acutodesmus obliquus SAG276-6, Scenedesmus sp. D3) was obtained from
the University of Texas (UTEX) Culture Collection of Algae. The earliest
description of this strain was in the discovery of a pathway for photo-
chemical production of hydrogen by Hans Gaffron and Jack Rubin [87].
It is unclear where this strain originated, but it is held in several culture
collections around the world. Additionally, this strain was recently
sequenced by Carreres et al. [88]. We conrmed the genus as Scene-
desmus via 18S rDNA identication. The strain was axenic with no
detected bacterial cohort, as determined by 16S rDNA identication.
The maximum specic growth rate, determined at 25 ◦C in DISCOVR
medium (Table S1), was higher at 5 PSU salinity (4.0 day
−1
) than at 0.4
PSU salinity (3.4 day
−1
) (Fig. 32A), indicating that this strain can be
cultivated in brackish-water growth medium. Greater than 5 PSU
salinity should be avoided, given that the maximum specic growth rate
declined to 2.6 day
−1
at 15 PSU salinity. No growth was observed at 35
PSU salinity. At 5 PSU salinity, healthy growth occurred within a tem-
perature tolerance range of 10 to 39 ◦C, and the optimum maximum
specic growth rate (4.6 day
−1
) was measured at 34 ◦C (Fig. 32B),
indicating that this strain should be suitable for spring, summer, and fall
season outdoor pond cultivation (Table 2 in Section 4). The activation
energy (E
a
), determined over a temperature range of 10.5–22.6 ◦C, was
869 J mol
−1
(Fig. S20).
There is no published temperature or salinity tolerance data for
Scenedesmus obliquus UTEX393. However, there is no shortage of specic
growth rate data for other Scenedesmus (Acutodesmus) obliquus strains,
presented here in chronological order. Martinez et al. [89] measured the
specic growth rate of Scendesmus obliquus 276-3a, obtained from the
Algae Collection of the University of G¨
ottingen (Germany), in waste-
water medium at 20, 25, 30, and 35 ◦C, and observed the optimum
growth rate of 1.05 day
−1
at 30 ◦C. Hodaifa et al. [90] cultured Scene-
desmus obliquus CCAP 276/3A in a photobioreactor with 298
μ
mol m
−2
s
−1
incident light intensity with temperatures ranging from 15 to 35 ◦C,
and measured an optimum growth rate of 0.58 day
−1
at ca. 29 ◦C. Ruiz-
Marin et al. [91] grew a locally isolated Scenedesmus obliquus strain in
articial wastewater with 135
μ
mol m
−2
s
−1
photon ux density and
measured a specic growth rate of 0.4 day
−1
at 25 ◦C. Guedes et al. [92]
tested the growth of Scenedesmus obliquus M2-1 in a factorial design as a
function of pH (6,7,8) and temperature (20, 25, 30 ◦C) using an incident
light intensity of 93
μ
mol m
−2
s
−1
, and observed the highest specic
growth rate of 0.29 day
−1
at 30 ◦C and pH 6. Xu et al. [93] measured
AB
C
Picochlorum oklahomensis CCMP2329
Fig. 27. Maximum specic growth rate of Picochlorum oklahomensis CCMP2329 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at salinity
of 35 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3).
M. Huesemann et al.
Algal Research 71 (2023) 102996
31
specic growth rates of Scenedesmus obliquus FACHB416 with 49
μ
mol
m
−2
s
−1
illumination at 14, 20, and 30 ◦C, and observed optimum
growth (0.24 day
−1
) at 30 ◦C. Feng et al. [56] reported a Scenedesmus
obliquus specic growth rate of 1.68 day
−1
at 25 ◦C, using an incident
light intensity of 250
μ
mol m
−2
s
−1
. Ansari et al. [94] grew Scenedesmus
obliquus in wastewater at 25 ◦C with 120
μ
mol m
−2
s
−1
illumination and
observed a specic growth rate of 0.42 day
−1
. Depr´
a et al. [95]
measured a specic growth rate of 0.96 day
−1
at 26 ◦C for Scenedesmus
obliquus CPCC05, employing a photon ux density of 150
μ
mol m
−2
s
−1
.
Trivedi et al. [96] cultured Scenedesmus obliquus CCAP276/3A at 30 ◦C
in a at panel photobioreactor (2.4 cm light path) using four photon ux
densities (50, 100, 150, and 200
μ
mol m
−2
s
−1
) and measured the
highest growth rate (0.35 day
−1
) at 150
μ
mol m
−2
s
−1
. Whitton et al.
[97] growth rate of 1.49 day
−1
at 20 ◦C for Scenedesmus obliquus 276/42,
using a photo ux density of 200
μ
mol m
−2
s
−1
. In conclusion, most of
these studies found optimum growth occurring around 30 ◦C, very
similar to our ndings. However, all of these studies reported much
lower specic growth rates, sometimes <1/10 of the growth rate
observed in our experiments, where special efforts were made to mea-
sure maximum specic growth rates. Possible reasons for this signicant
discrepancy are (a) strain specic differences, (b) use of sub-saturating
incident light intensities, (c) dense, self-shading cultures, (d) non-
optimal or inhibitory media (such as wastewater), and (e) potential
nutrient (N,P), trace-element, and CO
2
limitations.
There are several studies on salinity tolerance of Scenedesmus obli-
quus. Kaewkannetra et al. [98] measured the growth of Scenedesmus
obliquus in media containing NaCl ranging from 0 to 3 M and observed
optimum growth at 0 and 0.05 M NaCl (Note: 1 M NaCl =58.44 g/L,
thus 0.05 M =2.9 g/L or 2.9 ppt), with signicant reduction in growth
occurring at 0.3 M NaCl (ca. 17.5 ppt). No stable growth was observed at
concentrations of 0.6 M NaCl (ca. 35 ppt) or above. Pandit et al. [52]
cultured Scenedesmus obliquus KY741858 in media containing 0, 0.06,
0.08, 0.1, 0.3, and 0.4 M NaCl (ca. 0, 3.5, 4.7, 5.8, 17.5, and 23 pp.
salinity, respectively) and reported specic growth rates of 0.096, 0.123,
0.120, 0.114, 0.107, and 0.089 day
−1
, respectively. Similarly, Ji et al.
[99] grew Scenedesmus obliquus XJ002 in media with 0, 0.01, 0.1, 0.15,
and 0.2 M NaCl (ca. 0, 5.8, 8.8, and 11.7 ppt salinity, respectively) and
measured specic growth rates of 0.146, 0.132, 0.086, 0.071, and 0.056
day
−1
, respectively. In conclusion, these studies all found that optimum
growth occurs in low salinity (0–6 ppt) media, similar to our ndings
(Fig. 33).
3.29. Scenedesmus rubescens NREL46B-D3
Scenedesmus rubescens NREL46B-D3 (Halochlorella rubescens, Chlor-
ella fusca var. rubescens, Chlorella emersonii var. rubescens) was originally
C
AB
Fig. 28. Maximum specic growth rate of Picochlorum renovo NREL 39-A8 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at the
optimum salinity of 35 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3, with the exception of the
μ
max
measurement at 44.8 ◦C where n =1).
M. Huesemann et al.
Algal Research 71 (2023) 102996
32
isolated from a bioprospecting effort in the Southwest United States [5].
Specically, S. rubescens NREL46B-D3 was isolated from New Belgium
Brewery wastewater in Fort Collins, CO. The strains in the bio-
prospecting effort were then downselected using a screen of NREL's
resultant algal culture collection [47]. The genus and species were
determined via 18S sequencing by Dahlin et al. [47]. The strain was
assumed to be non-axenic, with a bacterial cohort consisting of Pro-
teobacteria, Betaproteobacteria, and Burkholderiales. This strain is
apparently euryhaline with maximum specic growth rates, determined
at 25 ◦C in DISCOVR medium (Table S1), remaining roughly the same
(2.6 to 2.8 day
−1
) over the entire salinity range (5 to 35 PSU) tested
(Fig. 34A). At 35 PSU salinity, vigorous growth occurred within a tem-
perature tolerance range of 11 to 44 ◦C, and the optimum maximum
specic growth rate (5.5 day
−1
) was measured at 34 ◦C (Fig. 34B),
indicating that this strain should be suitable for spring, summer, and fall
season outdoor pond cultivation (Table 2 in Section 4). The activation
energy (E
a
), determined over a temperature range of 10.9–28.5 ◦C, was
553 J mol
−1
(Fig. S21).
Temperature and salinity tolerance data for Scenedesmus rubescens
NREL46B-D3 was assessed by Dahlin [48], which determined a tem-
perature tolerance range from 10 to 30 ◦C and a broad salinity tolerance
from 17.5 ppt to 57.5 ppt. In the current characterization, we
determined the temperature optimum around 34 ◦C, which is signi-
cantly higher than the optimum of 30 ◦C observed by Dahlin [48].
Further, we observed consistent and stable growth even at temperatures
of ca. 39 and 44 ◦C, whereas Dahlin [48] reported no growth above 30
◦C. This is most likely due to different photoperiods used, which may
exacerbate temperature stress. The current study uses a 12:12 photo-
period to allow a dark recovery period as well as cell acclimation over
repeated dilution cycles, whereas Dahlin used constant (24 h day
−1
)
light in the reported characterization.
3.30. Scenedesmus sp. IITRIND2
Scenedesmus sp. IITRIND2 (Gene bank accession KT932960) was
isolated from a freshwater lake in Bhagwanpur, Uttarakhand, India
[100]. No 16S or 18S analyses were conducted, and the strain was
assumed to be non-axenic. The maximum specic growth rate, deter-
mined at 25 ◦C in DISCOVR medium (Table S1), was highest at 5 PSU
salinity (3.8 day
−1
), followed by 3.1 day
−1
at 15 PSU, and 1.2 day
−1
at
35 PSU (Fig. 35A), indicating that this strain can be cultivated in
brackish-water growth media. At 5 PSU salinity, vigorous growth
occurred within a temperature tolerance range of 12 to 46 ◦C, and the
optimum maximum specic growth rate (5 day
−1
) was measured at 35
AB
C
Picochlorum soleocismus DOE101
Fig. 29. Maximum specic growth rate of Picochlorum soloecismus DOE101 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at the
optimum salinity of 15 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3, with the exception of the
μ
max
measurements at 34.6 ◦C where n =2).
M. Huesemann et al.
Algal Research 71 (2023) 102996
33
◦C (Fig. 35B), indicating that this strain should be suitable for spring,
summer, and fall season outdoor pond cultivation (Table 2 in Section 4).
The activation energy (E
a
), determined over a temperature range of
12.3–29.4 ◦C, was 1317 J mol
−1
(Fig. S22).
There are no published temperature tolerance data for Scenedesmus
sp. IITRIND2. However, Arora et al. [101] measured biomass growth in
seawater media adjusted to four different salinities (0, 8.75, 17.5, and
35 g/L). It was found that while the exponential growth rate was similar
in all four media and thus not affected by salinity, the nal biomass
yield, measured as ash-free dry weight per L, declined with increasing
salinity, indicating a negative impact of salinity on biomass growth, as
was observed in our study.
3.31. Stichococcus minor CCMP819
Stichococcus minor CCMP819 was obtained from the National Center
for Marine Algae and Microbiota (NCMA). S. minor CCMP819 was
originally collected from a lagoon in Key Largo, Florida, by R. Lewin in
1985 and was noted as heat tolerant and recommended as a potentially
promising summer strain by J. Sexton, the former curator of the NCMA
algal collection. We were unable to conrm the genus as Stichococcus via
18S identication. The strain was non-axenic with an Alphaproteo-
bacterial cohort, which was determined by 16S identication. Robust
growth was observed in the DISCOVR medium (Table S1), over a wide
range of salinities (5 to 35 PSU) (Fig. 36A). At 25 ◦C, the highest
maximum specic growth rate (5.6 day
−1
) was measured at 15 PSU
salinity. At this salinity (15 PSU), vigorous growth occurred within a
temperature tolerance range of 16 to 40 ◦C, and the optimum maximum
specic growth rate (6 day
−1
) was measured at 34 ◦C (Fig. 36B), indi-
cating that this strain should be suitable for spring, summer, and fall
season outdoor pond cultivation (Table 2 in Section 4). The activation
energy (E
a
), determined over a temperature range of 17.8–35.2 ◦C, was
646 J mol
−1
(Fig. S23).
There is no published temperature or salinity tolerance data for Sti-
chococcus minor CCMP819.
However, Chen et al. [102] measured the temperature tolerance of
two psychrotolerant Stichococcus species, S. bacillaris NJ-10 and
S. minutus NJ-17, isolated from rock surfaces in Antarctica, as well as for
a temperate Stichococcus strain, S. bacillaris FACHB753. Both Antarctic
strains grew at temperatures between 4 and 25 ◦C. The temperate strain
was able to grow above 30 ◦C but could not survive at 4 ◦C, indicating
that the temperature tolerance range of Stichococcus strains depends on
the climate of the geographic location from where they were originally
isolated. Compared to the maximum specic growth rates measured in
our study, Chen et al. [102] reported their highest specic growth rate at
0.23 day
−1
(at 25 ◦C), which is at least one order of magnitude lower
AB
C
Porphyridium cruentum CCMP675
Fig. 30. Maximum specic growth rate of Porphyridium cruentum CCMP675 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at salinity of
35 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3).
M. Huesemann et al.
Algal Research 71 (2023) 102996
34
AB
C
Scenedesmus acutus LRB-AZ-0401
Fig. 31. Maximum specic growth rate of Scenedesmus acutus LRB-AZ-0401 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at salinity of
5 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3, with the exception of the
μ
max
measurements at 35 PSU where n =2).
M. Huesemann et al.
Algal Research 71 (2023) 102996
35
AB
C
Scenedesmus obliquus DOE0152.z
Fig. 32. Maximum specic growth rate of Scenedesmus obliquus DOE 0152.z as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at the
optimum salinity of 5 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3, with the exception of the
μ
max
measurement at 3.5 ◦C where n =2).
M. Huesemann et al.
Algal Research 71 (2023) 102996
36
than for our Stichococcus minor CCMP819 strain. Possible reasons for this
discrepancy are that Chen et al. [102] measured their specic growth
rates for different Stichococcus species, and that their cultures received
sub-saturating incident light intensities (100
μ
mol m
−2
s
−1
).
3.32. Stichococcus minutus CCALA727
Stichococcus minutus CCALA727 was obtained from the Culture
Collection of Phototrophic Organisms (CCALA). The strain was collected
from subglacial soil of the Midre Lovnbreen glacier on the Svalbard is-
land archipelago located within the Arctic circle and deposited in the
CCALA culture collection in 1970. We were unable to sequence the 18S
libraries prepared for Stichococcus minutus CCALA727 due to an unex-
pectedly large size of amplicons. The amplicon library most likely failed
due to non-specic binding, creating multiple peaks, making it difcult
to sequence. Robust growth was observed in DISCOVR medium (Table
S1), over a wide range of salinities (5 to 35 PSU) (Fig. 37A). At 25 ◦C, the
highest maximum specic growth rate (3.0 day
−1
) was measured at 15
PSU salinity. At this salinity (15 PSU), vigorous growth occurred within
a temperature tolerance range of 12 to 28 ◦C, and the optimum
maximum specic growth rate (3.6 day
−1
) was measured at 23 ◦C
(Fig. 37B), indicating that this strain should be suitable for spring and
fall season outdoor pond cultivation (Table 2 in Section 4). The activa-
tion energy (E
a
), determined over a temperature range of 11.4–23.6 ◦C,
was 575 J mol
−1
(Fig. S24).
There is no published temperature or salinity tolerance data for Sti-
chococcus minutus CCALA727. However, Chen et al. [102] measured the
temperature tolerance of Stichococcus minutus NJ-17, isolated from rock
surfaces in Antarctica, and reported specic growth rates increasing
from 0.08 day
−1
at 4 ◦C to 0.2 day
−1
at 25 ◦C, with no growth occurring
at 30 ◦C. These specic growth rates are about one order of magnitude
lower than measured in our study, most likely due to several key factors
in the experimental methodology, including the sub-saturating photon
ux density (100
μ
mol m
−2
s
−1
), lack of supplemental carbon dioxide,
and optical density measurements taken only once per day.
3.33. Synechococcus elongatus UTEX2973.1
Synechococcus elongatus UTEX2973.1 is a cyanobacterium obtained
from the University of Texas (UTEX) Culture Collection of Algae. Syn-
echococcus elongatus 2973.1 is mesophilic sub-isolate from Synechococcus
elongatus 2973. S. elongatus 2973 itself is described as a thermophilic
mutant of Synechococcus leopoliensis UTEX625, which was originally
isolated from Waller Creek in Austin, TX and studied extensively by
Kratz and Myers [103] and previously identied in these works as
Anacystis nidulans, strain Tx20. We conrmed the genus as Synecho-
coccus via 16S identication. The strain is non-axenic with a multi-
bacterial cohort including members of the Betaproteobacteria, Bur-
kholderiales, and Flavobacteria, which was determined by 16S identi-
cation. Robust growth was observed in the DISCOVR medium (Table
S1) at 5 and 15 PSU salinity, but no stable growth occurred at 35 PSU
(Fig. 38A). At 25 ◦C, the highest maximum specic growth rate (3.3
day
−1
) was measured at 5 PSU salinity, which was signicantly (statis-
tically) greater than the 2.7 day
−1
observed at 15 PSU. At the optimum
salinity (5 PSU), vigorous growth occurred within a temperature toler-
ance range of 19 to 39 ◦C, and the optimum maximum specic growth
AB
C
Scenedesmus obliquus UTEX393
Fig. 33. Maximum specic growth rate of Scenedesmus obliquus UTEX393 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at the optimum
salinity of 5 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥4).
M. Huesemann et al.
Algal Research 71 (2023) 102996
37
AB
C
Scenedesmus rubescens NREL46B-D3
Fig. 34. Maximum specic growth rate of Scenedesmus rubescens NREL46B-D3 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at the
optimum salinity of 35 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3, with the exception of
μ
max
measurements at 38.9 ◦C and 44.1 ◦C where
n =2).
M. Huesemann et al.
Algal Research 71 (2023) 102996
38
rate (5.5 day
−1
) was measured at 39 ◦C (Fig. 38B), indicating that this
strain should be suitable for summer season outdoor pond cultivation
(Table 2 in Section 4). The activation energy (E
a
), determined over a
temperature range of 11.4–23.8 ◦C, was 1970 J mol
−1
(Fig. S25).
There are no published salinity tolerance data for Synechococcus
elongatus UTEX2973.1 However, Kratz and Myers [103] measured the
maximum specic growth rate of the parent strain Synechococcus leo-
poliensis UTEX625 (at that time still named Anacystis nidulans) as a
function of temperature (25 to 45 ◦C), and reported the fastest doubling
time of 2 h at 41 ◦C, “the highest growth rate yet reported for an alga” (in
1955). This strain was also exceptionally heat tolerant, growing still
rapidly at 45 ◦C. Yu et al. [104] found that the strain originally deposited
by Kratz and Myers in the UTEX Culture Collection, i.e., Synechococcus
leopoliensis UTEX625, had over the years lost its ability to grow at 38 ◦C,
and they subsequently isolated a single colony able to grow at 38 ◦C. The
resulting culture was then deposited as Synechococcus elongatus
UTEX2973. Yu et al. [104] conrmed the exceptionally high growth rate
of this strain, which had a doubling time of only 2.1 h at 41 ◦C under
continuous light (500
μ
mol m
−2
s
−1
), the fastest growth rate (7.9 day
−1
)
compared to four other Synechoccoci strains that were studied.
3.34. Tetraselmis striata LANL1001
Tetraselmis striata LANL1001 (Platymonas) was isolated from a
contaminated outdoor pond culture of Nannochloropis salina CCMP1776
using ow cytometry via size exclusion [119]. We conrmed the genus
as Tetraselmis via 18S identication. The strain was non-axenic with an
unidentied bacterial cohort (Alphaproteobacteria), which was deter-
mined by 16S identication. The maximum specic growth rate,
determined at 25 ◦C in DISCOVR medium (Table S1), increased with
increasing salinity, from 3.7 day
−1
at 5 PSU to 4.6 day
−1
at 35 PSU
(Fig. 39A). At 35 PSU salinity, vigorous growth occurred within a tem-
perature tolerance range of 4 to 36 ◦C, and the optimum maximum
specic growth rate (4.4 day
−1
) was measured at 31 ◦C (Fig. 39B),
indicating that this strain should be suitable for fall, winter, and spring
season outdoor pond cultivation (Table 2 in Section 4). The activation
energy (E
a
), determined over a temperature range of 4.3–30.6 ◦C, was
839 J mol
−1
(Fig. S26).
Temperature and salinity tolerance data for Tetraselmis striata
LANL1001 were reported in the DOE-Regional Algae Feedstock Testbed
(RAFT) project closeout report [8]. Ogden et al. [8] showed a temper-
ature tolerance from ca. 15 to 35 ◦C, which generally agrees with the
data presented in this study, however the optimum temperature deter-
mined in Ogden is ca. 23 ◦C, whereas the current study determined that
AB
C
Scenedesmus sp. IITRIND2
Fig. 35. Maximum specic growth rate of Scenedesmus sp. IITRIND2 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at the optimum
salinity of 5 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3, with the exception of
μ
max
measurements at 45.6 ◦C where n =2).
M. Huesemann et al.
Algal Research 71 (2023) 102996
39
AB
C
Schococcus minor CCMP819
Fig. 36. Maximum specic growth rate of Stichococcus minor CCMP819 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at the optimum
salinity of 15 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3, with the exception of the
μ
max
measurement at 9.7 ◦C where n =2).
M. Huesemann et al.
Algal Research 71 (2023) 102996
40
AB
C
Schococcus minutus CCALA727
Fig. 37. Maximum specic growth rate of Stichococcus minutus CCALA727 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at the optimum
salinity of 15 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3).
M. Huesemann et al.
Algal Research 71 (2023) 102996
41
AB
C
Fig. 38. Maximum specic growth rate of Synechococcus elongatus UTEX2973.1 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at the
optimum salinity of 5 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3).
M. Huesemann et al.
Algal Research 71 (2023) 102996
42
the optimum growth for this strain was at 30 ◦C. Salinity tolerance re-
ported by Ogden et al. [8] ranges from 5 to 40 ppt, with an optimum
from 17 to 35 ppt. The wide salinity tolerance is conrmed by the cur-
rent study, showing statistically indistinguishable growth at 15 and 35
ppt. A classic report on a Hawaiian isolate of Tetraselmis (Platymonas)
was published by DOE's Solar Energy Research Institute [105], which
determined that Tetraselmis grew well in outdoor pond conditions under
a range of salinities from 15 to 35 ppt and at temperatures uctuating
from 20 to 34 ◦C. These values agree well with our temperature and
salinity characterizations.
Holdt et al. [106] cultured Tetraselmis striata CCAP 66/5 at pH 8 in a
bubble column reactor in 25 ppt salinity f/2 medium, and measured a
specic growth rate of 3.12 day
−1
at 20 ◦C, which is about the same
magnitude as was observed in our study. By contrast, Imamoglu et al.
[107] grew Tetraselmis striata in asks in f/2 medium (ca. 39 ppt
salinity) with 56
μ
mol m
2
-sec illumination and measured a specic
growth rate of only 0.23 day
−1
at 25.5 ◦C, which is about 17 times lower
than observed in our experiments. In addition to possible strain specic
differences, the sub-saturation light intensities used by Imamoglu et al.
[107] are most likely the reason for this discrepancy.
Regarding salinity tolerance, Abu-Rezq et al. [108] observed robust
growth of Tetraselmis sp. in salinities ranging from 5 to 40 ppt, with
optimum growth occurring between 25 and 35 ppt, similar to what was
observed in our study. Khatoon et al. [109] cultured Tetraselmis sp. in
continuous light (12
μ
mol m
−2
s
−1
) at 25 ◦C at 20, 30, and 40 ppt salinity
media, and observed the highest specic growth rate (0.73 day
−1
) at 30
ppt. Their specic growth rate value is much lower than in this study at
35 ppt (4.6 day
−1
), most likely due to the very low, sub-saturating light
intensities that were used in their experiments. Finally, Kim et al. [110]
determined growth rates of Tetraselmis sp. KCTC 12236BP in salinities
ranging from 0 to 70 ppt. Robust growth was observed at all tested sa-
linities, with optimum growth occurring at 22 ppt. No growth could be
maintained in freshwater (0 ppt).
3.35. Tisochrysis lutea CCMP1324
Tisochrysis lutea CCMP1324 (Tahitian strain of Isochrysis (T-Iso),
NEPCC601, CCAP927/14, CS-177) was obtained from the National
Center for Marine Algae and Microbiota. This strain was originally
collected from the South Pacic, specically Mataiva, Tahiti. This strain
is widely used as an aquaculture feed and was initially presumed to be a
strain of Isochrysis galbana, but was later reclassied as a distinct genus
[111]. We conrmed the genus as Tisochrysis via 18S identication. The
strain was non-axenic with a multi-bacterial cohort consisting of
Alphaproteobacteria, Gammaproteobacteria, and Sphingobacteriales,
which were determined by 16S identication. This strain required
thiamin and/or biotin in addition to cyanocobalamin to grow stably in
ask cultivation studies. Cultivation with only cyanocobalamin was
unstable. The maximum specic growth rate, determined at 25 ◦C in
DISCOVR medium (Table S1), increased from 0.6 day
−1
at 5 PSU salinity
to 1.1 day
−1
at 15 PSU salinity (Fig. 40A). Increasing the salinity to 35
PSU resulted in a slightly lower, but not statistically signicant,
maximum specic growth rate of 0.9 day
−1
(Fig. 40A). At 35 PSU
salinity, vigorous growth occurred within a temperature tolerance range
of 17 to 35 ◦C, and the optimum maximum specic growth rate (3
day
−1
) was measured at 29 ◦C (Fig. 40B), indicating that this strain
AB
C
Tetraselmis striata LANL1001
Fig. 39. Maximum specic growth rate of Tetraselmis striata LANL1001 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at the optimum
salinity of 35 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3).
M. Huesemann et al.
Algal Research 71 (2023) 102996
43
AB
C
Tisochrysis lutea CCMP1324
Fig. 40. Maximum specic growth rate of Tisochrysis lutea CCMP1324 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at salinity of 35
PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3).
M. Huesemann et al.
Algal Research 71 (2023) 102996
44
AB
C
Tribonema minus UTEXB3156
Fig. 41. Maximum specic growth rate of Tribonema minus UTEXB3156 as a function of salinity (PSU) at 25
◦C (A) and as a function of temperature at the optimum
salinity of 5 PSU (B). Micrograph (C). Error bars are one standard deviation (n ≥3, with the exception of
μ
max
measurements at 35 PSU and 34.7 ◦C where n =1).
M. Huesemann et al.
Algal Research 71 (2023) 102996
45
should be suitable for spring, summer, and fall season outdoor pond
cultivation (Table 2 in Section 4).
There are no published salinity tolerance data for Tisochrysis lutea
CCMP1324. However, da Costa et al. [112] measured the specic
growth rate of this strain at 20–23 ◦C and 34–35 PSU (ca. 35 ppt salinity)
and reported a value of 1.2 ±0.1 day
−1
, which is slightly less than
determined in this study.
3.36. Tribonema minus UTEXB3156
Tribonema minus UTEXB3156 was isolated by research staff from
MicroBio Engineering, Inc. from a polyculture growing in reclaimed
wastewater in San Luis Obispo, California. The isolated strain was
identied as Tribonema minus and provided by Dr. Aubrey Davis [113].
The annotated genome sequence for this strain has been published by
Mahan et al. [114]. No 16S or 18S analyses were conducted, and the
strain was assumed to be non-axenic with an unknown bacterial cohort.
Due to the lamentous nature of this strain, ca. 0.5 g of 3 mm sterile
glass beads were added to the shaker asks during cultivation to limit
lament size and facilitate optical density measurements. The maximum
specic growth rate, determined at 25 ◦C in DISCOVR medium (Table
S1), declined with increasing salinity, from 2.9 day
−1
at 5 PSU to 2.2
day
−1
at 15 PSU (Fig. 41A). Only very slow growth (0.2 day
−1
) was
observed at 35 PSU salinity. At 5 PSU salinity, healthy growth occurred
within a temperature tolerance range of 12 to 28 ◦C, and the optimum
maximum specic growth rate (3 day
−1
) was measured at 23 ◦C
(Fig. 41B), indicating that this strain should be suitable for spring and
fall season outdoor pond cultivation (Table 2 in Section 4).
There is no published temperature or salinity tolerance data for
Tribonema minus UTEXB3156. However, the temperature tolerance of
another Tribonema minus strain, obtained from the Culture Collection of
Algae at G¨
ottingen University in Germany, was determined by Wang
et al. [115] who found that this strain grew well between 5 and 35 ◦C, a
wider range than observed in our study. Butterwick et al. [116]
measured the growth of a Tribonema sp. isolated from Farnmoor reser-
voir near Oxford, UK, in cultures with temperatures ranging from 2 to
35 ◦C, and observed the highest division rates (ca. 0.55 divisions/day,
equivalent to a specic growth rate of ca. 0.38 day
−1
) between 14 and
25 ◦C. No growth was observed at 30 ◦C or above. Huo et al. [117]
cultured an unidentied Tribonema sp. in petrochemical wastewater in a
photobioreactor with 300
μ
mol m
2
-sec lighting at 25 ◦C, and observed a
specic growth rate of 0.47 day
−1
, about six times lower than in our
study, which is most likely due to sub-saturating light intensity condi-
tions and possible inhibition by petrochemicals.
4. Summary and conclusions
To identify high productivity strains for microalgal biofuels gener-
ation, 38 of the initial 55 strains were characterized in terms of their
salinity and temperature tolerance to identify, respectively, the most
suitable medium salinity and the best growing season for outdoor pond
cultivation in Arizona. Table 2 summarizes the recommended growing
Table 2
Suitable growing seasons (SP =spring, SU =summer, FA =fall, WI =winter) in Arizona outdoor ponds (see Fig. 1), optimum
medium salinity, and highest value of the maximum specic growth rate observed at respective temperature for each strain
evaluated (see Section 3 for details). All strains marked with an asterisk were selected for subsequent Tier 2 screening in the LEAPS
climate simulation photobioreactors ([2] this issue).
Alga strain Growing seasons Opt. salinity
PSU
μ
max
(T
opt
)
d
−1
(◦C)
Agmenellum quadruplicatum UTEX2268* SU 35 7.2 (41)
Anabaena sp. ATCC33081 SU 5 3.6 (40)
Arthrospira fusiformis UTEX2721 SP, SU, FA 5 2.9 (32)
Arthrospira platensis UTEX3086 SP, SU, FA 5 1.8 (32)
Chlorella autotrophica CCMP243 SP, SU, FA 35 3.2 (35)
Chlorella sorokiniana DOE1044 SP, SU, FA 15 5.6 (35)
Chlorella sorokiniana DOE1116/UTEXBP15* SP, SU, FA 5 4.7 (29)
Chlorella sorokiniana DOE1412* SP, SU, FA 5 5.2 (36)
Chlorella vulgaris NREL4-C12* SP, SU, FA, WI 5 4.1 (23)
Chlorella vulgaris AZ-1201* SP, SU, FA 15 4.4 (30)
Chlorococcum littorale UTEX117 SP, SU, FA 15 2.8 (36)
Chlorococcum sp. DOE1426/UTEX BP7 SP, SU, FA 0.4 4.0 (29)
Chloromonas reticulata CCALA870 SP, FA, WI 5 2.6 (19)
Coelastrella sp. DOE0202 SP, SU, FA 15 3.5 (28)
Cyanobacterium sp. AB1 SU 15 7.0 (48)
Micractinium sp. NREL14-F2* SP, FA, WI 5 3.9 (28)
Microchloropsis gaditana CCMP1894 SP, FA 35 2.5 (30)
Microchloropsis salina CCMP1776 SP, FA 35 1.8 (24)
Monoraphidium minutum 26B-AM* SP, SU, FA, WI 5 3.4 (28)
Monoraphidium sp. MONOR1* SP, SU, FA 15 3.4 (29)
Nannochloropsis oceanica CCAP849/10* SP, FA, WI 35 2.9 (30)
Oscillatoria cf. priestleyi CCMEE5020.1-1 SP, FA 5 1.4 (24)
Picochlorum celeri TG2-CSM/EMRE* SP, SU, FA 35 6.8 (35)
Picochlorum oklahomensis CCMP2329* SP, SU, FA 35 6.3 (34)
Picochlorum renovo NREL39-A8* SP, SU, FA 35 5.1 (40)
Picochlorum soleocismus DOE101* SP, SU, FA 15 5.3 (28)
Porphyridium cruentum CCMP675* SP, SU, FA 35 2.8 (24)
Scenedesmus acutus LRB-AZ-0401* SP, SU, FA 5 4.4 (35)
Scenedesmus obliquus DOE0152.z* SP, SU, FA 5 4.4 (29)
Scenedesmus obliquus UTEX393* SP, SU, FA 5 4.6 (34)
Scenedesmus rubescens NREL46B-D3* SP, SU, FA 35 5.5 (34)
Scenedesmus sp. IITRIND2 SP, SU, FA 5 5.0 (35)
Stichococcus minor CCMP819* SP, SU, FA 15 6.0 (34)
Stichococcus minutus CCALA727* SP, FA 15 3.6 (23)
Synechococcus elongatus UTEX2973.1 SU 5 5.5 (39)
Tetraselmis striata LANL1001* SP, FA, WI 35 4.4 (31)
Tisochrysis lutea CCMP1324 SP, SU, FA 35 3.0 (29)
Tribonema minus UTEXB3156 SP, FA 5 3.0 (23)
M. Huesemann et al.
Algal Research 71 (2023) 102996
46
seasons, the best medium salinity and the highest maximum specic
growth rates measured for each of the 38 strains characterized in this
study.
The identity and the presence of bacterial cohorts was determined for
each strain using 18S and 16S rDNA sequencing, respectively. In many
cases, the maximum specic growth rates measured in this study for the
different strains were higher than reported in the literature, indicating
that published growth rates were likely measured under conditions
limiting growth, such as, for example, sub-saturating light intensities
and carbon limitation. The fastest growing strains were down-selected
for subsequent biomass productivity measurements in climate-
simulation photobioreactors, as reported in the next paper in this issue.
CRediT authorship contribution statement
Michael Huesemann: Funding acquisition, Conceptualization,
Experimental Design, Project Supervision, Project Administration,
Writing – Original Draft, Writing – Review & Editing;
Scott Edmundson: Conceptualization, Experimental Design, Project
Supervision, Investigation- Performing Experiments, Data Analysis,
Writing – Original Draft, Writing – Review & Editing;
Song Gao: Investigation – Performing experiments, Data analysis,
Writing – Original Draft, Writing – Review & Editing;
Sangeeta Negi: Investigation – Performing experiments, Data anal-
ysis, Writing – Original Draft, Writing – Review & Editing;
Taraka Dale: Funding acquisition, Conceptualization, Experimental
Design, Project Supervision, Project Administration, Writing – Review &
Editing;
Andrew Gutknecht: Investigation – Performing experiments, Data
analysis;
Hajnalka E. Daligault: Investigation – Performing experiments, Data
analysis;
Carol K. Carr: Investigation – Performing experiments, Data analysis;
Jacob Freeman: Investigation – Performing experiments, Data
analysis;
Theresa L. Kern: Investigation – Performing experiments, Data
analysis;
Shawn R. Starkenburg: Methodology, Formal analysis;
Cheryl D. Gleasner: Investigation – Performing experiments, Data
analysis;
William Louie: Investigation – Performing experiments, Data
analysis;
Robert Kruk: Investigation – Performing experiments, Data analysis;
Sean McGuire: Investigation – Performing experiments, Data
analysis;
Declaration of competing interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgements
We thank Dr. Joseph Weissman (Exxon Mobil) and Dr. Matthew
Posewitz (Colorado School of Mines) for providing Picochlorum celeri
TG2-WT-CSM/EMRE. We also thank Dr. Sherry Cady, curator of the
Culture Collection of Microorganisms from Extreme Environments
(CCMEE), to supply Oscillatoria cf. priestleyi CCMEE5020.1-1. We would
like to acknowledge Kim Mcmurry for 16S and 18S sequencing support.
The DISCOVR consortium is sponsored by the U.S. Department of Energy
(DOE) Ofce of Energy Efciency and Renewable Energy (EERE),
Bioenergy Technologies and Vehicle Technologies Ofces. The views
and opinions of the authors expressed herein do not necessarily state or
reect those of the U.S. Government or any agency thereof. Neither the
U.S. Government nor any agency thereof, nor any of their employees,
makes any warranty, expressed 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. Pacic Northwest
National Laboratory (PNNL) is operated by the Battelle Memorial
Institute for the U.S. Department of Energy under contract No. DE-AC05-
76RL01830. This research was conducted at PNNL as part of the DIS-
COVR national laboratory consortium project sponsored by the U.S.
Department of Energy (DOE) Ofce of Energy Efciency and Renewable
Energy (EERE), Bioenergy Technology Ofce, under Award No.
NL0032208. This work was supported by the U.S. Department of Energy
through the Los Alamos National Laboratory. Los Alamos National
Laboratory is operated by Triad National Security, LLC, for the National
Nuclear Security Administration of U.S. Department of Energy (Contract
No. 89233218CNA000001). This material is based upon work supported
by the U.S. Department of Energy, Ofce of Energy Efciency and
Renewable Energy (EERE), specically the Bioenergy Technologies Of-
ce DISCOVR project, under contract No. NL0032174.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.algal.2023.102996.
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