J Appl Phycol

DOI 10.1007/s10811-015-0560-2

Isolation and screening of euryhaline Tetraselmis spp. suitable

for large-scale outdoor culture in hypersaline media for biofuels
S. Fon-Sing & M. A. Borowitzka

Received: 26 September 2014 / Revised and accepted: 1 March 2015

# Springer Science+Business Media Dordrecht 2015

Abstract Natural saline lakes in Western Australia were sam- shown by the outdoor cultivation of diatom, Halamphora
pled for microalgae species and strains with potential for coffeaeformis MUR 158, in parallel with Tetraselmis sp.
large-scale outdoor cultivation over a wide range of salinities MUR 167 which resulted in the diatom being outcompeted
for biofuels production. Using a rational isolation and screen- by the green alga. Our results demonstrate the high commer-
ing process, several Tetraselmis strains (Chlorophyta, cial potential of euryhaline Tetraselmis spp. for cultivation
Chlorodendrales) with a broad range of salinity tolerance were over a broad range of salinity in outdoor cultures.
identified and were characterised further for their potential for
biofuels production. Specific growth rates increased from 0.8
Keywords AFDW productivity . Total lipid productivity .
to 1.2 days−1 when the medium salinity was decreased from
Salinity tolerance
11 to 3 % (w/v) NaCl (1.88 to 0.51 M NaCl) in batch cultiva-
tion mode, thereby indicating quick adaptation to large salin-
ity changes. In general, ash-free dry weight (AFDW), total
lipid, protein and carbohydrate contents per cell were highest
in the early stages of growth. Salinity increases led to an in- Introduction
crease in cell AFDW, with the highest mean maximum of
2555±659 pg AFDW.cell−1 at 11 % (w/v) NaCl in the strains The cultivation of microalgae as a source of sustainable bio-
Tetraselmis MUR 167 and MUR 219 which had been in cul- mass for biofuels is currently attracting considerable attention
ture for many years, as compared to the mean maximum of (Wijffels and Barba 2010). The potential benefits that
981±141 pg AFDW.cell−1 the in newly isolated strains MUR microalgae offer over conventional land plants are compelling
230, 231, 232 and 233. Similar observations on total lipid, (e.g. higher areal productivities, high lipid content, the ability
protein and carbohydrate content per cell were made between to use nonarable land for cultivation), and there are extensive
the two groups of strains. Overall, all strains yielded high efforts worldwide to develop a viable industry. In particular,
biomass and total lipid productivities over a very wide range one of the greatest advantages of using microalgae as feed-
of salinities without large variation in their gross biochemical stock compared to land plants is the possibility of using saline
composition and growth pattern. Based on AFDW and total water for cultivation. Mass cultivation of microalgae for
lipid productivity data, the order of preference for selecting biofuels requires extremely large amounts of water
strains for further investigation for large-scale culture was (Borowitzka and Moheimani 2013; Fon Sing et al. 2013)
MUR 231>MUR 233>MUR 219>MUR 230>MUR 232> which directly competes with food crops for this limited re-
MUR 167. The Tetraselmis spp. were also very competitive as source. The use of saline water, either from the sea or from
inland saline groundwater sources, greatly enhances the eco-
nomics and environmental sustainability of this prospective
source of biofuels (Pate et al. 2011; Yang et al. 2011;
Resurreccion et al. 2012). Furthermore, cultivation in saline
S. Fon-Sing (*) : M. A. Borowitzka
water also reduces the risk of contamination by other species
Algae R&D Centre, School of Veterinary and Life Sciences,
Murdoch University, Murdoch, Western Australia 6150, Australia and can make the long-term culture potentially more reliable.
e-mail: [email protected] However, microalgae cultivation in saline media, especially in
 J Appl Phycol

open ponds, can be severely impacted by changes in salinity the environmental water samples (Table 1). The medium on
which arise due to evaporation or rain, and the salinity of the half of the agar plates had 10 mg.L−1 GeO2 added to inhibit
medium will depend on whether fresh water or saline water is diatom growth (Lewin 1966). The plates were incubated at
used to replace evaporative losses. If fresh water is used to 30 μmol photons.m−2.s−1 on a 12:12-h L:D cycle at 25 °C
replace evaporative losses, then very large amounts of fresh (this temperature was selected as representing the average
water are required, whereas if saline water is used, the salinity temperature that would be encountered in outdoor algae cul-
in the ponds will rise. Thus, for successful and sustainable tures), and they were regularly checked for the presence of
long-term cultivation in saline water, microalgae strains with microalgae or other microorganisms. Plates with heavy bacte-
a wide tolerance to salinity are highly desirable (Borowitzka rial contamination were re-streaked on new plates to separate
and Moheimani 2013). For biofuel production and reliable microalgae cells. After seventeen days of incubation, small col-
long-term culture outdoors, such strains must also demonstrate onies could be seen, and selected colonies were re-streaked on
additional traits such as high lipid and biomass productivities, new agar plates enriched with twice the amount of nitrate and
fast growth rates and an appropriate temperature tolerance phosphate. Colonies obtained through repeated streaking and
(Borowitzka 2013). The extensive microalgae isolation and single cells isolated by micropipetting were then transferred
screening conducted in the 1980s to 1990s in the USA by using a sterile loop from the agar plates into liquid F/2 and F
the Solar Energy Research Institute (SERI) (Barclay et al. medium (Guillard and Ryther 1962) of the appropriate salinity
1985; Sheehan et al. 1998) demonstrated the great potential in 10-mL McCartney bottles for further growth. The liquid cul-
of highly halotolerant microalgae (e.g. Dunaliella, Amphora, tures were kept at 70 μmol photons.m−2.s−1 on a 12:12-h L:D
Synechococcus) for large-scale cultivation for biofuels. This cycle at 25 °C and were shaken manually once a day.
comprehensive study however also highlighted the nonuni-
form response in growth and biochemical composition be- Characterisation and preliminary selection
tween different species and strains across the range of salinities
investigated. Such differences in growth and biochemical Microalgae cells from healthy agar and liquid cultures were
composition are well-documented in the literature (e.g. characterised according to their size, shape and motility
Renaud and Parry 1994; Fábregas et al. 1985) and emphasises (Table 1). Identification was based on cell shape (filamentous,
the need for thorough screening procedures to obtain the best coccoid, rod-like), presence/absence of flagella, pyrenoids and
strain for improved productivity (Barclay et al. 1987; other distinctive features using standard references (Butcher
Borowitzka 2013). 1959; John 1983; Round et al. 1990; Carmelo and Grethe
Here, we report on the rational isolation and screening of 1997; Borowitzka and Siva 2007). Diatoms were cleaned
microalgae from Australia which can be grown over a very using the nitric acid method, and the valves were mounted
broad range of salinities (3 to 11 %w/v NaCl; =0.51 to 1.88 M in Depex resin for examination (Round et al. 1990).
NaCl) suitable for long-term high productivity in large-scale Viable liquid cultures were scaled up and grown through
culture outdoors for the production of biofuels. A short out- one growth cycle in 75 mL of F medium in 100-mL
door cultivation trial to evaluate the competitive ability of Erlenmeyer flasks at 70 μmol photons.m−2.s−1 on a 12:12-
selected microalgae strains was also carried out. h L:D cycle at 25 °C and at 7 % (w/v) NaCl. Their growth
rate, lipid content and ease of culturing were then determined
for further selection.
Materials and methods Cells from clumpy and settling cultures, such as the diatom
cultures and some green algae cultures, were regularly shaken
Isolation of algae strains by hand, and 4-mm glass beads were added to the flasks to
enhance cell dislodgement from the glass surface. Prior to
Water samples were collected from the periphery of three counting with a haemocytometer, cells clumps were thorough-
meromictic saline lakes (Lake Bagdad, Herschel Lake, ly homogenised either by using a glass homogeniser with a
Government House Lake; Bunn and Edward 1984) on loose fitting Teflon plunger or by blowing small air bubbles
Rottnest Island (31° 59′ S, 115° 32′ E), Western Australia, vigorously through the cell sample with compressed air
Australia, in October 2005. The lake water salinity and tem- through a narrow bore plastic pipette tip.
perature were measured at the time of collection with a hand-
held refractometer (Atago) and a digital thermometer. Initial Outdoor cultivation
isolation of algae was achieved by streaking on agar plates
following Andersen and Kawachi (2005). The growth medi- Two 1 m2 (2×0.5×0.2 m, L×W×D) fibreglass raceway ponds
um used for agar plating consisted of charcoal-filtered seawa- were used for outdoor cultivation. The water was circulated in
ter enriched with 75 g.L−1 NaNO3 and 5 g.L−1 NaH2PO4 ·H2O the pond by paddlewheels with a mixing speed of 20 cm.s−1.
with the salinity adjusted with NaCl to match the salinity of Prior to cultivation, the ponds were sanitised with sodium
J Appl Phycol

hypochlorite, rinsed with fresh water and left to dry in the sun. this experiment, aliquots of the selected microalgae cultures
Seawater was sterilised with 12.5 % NaClO overnight in sep- were transferred into 200 mL F medium at 3, 5, 7, 9 and 11 %
arate drums before use. The seawater was enriched with F (w/v) NaCl (i.e. 0.51, 0.86, 1.20, 1.54 and 1.88 M NaCl) in
medium nutrients, and the salinity was adjusted to the desired 250-mL Erlenmeyer flasks. The cultures were regularly
salinity with commercial pool salt. shaken manually and kept at 70 μmol photons−1.m−2.s−1
provided by cool-white fluorescent lamps on a 12:12-
Effect of salinity h L:D cycle and at 25±0.2 °C. Samples for biochemical
analysis were taken at the late exponential phase (day 7),
Selected newly isolated strains were maintained in F medium stationary phase (day 18) and late stationary phase (day 34)
at 7 % (w/v) NaCl salinity prior to the salinity experiment. For of growth.

Table 1 Characteristics of lake conditions and cell features of microalgal isolates originating from Rottnest Island, Western Australia

Location Salinity (% NaCl) Water temperature (°C) ISO no. Classification Size (μm) (W×L) Motility

Chlorophyta
Herschel Lake 6.4 21.6 48 Nannochloropsis 3×3 −
Herschel Lake 6.4 21.6 49 Tetraselmis 10×15 +
Herschel Lake 6.4 21.6 2 Tetraselmis 10×15 −
Herschel Lake 6.4 21.6 5 Tetraselmis 10×13 +
Lake Bagdad 7.3 19.7 1 Tetraselmis 20×40 +
Lake Bagdad 7.3 19.7 17 Tetraselmis 10×15 −
Lake Bagdad 7.3 19.7 35 Tetraselmis 10×15 +
Lake Bagdad 7.3 19.7 36 Tetraselmis 10×20 −
Lake Bagdad 9.7 20.1 38 Dunaliella 5×10 −
Lake Bagdad 9.7 20.1 39 Tetraselmis 10×15 +
Lake Bagdad 9.9 20.1 8 Tetraselmis 10×20 +
Lake Bagdad 9.9 20.1 18 Dunaliella 5×10 −
Lake Bagdad 12.4 21.4 43 Dunaliella 5×10 −
Lake Bagdad 12.4 21.4 26 Dunaliella 5×10 +
Herschel Lake 12.6 22.0 16 Tetraselmis 10×15 +
Herschel Lake 12.6 20.8 25 Nannochloropsis 3×3 −
Government House Lake 12.6 20.8 27 Tetraselmis 10×15 +
Government House Lake 12.6 20.8 34 Dunaliella 5×10 +
Herschel Lake 13.1 22.0 15 Tetraselmis 10×15 +
Bacillariophyceae
Herschel Lake 6.4 21.6 40 Amphora/Cymbella 6×8 −
Herschel Lake 6.4 21.6 52 Amphora/Cymbella 3×12 −
Lake Bagdad 7.3 19.7 3 Amphora/Cymbella 5×10 −
Lake Bagdad 7.3 19.7 29 Amphora/Cymbella 5×15 −
Lake Bagdad 7.3 19.7 32 Amphora/Cymbella 5×15 −
Lake Bagdad 9.7 20.1 41 Amphora/Cymbella 7×10 −
Lake Bagdad 9.7 20.1 42 Amphora/Cymbella 4×6 −
Lake Bagdad 9.9 20.1 24 Nitzschia 5×15 −
Lake Bagdad 9.9 20.1 28 Navicula 5×15 −
Lake Bagdad 9.9 20.1 31 Navicula 4×10 −
Lake Bagdad 12.2 21.4 10 Nitzschia 7×25 −
Lake Bagdad 12.2 21.4 21 Amphora/Cymbella 5×10 −
Lake Bagdad 12.4 21.4 23 Amphora/Cymbella 5×12 −
Lake Bagdad 12.4 21.5 51 Amphora/Cymbella 8×18 −
Herschel Lake 12.6 22.0 45 Amphora/Cymbella 5×10 −
Herschel Lake 13.1 22.0 46 Amphora/Cymbella 5×15 −
 J Appl Phycol

Analytical procedures pennate diatoms Amphora/Cymbella (12 strains), Nitzschia

(2 strains) and Navicula (2 strains) (Table 1).
Procedures for cell counts, specific growth rate calculation, Several of the Tetraselmis strains were by far the easiest to
sample preparation prior to analysis and methods for the de- grow and maintain because the cells were fully motile and
termination of proximate composition (total lipids, carbohy- homogeneously distributed throughout the water column.
drates and proteins), dry weight and ash-free dry weight Even if they settled and adhered to the flask surface, they were
(AFDW) were as detailed in Moheimani et al. (2013). In brief, easily dislodged with the aid of the glass beads. On the other
cell counts were carried out using an improved Neubauer hand, the diatom isolates adhered more strongly and uniform-
haemocytometer and specific growth rate was calculated by ly to the flask surface, requiring continuous and vigorous
dividing the natural logarithm of two by the doubling time; the shaking (which often damaged the cells) to keep the cells in
doubling time was obtained from the slope of the growth suspension. The Dunaliella and Nannochloropsis isolates
curve at exponential growth plotted on a semi-log graph. grew poorly in liquid medium, and this was obvious by the
Total lipids were determined gravimetrically using a modified delayed growth and very low cell numbers. Given their poor
Folch method (Folch et al. 1957), total proteins were analysed growth, coupled with their small size relative to the
using Lowry’s method (Lowry et al. 1951) as adapted by Tetraselmis and the diatoms, they were deemed unsuitable
Dorsey et al. (1978), and total carbohydrates were estimated for further study, and further screening was focused on the
using the method developed by Kochert (1978) and Ben- Tetraselmis and diatom isolates.
Amotz et al. (1985) as optimised by Mercz (1994). Dry weight After consistent reliable growth was attained in liquid me-
and ash-free dry weight analysis were carried out by consec- dium, the Tetraselmis and diatom isolates were grown in batch
utive drying and combustion of filtered samples (Moheimani mode for 25 days, during which the growth rate and lipid
et al. 2013). content were determined. Daily observation of the cultures
in the first few days indicated good growth for all isolates.
However, the tendency of the diatoms to stick and clump
Statistical analysis
became more apparent as the cultures aged and sampling
and cell counts became progressively more difficult and inac-
To compare the individual and combined effects of salinity
curate; stronger homogenisation in an attempt to dissociate the
and growth stage on AFDW content and biochemical compo-
diatom cells from each other often resulted in cell breakage
sition, two-way repeated measures ANOVA at α-level=0.05
and bubbling air through the culture was inefficient. As a
were performed using the statistical package in the Sigmaplot
consequence, no reliable growth rate data could be obtained
software (Systat). One-way ANOVA was used to compare the
from the diatom cultures. Tetraselmis cultures, on the other
AFDW and lipid productivity of each strain over the salinity
hand, were easier to handle and grew well from inoculum
range, as well as between strains at each salinity. The Holm-
densities starting from as low as 2×104 cells.mL−1, with spe-
Sidak pairwise comparison method based on ranks was ap-
cific growth rates ranging between 0.17 and 0.69 day−1.
plied when normality and equal variance tests failed.
Lipid content at the early stationary phase differed between
the two phyla, with total lipid content in the Tetraselmis strains
being on average half of that found in diatoms which
Results contained between 20 and 40 % total lipids on a dry weight
basis. However, despite the higher total lipid content, the dia-
Isolation, characterisation and preliminary selection tom isolates were deemed not suitable for further investigation
because of their very sticky nature which can make reliable
At the time of sampling, the salinity at Lake Bagdad, Herschel cultivation in open ponds on a large-scale difficult.
Lake and Government House Lake ranged between 6.4 and
13.1 % (w/v) NaCl, and the average water temperature was Preliminary outdoor culture trials with Tetraselmis
20 °C. Plating of the lake water samples on nitrate- and and Halamphora
phosphate-enriched agar resulted in the growth of various al-
gae, with green algae, diatoms and cyanobacteria dominating. In parallel with the indoor screening to isolate new strains,
Upon transfer from the agar plates to liquid medium, many outdoor cultivation investigations were also carried out using
strains showed poor growth and were therefore discarded. No previously isolated strains from our culture collection. Earlier
cyanobacteria were isolated into unialgal culture as laboratory studies by Mercz (1994) on microalgae isolated
cyanobacteria have only a low lipid content and were there- from the same sites on Rottnest Island in 1990 had identified
fore of no interest. The 35 unialgal cultures finally isolated two promising lipid-rich and fast-growing strains for potential
consisted of the green algae Tetraselmis (12 strains), biofuels production. As these strains from the Murdoch
Dunaliella (5 strains), Nannochloropsis (2 strains), and the University Culture Collection (Halamphora (Amphora)
J Appl Phycol

coffeaeformis MUR158 and Tetraselmis sp. MUR167) were the diatom culture as compared to that of the Tetraselmis cul-
of the same genera as those being newly isolated, it was de- ture. However, despite the diatom’s high rate of growth,
cided to compare their growth in outdoor culture in two 1 m2 Tetraselmis sp. MUR 167 proved to be the more robust and
outdoor raceway ponds. Tetraselmis MUR 167 at 7 % (w/v) competitive of the two species at both low (3.5 % NaCl) and
NaCl and H. coffeaeformis MUR 158 at 3.5 % (w/v) NaCl high (7.0 % NaCl) salinities. This led to the conclusion that the
were grown in F medium in adjacent ponds in summer Tetraselmis sp. strains so far isolated were potentially better
(February). Both cultures were maintained in batch mode until candidates for cultivation for biofuel production than the dia-
cell density started to decline, upon which time semi- toms. Consequently, further work concentrated on the
continuous culture mode was initiated by partial harvesting Tetraselmis sp. isolates only.
and replacement with fresh medium (Fig. 1). By the end of
2 months of cultivation, the diatom culture showed extensive Screening of Tetraselmis sp. strains for salinity tolerance
clumping of the cells and collapsed, whilst, on the other hand,
the Tetraselmis culture continued to grow, albeit at a slow rate, Growth Following the initial isolation and screening trials
until the culture gradually collapsed as a result of amoeba above as well as the outdoor culture trial comparing the dia-
contamination. A second attempt to grow the diatom lasted tom Halamphora and Tetraselmis sp., further screening of the
only 1 month as Tetraselmis MUR 167 cells from the adjacent Tetraselmis strains was carried out to determine the effects of a
pond contaminated the culture and eventually outcompeted range of salinities. In these trials, four newly isolated
the diatom culture (Fig. 1a). A third attempt to grow the dia- Tetraselmis sp. strains (MUR230, MUR231, MUR232 and
tom only, this time in winter (July–September), resulted in a MUR233) and two previously isolated strains from the
long lag phase, and after 1 month, the culture was once again Murdoch University Algal Culture Collection (MUR167 and
overtaken by Tetraselmis MUR 167 cells, although no MUR 219) were used. Inocula of all six strains of Tetraselmis
Tetraselmis was being cultivated in the adjacent pond at that sp. maintained at 7 % (w/v) NaCl were transferred to F medi-
time, and both ponds had been carefully bleached and washed um at 3, 5, 7, 9 and 11 % (w/v) NaCl. Despite the absence of a
and left dry for 2 months. The origin of the contaminating salt-acclimation step upon transfer from 7 % NaCl to both
Tetraselmis is unclear, but was most likely from the adjacent lower and higher salinities, all strains grew at the various
environment (soil) where some Tetraselmis from the earlier salinities for the entire 34-day cultivation period (Fig. 2). In
culture effort could have survived in a resting stage. general, cultures of all strains at 3 and 5 % NaCl were the least
In general, the growth rate of H. coffeaeformis MUR 158 affected by the change in salinity and had a similar growth
was up to 4.5 times faster than that of Tetraselmis sp. MUR pattern to the cultures at 7 % NaCl, progressing through a
167 (μH.coffeaeformis =0.99 and 0.77 days−1, μTetraselmis =0.22 well-defined period of exponential growth to yield high cell
and 0.29 days−1 on the first and second culture attempts, re- densities. In contrast, growth at 9 and 11 % NaCl was slower
spectively), which could have been due to the lower salinity in in most strains, and some strains were clearly less tolerant to

Fig. 1 Culture of Halamphora

coffeaeformis MUR 158 (a) with
Tetraselmis sp. MUR 167 as
contaminant, and Tetraselmis sp.
MUR 167 (b) in outdoor raceway
ponds. H. coffeaeformis MUR
158 (open diamond), Tetraselmis
sp. MUR 167 (closed circle)
 J Appl Phycol

Fig. 2 Growth curves of

Tetraselmis strains MUR 167,
219, 230–233 at 3 % NaCl (filled
circle), 5 % NaCl (open circle),
7 % NaCl (inverted filled
triangle), 9 % NaCl (open
triangle) and 11 % NaCl (filled
square). Data are mean (n=2)

the high salinity than others. For instance, cell division for salinity. Such a rapid adaptation to changes in salinity is high-
strains MUR 167, MUR 219 and MUR 231 was noticeably ly advantageous and desirable during heavy rain episodes, as
impacted at 11 % NaCl as the cultures had a long lag phase of
growth, eventually yielding some of the lowest cell densities
at the end of the experiment. Based on the overall performance
over the salinity range investigated, it appeared that interspe-
cific differences existed between the six strains of Tetraselmis,
and that strains MUR 230, MUR 232 and MUR 233 were the
most promising candidates for cultivation over a broad range
of salinities.

Growth rates Trends in the specific growth rates between all

Tetraselmis strains were similar over the range of salinities
tested, i.e. specific growth rates were highest at lower salin-
ities and declined with increasing salinity. Tetraselmis MUR
231 was the fastest growing strain at 3 % NaCl with 1.2 cell
divisions.day−1 (Fig. 3). Strain variation, as interpreted by the
difference in the specific growth rate profile of each strain over
the salinity range, was clearly apparent, with strains MUR
231, MUR 233, and to some extent MUR 219, displaying Fig. 3 Mean specific growth rates of Tetraselmis strains grown over a
the strongest and best growth in response to a decrease in range of salinities. Mean of 2 replicate cultures
J Appl Phycol

this would ensure uninterrupted and better growth as the cul- Proximate composition The bulk of the organic matter of the
tures progressively become more diluted due to rain. cultures at any point in time during growth mainly consisted
of proteins and lipids, with carbohydrates representing on av-
AFDW per cell The total AFDW content at the exponential, erage less than 14.2±1.3 % of the cell constituents at all times
stationary and late stationary phases of growth for all of the (Fig. 5). The amount of lipid, protein and carbohydrate per cell
strains is shown in Fig. 4. Overall, cell AFDW was highest in was highest in the early stages of growth and subsequently
the early stages of growth in all cultures and in cultures grown declined with culture age.
at high salinity, especially at 11 % NaCl (Fig. 4). At this When the overall gross biochemical make-up of the
salinity, the AFDW per cell of strains MUR 167 and MUR cells of all strains was evaluated with respect to salinity,
219 which had been in culture for several years was very high it was found that the per cell content of total proteins,
at all stages of growth in comparison with the other strains. Of lipids and carbohydrates of all six strains increased from
the six strains, strain MUR 230 showed the least significant low to high salinity. This trend was particularly promi-
difference (P<0.05) in its AFDW content per cell over the nent at the early stage of growth in all cultures and is
salinity range of 3 to 9 % NaCl, which indicates that this strain even more pronounced in the lipid trend at 11 % NaCl
can potentially maintain a steady level of organic biomass in strains MUR 167 and MUR 219 which contained
production when grown across this broad salinity range. twice as much lipids as the other strains.

Fig. 4 Cell AFDW content of

Tetraselmis strains MUR 167,
219, 230–233 at late exponential,
stationary and late stationary
phases of growth. Note that the
different y-axis scales are used for
strains MUR 167 and MUR 219.
Mean±S.E., n=4
 J Appl Phycol

Fig. 5 Proximate composition

per cell of Tetraselmis strains over
a range of salinity at late
exponential (day 7), stationary
(day 18) and late stationary (day
34) phases of growth. Total
carbohydrates (black), total
proteins (light grey) and total
lipids (dark grey). n=2

AFDW and total lipid productivities To assess the commercial densities, even though the per cell AFDWs were higher than
potential of these Tetraselmis strains for cultivation in saline for the cells grown at lower salinities (Fig. 4). This is clearly
water at various salinities, the AFDW and total lipids produc- exemplified by strains MUR 167 and MUR 219 which had
tivity of each strain were also determined. The productivities, particularly high per cell AFDW at 11 % NaCl, but very slow
calculated as the product of the biomass yield and the specific rates of growth with distinctly lower cell densities than the rest
growth rate during the exponential phase of growth are given of the cultures.
in Table 2. The productivities generally were highest at salin- None of the strains maintained a constant AFDW produc-
ities ≤7 % NaCl, with the highest value being recorded 3 % tivity across the salinity range. Instead, most strains had sta-
NaCl for MUR 231. This was a direct result of its exception- tistically significantly similar AFDW productivities between 3
ally high rate of growth at this salinity (Fig. 3). The most and 5 % NaCl, but then showed different and declining pro-
significant drops in biomass and total lipid productivity were ductivities as the salinity was increased from 9 to 11 % NaCl.
observed at 11 % NaCl where productivities of less than AFDW productivities were clearly dissimilar at each salinity
50 mg.L−1.day−1 AFDW, and less than 18 mg.L−1.day−1 total level for strains MUR 219 and MUR 233, indicating that the
lipids were recorded due to lower growth rates and lower cell productivity of these strains is potentially more affected by
J Appl Phycol

Table 2 Total lipid and biomass productivities on an AFDW basis of six strains of Tetraselmis grown in batch mode over a range of salinities

Salinity (%) (w/v) NaCl

Strain 3 % NaCl 5 % NaCl 7 % NaCl 9 % NaCl 11 % NaCl

AFDW productivity (mg.L−1.day−1)

MUR 167 99.1±5.7Ab 95.0±4.0Abcd 58.0±6.0Bc 42.6±2.7Caf 6.2±1.6De
(142.8) (143.1) (86.1) (90.7) (19.9)
MUR 219 86.4±3.3Abcd 142.8±5.7Be 100.9±2.1Ca 49.6±2.3 Dadef 17.5±1.9Ea
(94.1) (193.8) (140.9) (95) (60.3)
MUR 230 96.4±2.5ADbcd 101.4±3AEab 90.9±6.5Adad 73.2±2.9BFb 34.6±4.2CGd
(142.2) (175.9) (173.0) (143.3) (84.9)
MUR 231 201.0±6.1Ca 131.2±5.8Bae 82.2±1.6Aabd 69.5±3.4Abc 14.2±2.3 Da
(243.0) (182.9) (137.6) (168.9) (51.4)
MUR 232 96.4±8.7Cbc 99.9±7.1Cabc 68.4±1.8Abc 58.1±5.8ABabcd 47.5±1.5ABb
(126.2) (150.9) (130) (132.6) (127.8)
MUR 233 167.9±4.1Aa 75.4±3.0Bbcd 133.1±4.3Ce 57.7±2.8Dabcde 25.9±1.4Ec
(239.5) (119.1) (226.9) (119.4) (58.1)
Total lipid productivity (mg.L−1.day−1)
MUR 167 25.8±1.5CDd 22.3±1.5ABDc 16.3±0.3ACa 10.8±0.4BDb 18.3±0.0Ba
MUR 219 30.3±4.4BCbcd 42.1±5.4Cb 33.1±6.0ABDbd 21.6±4.0ABCDac 7.7±0.4 Da
MUR 230 43.2±4.4ABcd 35.2±2.7ACabcde 47.6±8.5Bc 35.5±3.8Aacde 16.5±0.59BCab
MUR 231 85.5±3.1Ed 62.9±2.3Ed 31.3±2.3ABDa 33.8±3.5ACa 13.5±0.8Ab
MUR 232 25.8±0.9CDab 34.2±5.2ABCb 18.5±0.5ACac 17.8±0.8BDac 16.0±0.5ABCac
MUR 233 58.0±6.5Ac 26.2±1.7ABDa 41.3±3.0BDb 23.9±1.1ABCa 10.4±1.2Ec

Dry biomass productivities (mg.L−1 .day−1 ) are shown in brackets. Each value is presented as mean±SE (n=4)
Means within each row with same letters (A–G) do not differ significantly (P<0.05)
Means within each column with same letters (a–f) do not differ significantly (P<0.05)

salinity changes. Based on these results, the order of prefer- Discussion

ence for strains with the best potential to yield high biomass
productivities over the whole salinity range would be MUR The aim of this study was to isolate and identify broadly
231>MUR 233>MUR 219>MUR 230>MUR 232>MUR halotolerant microalgae species possessing the following fea-
167. tures for large-scale cultivation in saline water: ease of culture
Total lipid productivity trends were similar to those ob- and scale-up, fast growth in a relatively simple growth medi-
served for AFDW biomass productivities, with no statistical um, tolerance to a wide range of salinity and the ability to
significance in the differences found at 3–5 % NaCl and 9– grow reliably in outdoor conditions. Similar to the outcomes
11 % NaCl. Of the six strains, MUR 230 and MUR 231 were obtained by Mercz (1994) who also isolated microalgae from
the only strains that maintained total lipid productivities in saline lakes, our results demonstrate that halotolerant, fast-
excess of 30 mg.L−1.day−1, at salinities up to 9 % NaCl. growing and lipid-rich Tetraselmis species were the most ro-
Thus, in terms of strain selection for high lipid productivities bust isolates identified in the screening process. Furthermore,
over a broad range of salinities, these two strains would be the our findings confirm that several of the isolated strains can be
most suitable candidates, followed by MUR 233, MUR 219, grown at salinities ranging from 3 to 11 % NaCl with high
MUR 232 and MUR 167 (in descending order). productivities, and that at least one of them could be cultivated
Finally, to evaluate the performance of the domesticated outdoors as the potentially dominating species in a mixed
strains MUR 167 and MUR 219 against that of the newly culture. Hence, through a careful selection of sampling loca-
isolated strains, a comparison of the AFDW and total lipid tion and screening methodologies, we have shown that high-
productivities was performed at each salinity level. Strain performing strains with commercial potential can be acquired
MUR 167 was the least productive of all strains with regards from natural habitats with a reliable degree of repeatability.
to AFDW productivity at each salinity, whilst MUR 219 was
as productive as the other four strains. Total lipid productiv- Sampling environment The first step in our screening ap-
ities of the domesticated strains were statistically similar to proach was to sample saline lakes. Saline lakes are highly
those obtained for the newly isolated strains. selective aquatic environments often characterised by variable
 J Appl Phycol

and high salinities (>seawater salinity), high temperatures Tetraselmis —a promising species for commercial cultivation
(20–30 °C), high pH (7.8–10.6) and alkalinity, high phosphate in hypersaline water Tetraselmis, on the other hand,
and silicate, and low nitrate content (Hammer 1981). Despite displayed several desirable features which makes it one of
their seemingly hostile and barren environment, salt lakes the most suitable candidates for outdoor large-scale cultiva-
contain a well-adapted microflora (Borowitzka 1981; tion in saline water. Tetraselmis is a quadriflagellate naked
Sheehan et al. 1998) which can sustain occasionally high pri- chlorophyte belonging to the Chlorodendrales. Compared to
mary productivity at high photosynthetic efficiencies (up to other green algae flagellates, it is more robust and often dom-
8 % of photosynthetic active radiation) in the euphotic zone inates in laboratory-mixed cultures (Regan and Ivancic 1984;
(Hammer 1981). Okauchi and Kawamura 1997), and because of its simple nu-
The dominant microalgae genera inhabiting most saline trient requirements (no vitamins required and different sources
lakes around the world (USA, Southern Europe, Central of nitrogen can be used), ease of culture, fast growth and high
Asia, Africa, Australia) are the green algae, especially the total lipid content, it is commonly used as a food source for
flagellated types (e.g. Dunaliella), a number of benthic dia- zooplankton, shellfish and crustacean larvae (Dunstan et al.
toms and a range of cyanobacteria (Thomas et al. 1984; 1992; Fábregas et al. 1996; Wikfors et al. 1996; Ferreira et al.
Barclay et al. 1986; Mercz 1994; Kawabata et al. 1997; 2009). Most Tetraselmis spp are euryhaline and are common-
López-González et al. 1998). Diatoms, especially benthic pen- ly found in fresh, brackish and marine environments (Butcher
nate diatoms such as Nitzschia, Navicula, Amphora and 1959; Van den Hoek et al. 1995) as well as in hypersaline
Amphiprora are the most frequently encountered, making up water bodies (Brand 1981; Kawabata et al. 1997; López-
almost 60 % of the entire salt lake phytoplankton population at González et al. 1998; Arora et al. 2013), and it can tolerate
times (Sheehan et al. 1998), possibly due to the high silicate temperatures between 2 and 35 °C (Regan 1988). Its wide
load. salinity tolerance is mainly due to its ability to osmoregulate
The microalgae assemblage obtained from Rottnest Island by producing the compatible solute mannitol (Kirst 1977b,
corresponded to the phytoplankton community of saline lakes 1988). Additionally, beside active osmolyte production,
described above, but like Mercz (1994), the only persisting Tetraselmis species possess a highly efficient and active Na+
species which grew well on both agar and liquid medium were efflux pump via an Na+-ATPase, the activity of the latter being
the Tetraselmis spp. and a large number of pennate benthic enhanced by high internal [Na+] (Kirst 1977a; Strizh et al.
diatoms. The diatoms showed good lipid production potential 2004). It is also possible that Tetraselmis produces dimethyl
by virtue of their high lipid content. However, being benthic, sulfoniopropionate (DMSP), a dimethyl sulfide (DMS) pre-
they also showed strong adhesion to the culture vessel wall cursor, together with mannitol during long periods of stress
and/or formed thick clumps with no free cells in suspension in and especially under high salinities (Dickson and Kirst 1986)
the water column. Although some of these diatoms, such as and in nitrogen-deficient medium (Gröne and Kirst 1992).
Cyclotella and Amphora have been cultured successfully with Thus, Tetraselmis has the right attributes for large-scale culti-
high short-term biomass productivities in outdoor raceway vation in saline water of variable salinity. Moreover, the mere
ponds (Weissman and Goebel 1986), our experience with fact that it has been repeatedly and successfully isolated from
the H. coffeaeformis MUR 158 outdoor raceway culture various saline and hypersaline environments, and maintained
showed that cell clumping remains a concern which can make on defined growth media, makes it one of the most accessible
culture production and maintenance very challenging. Cell species to work with.
adhesion potentially could be controlled through the alteration
and reduction of specific medium constituents such as calcium Outdoor cultivation trial Whilst growth rates displayed by
or iron salts (Cooksey 1981; Walach and Pirt 1986; Geesey H. coffeaeformis MUR 158 in the outdoor ponds were supe-
et al. 2000), but this would most likely lead to reduced growth rior to that of Tetraselmis MUR 167 on several occasions, the
rates, as was shown upon a reduction in iron concentration for inability for the diatom to compete and retain high biomass
Chlorella (Walach and Pirt 1986), and for Nitzschia communis concentrations in the presence of Tetraselmis MUR 167 clear-
when the calcium chloride concentration was reduced from ly indicated that the latter alga would almost always dominate,
7.6 to 0.037 g.L −1 (Dempster and Sommerfeld 1998). regardless of the salinity level. Furthermore, there are some
Alternatively, addition of anti-fouling agents such as cyclo- doubts in the diatom’s usefulness and commercial potential for
heximide or tunicamycin—which are adhesion inhibitors several reasons. In comparison to green algae, diatoms gener-
(Cooksey and Cooksey 1986)—to the medium could be con- ally seem to have a relatively low saturating irradiance Ik
sidered but would be undesirable from an economical as well (Ryther 1956; Falkowski and Owens 1980; Richardson et al.
as environmental point of view. Therefore, based on practical 1983). Thus, in areas of high insolation, unless culture condi-
considerations, benthic diatom strains obtained from the cur- tions are exclusively set up to maintain dominance by the
rent screening process were unsuitable for large-scale diatoms, it would appear that the diatoms would be more
cultivation. rapidly and strongly light-inhibited and therefore less
J Appl Phycol

productive than green algae, which could then lead to a rapid reported by Clavero et al. (2000) for 34 euryhaline diatom
onset of contamination by other algae with better tolerance to isolates which were grown over a similar salinity range. The
high light. Additionally, whilst the clumping tendency of the reason for the high growth rates at lower salinity in the
diatom would have been desirable for harvesting, it would Tetraselmis strains is unknown, but it is likely that osmoregu-
have made mass-cultivation more challenging due to fouling lation at higher salinities requires more energy thus reducing
of the pond walls and paddlewheel, to inefficient use of light the growth rate (Hellebust 1976; Vonshak and Richmond
and nutrients, and to heavy and rapid settling in quiescent 1981). For strains MUR 167, MUR 219 and MUR 231, this
zones. Additionally, as pointed out earlier, cultivation of dia- osmotic stress at 11 % NaCl must have been large enough to
toms would most likely require a silicate source which would significantly impede cell division over a few days so that cells
impose additional costs. In contrast, Tetraselmis has minimal became heavier and accumulated more lipids as storage prod-
nutrient requirements and is therefore comparatively more ucts. It could also be inferred that more cellular energy was
suited for large-scale cultivation. Subsequent long-term out- being diverted for the production of osmolytes such as man-
door cultivation of several of the newly isolated Tetraselmis nitol to adjust to the high salinity, which could have occurred
strains at high salinity was successful, which confirmed our in all strains, according to the increase in total carbohydrates
interpretations of the data, as well as the high commercial with increasing salinity.
potential of this particular species (Fon Sing 2011, Isdepsky It was interesting to note that the cell lipid contents of all
unpublished data). strains were highest in the early stage of growth when presum-
ably the cells were still nutrient-replete as compared to those
The effect of salinity on growth, biochemical composition and in stationary or late stationary phases of growth. Similarly,
productivities In this study, six strains of Tetraselmis isolated Sheehan et al. (1998) and Reitan et al. (1994) have reported
from saline lakes were successfully cultivated over a broad higher lipid contents in nutrient-replete Tetraselmis cultures,
range of salinities up to 11 % NaCl with high productivities. and Pusceddu and Fabiano (1996) demonstrated that lipid
Undoubtedly, this is a reflection of their natural adaptation to content was higher in the early days of cultivation. On the
the wide range of salinities that occur in the saline lakes as a other hand, several studies have also shown that nutrient de-
result of seasonal influences (Hodgkin 1959; Bunn and ficiency encourages lipid accumulation in Tetraselmis
Edward 1984; Brearley 2005) and emphasises the relevance (Griffiths and Harrison (2009) and references therein). The
of sampling such habitats for euryhaline microalgal species. fact that the strains in our study are more lipid-rich at the early
At the time of sampling from the saline lakes, the majority stage of growth is highly advantageous as this would mean
of the Tetraselmis isolates were found in water samples with high lipid productivities (since lipid productivity is the prod-
salinity ~7 % NaCl, which subsequently was chosen as the F uct of lipid content and growth rate) within shorter cultivation
medium salinity at which the six chosen strains would be timeframes, especially in continuous cultures.
grown (strains MUR 167 and 219 were already being main-
tained at 7 % NaCl in the culture collection). In comparison to Strain selection within the Tetraselmis strains Differences be-
most of Tetraselmis cultures grown elsewhere, this cultivation tween the new isolates were not as marked as the differences
salinity is high for this genus, and to our knowledge, this is the between these new isolates and the domesticated strains which
first report of such cultivation practice (in combination with had been maintained indoors at constant conditions for many
the study of Mercz (1994)). Most studies of salinity tolerance years. Strain variation arises from genotypic and phenotypic
in Tetraselmis spp. have not exceeded a salinity of 5 % NaCl selective pressures and can lead to a wide array of physiolog-
(e.g. Kirst and Keller 1976; Sigaud and Aidar 1993; Khatoon ical traits within one genus. Strain variation also arises through
et al. 2014). Whilst there is no real immediate advantage for anthropogenic-imposed selective growth conditions as is seen
this salinity to be used in indoor cultures, inoculation and in algal culture collections where some algae can undergo
cultivation outdoors at such high salinity provides an instant changes in the life history or physiology such as changes in
benefit as less salt-tolerant contaminating organisms are maximum growth rate or decline in photosynthetic efficiency
quickly outcompeted and potentially eliminated, thereby en- (Lakeman et al. 2009). This was observed to some extent for
suring dominance by this alga. Data from the outdoor work Tetraselmis MUR 167 and MUR 219 which differed from the
where no contamination by H. coffeaformis MUR 158 was rest of the strains in terms of growth kinetics and the
found in the adjacent 7 % NaCl Tetraselmis sp. MUR 167 proportion of AFDW and total lipids. This is further
culture seems to confirm this. accentuated by the fact that by the time strain MUR 167 was
An overall assessment of the performance of all six acquired from the culture collection for this study, no motile
Tetraselmis strains over the salinity range indicates that 7 % cells were found, which contrasts with the motile status
NaCl may represent the critical physiological point above reported by Mercz (1994) who isolated the strain. Hence, it
which growth starts to be impaired and below which growth is hypothesised that the acute differences displayed by these
is greatly enhanced. This observation corroborates with those two strains could have resulted from man-made selection
 J Appl Phycol

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