J Appl Phycol (2014) 26:1431–1437
DOI 10.1007/s10811-013-0177-2
pH effects on growth and lipid accumulation of the biofuel
microalgae Nannochloropsis salina and invading organisms
Meridith L. Bartley & Wiebke J. Boeing &
Barry N. Dungan & F. Omar Holguin & Tanner Schaub
Received: 3 May 2013 / Revised and accepted: 27 September 2013 / Published online: 5 November 2013
# Springer Science+Business Media Dordrecht 2013
Abstract Biofuels derived from non-crop sources, such as
microalgae, offer their own advantages and limitations. De-
spite high growth rates and lipid accumulation, microalgae
cultivation still requires more energy than it produces. Fur-
thermore, invading organisms can lower efficiency of algae
production. Simple environmental changes might be able to
increase algae productivity while minimizing undesired or-
ganisms like competitive algae or predatory algae grazers.
Microalgae are susceptible to pH changes. In many production
systems, pH is kept below 8 by CO2 addition. Here, we uncou-
ple the effects of pH and CO2 input, by using chemical pH
buffers and investigate how pH influences Nannochloropsis
salina growth and lipid accumulation as well as invading
organisms. We used a wide range of pH levels (5, 6, 7, 8, 9,
and 10). N. salina showed highest growth rates at pH 8 and 9
(0.19±0.008 and 0.19±0.011, respectively; mean ± SD). Max-
imum cell densities in these treatments were reached around
21 days into the experiment (95.6×106 ±9×106 cells mL−1 for
pH 8 and 92.8×106 ±24×106 cells mL−1 for pH 9). Lipid
accumulation of unbuffered controls were 21.8±5.8 % fatty
acid methyl esters content by mass, and we were unable to
trigger additional significant lipid accumulation by manipulat-
ing pH levels at the beginning of stationary phase. Ciliates
(grazing predators) occurred in significant higher densities at
pH 6 (56.9±39.6×104 organisms mL−1) than higher pH treat-
ments (0.1–6.8×104 organisms mL−1). Furthermore, the addi-
tion of buffers themselves seemed to negatively impact diatoms
(algal competitors). They were more abundant in an unbuffered
M. L. Bartley : W. J. Boeing (*)
Department of Fish, Wildlife and Conservation Ecology,
New Mexico State University, Las Cruces, NM 88003, USA
e-mail: wboeing@nmsu.edu
B. N. Dungan : F. O. Holguin : T. Schaub
Chemical Analysis and Instrumentation Laboratory,
New Mexico State University, Las Cruces, NM 88003, USA
control (12.7±5.1×104 organisms mL−1) than any of the pH
treatments (3.6–4.7×104 organisms mL−1). In general, pH
values of 8 to 9 might be most conducive to increasing algae
production and minimizing invading organisms. CO2 addition
seems more valuable to algae as an inorganic carbon source and
not as an essential mechanism to reduce pH.
Keywords Algae biodiesel . Nannochloropsis salina . FAME
analysis . pH . Oil accumulation . Invaders
Introduction
The increase in atmospheric CO2 due to fossil fuel combustion
contributes to global climate change, which has created envi-
ronmental problems (Karl et al. 2009). Despite demands for
fossil fuel consumption reductions, renewable energy sources,
including biofuels, account for only 1.8 % of total world
energy consumption (BP 2011). Growing controversy about
the use of food crops for biofuel production (Brennan and
Owende 2010; Tufvesson et al. 2013) has turned attentions to
new sources. Microalgae are capable of year-round produc-
tion, require less water than terrestrial crops, and produce only
point-source wastewater and valuable co-products (Chisti
2007; Rodolfi et al. 2008; Mata et al. 2010). In addition,
microalgae are potentially valuable biological mitigators of
carbon dioxide due to their ability to convert CO2 (and nutri-
ents) into biomass that may in turn be utilized to obtain
biodiesel (Wang et al. 2008; Franchino et al. 2013). However,
the commercial viability of algal biofuel is limited by current
production systems, typically open ponds that are inexpensive
but often fail to maintain selected species for more than a few
weeks or months due to contamination by invading organ-
isms, both competitors and predators (Rodolfi et al. 2008).
Major contaminants that pose risks to these systems include
unwanted algae, yeast, molds, fungi, bacteria, and predators
1432
(Becker 1994; Borowitzka 1998). Optimized systems yielding
productive cultures that are resilient and resistant to invading
organisms are essential to the future of the biofuel industry.
Microalgae cultivation can be optimized by regulating
environmental parameters, such as salinity (Doan et al.
2011; Moazami et al. 2012; Bartley et al. 2013), temperature
(Sukenik et al. 1993; Rocha et al. 2003; Van Wagenen et al.
2012), and pH (Rocha et al. 2003; Moheimani and
Borowitzka 2011). However, how these parameters impact
lipid accumulation and invading organisms has been less well
studied. Research (Richmond and Becker 1986) demon-
strated that subjecting the culture to a temporary change
of environmental factors such as pH can decrease
unwanted organisms and improve final culture densities. An-
ecdotally, invading diatoms and ciliates (competitor and pred-
ator, respectively) of outdoor cultures of Nannochloropsis
salina were concurrent with low algae culture densities due
to nutrient limitation and predation (Boussiba et al. 1987).
Other research (Richmond et al. 1990) reported contamination
of a Spirulina platensis raceway pond with Dunaliella sp. and
Chlorella sp., which lowered productivity by up to 35 %.
Thus, contamination from unwanted species is still one of the
major challenges in algae cultivation (Das et al. 2011).
Members of the genus Nannochloropsis have been the
subjects of many studies exploring their promise for biofuel
development, as well as a source of important ω-3 polyunsat-
urated fatty acids for human consumption (Roessler 1990;
Cheng-Wu et al. 2001; Rocha et al. 2003) and feed for rotifers
in fish hatcheries (Chini Zittelli et al. 2000; Rebolloso-Fuentes
et al. 2001; Chini Zittelli et al. 2003). The marine microalga
N . salina (Eustigmatophyceae) has been shown to exhibit
high growth rates, lipid productivity, and a wide tolerance
range for different environmental parameters (Richmond and
Cheng-Wu 2001; Menke et al. 2012; Huang et al. 2013).
Day/night pH fluctuations driven by photosynthesis and
respiration create an environment that exhibits changing pH
ranges. During the day, photosynthesis and use of CO2 raise
pH levels. Respiration during the dark reverses this process
and lowers pH levels again. Inorganic carbon is an essential
microalgae macronutrient, and previous research (Yamasaki
and Hirata 1995; Arudchelvam and Nirmalakhandan 2012)
suggests that a carbon supply can increase densities of
Nannochloropsis spp. Abu-Rezq et al. (1999) could maintain
the pH of algae cultures in the pH range of 6.75–7.25 with
CO2 injections, while untreated cultures exhibited pH levels
as high as pH 8.28 by day 15. In a recent study, Moheimani
(2013) found an optimum pH for biomass and lipid produc-
tion of Tetraselmis suecica and Chlorella sp of pH 7.5 and 7,
respectively. Sodium bicarbonate and HCl were used to ma-
nipulate pH. These two compounds can form carbonic acid,
which can break down into water and CO2. Bicarbonate can
also be used directly by many microalgae as an inorganic
carbon source for photosynthesis (Giordano et al. 2005;
J Appl Phycol (2014) 26:1431–1437
Price et al. 2008; Maberly et al. 2009; Chi et al. 2011). To
date, effects of supplying inorganic carbon versus pH levels
have not fully been separated (but see Spolaore et al. 2006)
and the pH effect has mainly been simulated in models (James
et al. 2013). Further, pH may affect the lipid composition of
N. salina (Griffiths and Harrison 2009; Rodolfi et al. 2008;
Moheimani 2013). Lipid accumulation is maximized when
Nannochloropsis cells are stressed by unfavorable conditions
so that growth slows and energy is stored as lipids (Abu-Rezq
et al. 1999; Wang et al. 2009).
To our knowledge, no research has been conducted on N.
salina to separate the effects of CO2 and pH on growth rates,
lipid accumulation, and invading organisms simultaneously.
The present study explores the use of pH manipulations to
enhance growth rates of N. salina while minimizing undesir-
able organisms. In addition, this project explored the use of
pH stress to increase the lipid content within Nannochloropsis
cultures. This research is novel because of its focus on invad-
ing organisms and its unique attempt to utilize pH change as a
stressor for increased lipid accumulation. The implications of
this work may provide cost-effective, viable, and sustainable
development of algal biofuels industry.
Materials and methods
Nannochloropsis salina 1776, a marine microalga (Class:
Eustigmatophyceae), was obtained from the Provasoli–Guillard
National Center for Culture of Marine Phytoplankton via the Los
Alamos National Laboratory. The culture medium used was f/2
medium (Guillard and Ryther 1962), made with Instant Ocean in
non-sterile deionized water, bringing the salinity to a standard 34
PSU. All experiments were conducted in a greenhouse located at
New Mexico State University (Las Cruces, New Mexico, USA)
where open aquaria were subjected to natural light and temper-
ature conditions, as well as invading organisms. Algae culture
circulation was achieved with air stone aerators using natural air,
which also supplied around 400 ppm CO2. One experiment
examined the effect of a range of pH levels (pH 5–10) on growth
rate and abundance of N. salina and abundance of invading
organisms. The second experiment examined the effect of induc-
ing stress with varying levels of pH change (pH 7–9) on lipid
content of algae. To maintain desired pH levels within the media,
aquaria contained 25 mM of either (1) trisodium citrate for pH 5,
(2) 2-(N-morpholino)ethanesulfonic acid (Mes) for pH 6, (3)
4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (Hepes)
for pH 7, (4) N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine
(Tricine) for pH 8, or (5) N-tris[hydroxymethyl]methyl-3-amino-
propanesulfonic acid for pH 9 and 10. To separate effects of CO2
and pH, we utilized buffers and not CO2 injections to manipulate
pH. We maintained stock cultures of N. salina in an outdoor
raceway under standard cultivation conditions and then trans-
ferred aliquots to the greenhouse aquaria for experiments. Both
J Appl Phycol (2014) 26:1431–1437
experiments were conducted in aquaria with 15 L working
volume at a salinity of 34 PSU in f/2 medium. Temperature,
pH, and salinity readings were monitored mid-day daily with a
Hach Hydrolab (model MS 5, Hach Hydromet, USA) and
absorbance readings with a Thermo Spectronic spectrophotom-
eter. pH was also monitored on random nights to ensure that it
remained constant. pH was adjusted when measurements devi-
ated by 0.2 units or more. Samples for algae counts were pre-
served with Lugol’s solution and stored in dark, cool locations.
Growth rates and invading organisms (experiment 1)
Nannochloropsis salina cultures were grown at six different
pH levels (pH 5, 6, 7, 8, 9, and 10) in six replicates, in the
experimental aquaria described above. All aquaria were inocu-
lated with 250 mL of algae so that the initial N. salina abun-
dance in all tanks was approximately 1.750×106 cells mL−1.
We ran the experiment for 30 days in March and April 2011,
providing us with an average photoperiod of 12 h and
11 min of natural light within an unshaded greenhouse.
Temperature of the aquaria over the duration of the
experiment ranged from 17.2 to 25.7 °C. Sampling occurred
three times each week for algal density and on the last sam-
pling date for invading organisms (see “Algal Density Mea-
surements and Quantification of Invading Organisms”). All
samples were preserved with Lugol’s solution. Previous re-
search shows that this method preserves the greatest cell num-
bers (Leakey et al. 1994), but may shrink or distort cell mor-
phology (Stoecker et al. 1994).
Lipid accumulation (experiment 2)
The purpose of this experiment was to see if we would be able
to grow algae to maximum density and then change up the pH
as an additional stressor to trigger higher lipid accumulation.
We grew 24 algae cultures at an uncontrolled pH in aquaria as
described above until they reached stationary phase in De-
cember 2011. pH fluctuated between pH 7.81 and 9.70. Av-
erage photoperiod during that time was 10 h and 7 min and
temperature of the aquaria over the duration of the experiment
ranged from 14.3 to 21.2 °C. Stationary phase indicated
nutrient or light limitation of algae cultures. Changing pH of
an already buffered algae medium proved difficult, which led
to our decision to grow cultures with no initial buffering that
may have interfered with subsequent pH control. Aquaria
were inoculated with 1 L of stock algae, resulting in an initial
N. salina abundance of approximately 5.575×106 cells mL−1.
We used a higher starting cell density than in experiment 1, as
we were not interested in observing growth rates and to reach
stationary phase faster. Algae density was monitored daily
with absorbance and cell counts to identify when stationary
phase was reached, 16 days later. On day 17, we left the one
treatment unbuffered (control) and altered pH to 7, 8, and 9 for
1433
the other treatments (six replicates each) using the buffers
described above. One-liter samples were collected 2 weeks
after pH levels were altered for subsequent measurements of
algal density and lipid content (see sections below).
Algal density measurements and quantification of invading
organisms
Two metrics of algal density, absorbance at 740 nm and
cell number, were used to track population dynamics.
Absorbance was measured within 1 h of collection with
a Spec 20 (model D+; Thermo Fisher Scientific, USA)
and cell count samples were preserved with Lugol’s solu-
tion and stored in dark, cool locations for less than
3 months. Cell counts were conducted by counting at least
400 N. salina cells within a Neubauer hemocytometer. A
1-mL gridded Sedgewick–Rafter chamber was used to
count larger invading organisms (>30 μm). Invading or-
ganisms were counted within the entire chamber, provid-
ing us with direct estimates of organisms per milliliter.
Smaller organisms were counted using a hemocytometer.
At least 100 invading organisms were counted for each
sample. Growth rates were calculated from cell counts of
experiment 1 from inoculation until all treatments had
approximately reached their maximum densities (day 0–
day 21) and showed no more positive population growth
with the following formula:
lnðd n Þ−lnðd 0 Þ
r¼
Δt
where d = density, n = day of experiment, and Δt = time in
days
Fatty acid methyl ester analysis
Lipid content was monitored by transesterification and analy-
sis of fatty acid methyl esters (FAME) by gas chromatography
mass spectrometry (GC/MS) for each treatment. Algae from
each treatment were collected on a pre-weighed disk filter,
dried, and placed in a pre-weighed 60-mL glass vial to which
30 mL 0.2 N KOH in methanol was added and allowed to
stand for 30 min at 40 °C in a water bath. An internal standard,
glyceryl tritridecanoate, was included in the reaction to mon-
itor conversion efficiency. Each sample vial was vortexed in
10-min intervals. The reaction was quenched after 30 min
with 1 mL of 1 M glacial acetic acid. Each sample was back
extracted with 10 mL hexane with methyl tricosanoate C23:0,
an internal standard for quantification/internal standard cali-
bration. The hexane fraction was further diluted and analyzed
by GC/MS under conditions as described by Patil et al.
(2012). Briefly, all samples were analyzed with a Hewlett
Packard 5890 gas chromatograph with a 5972 Mass Selective
1434
Detector and 7673 Autosampler. Two-microliter injections
were loaded onto a 30 m×0.25 mm diameter × 0.25 μm film
Agilent DB-23 capillary column with helium carrier gas. The
initial temperature was 80 °C and ramped 20 °C min−1 to
220 °C and held for 6 min for a total run time of 13.3 min.
Chromatographic signals were matched to a standard mix
(Supelco 37 Comp. FAME mix 10 mg mL−1 in methylene
chloride) and internal calibration was achieved using a C23:0
internal standard spiked at 50 μg mL−1.
Statistical analyses
Linear regression was used to evaluate the effect of pH on
maximum cell density, and growth rate. Optimum conditions
were estimated for each of these response variables. Data were
checked for equal variance and normality using residual plots.
Analyses were conducted in R software, and α was set to
0.05. One-way ANOVAs were used to test the effect of total
lipid content, and invading organism abundance. Data were
checked for equal variance and normality using Levene’s and
Shapiro–Wilk tests. Analyses were conducted in SAS 9.2, and
α was set to 0.05. Data in Fig. 1 were smoothed with a three-
sampling-date average for better presentation of growth
curves and maxima reached in each treatment.
Results
Nannochloropsis growth (experiment 1)
Maximum cell densities observed were in pH 8 and 9 and
were reached around 21 days into the experiment (95.6×106 ±
9×106 cells mL−1 for pH 8 and 92.8×106 ±24×106 cells mL−1
for pH 9). Maximum cell densities can be predicted from pH
by the following formula (Fig. 1; p <0.0001, R 2 =0.72):
100
5
Nannochloropsis Cell Density * 106 (mL-1)
6
7
80
8
9
60 10
40
20
0 Fig. 2 Average % FAME accumulation ± SE (n =3) in Nannochloropsis
0 5 10
Experiment Duration (days)
Fig. 1 Nannochloropsis mean cell density ± SE (n =6) in six different
pH levels (5, 6, 7, 8, 9, and 10)
J Appl Phycol (2014) 26:1431–1437
Y Density ¼ −755; 029 þ 216; 883  pH−13; 958  pH2
The optimum pH for cell density was estimated to be pH 7.8.
pH also can be used to predict the growth rates of N. salina
(p <0.0001, R 2 =0.86).
Y GRate ¼ −1:61 þ 0:47  pH−0:03  pH2
The optimum pH for growth rate was estimated to be
pH 7.7. Growth rates were highest for pH 8 and 9 (both
0.19), followed by pH 7 (0.18).
Lipid accumulation (experiment 2)
Lipid accumulation appeared to be unaffected by pH; however,
the greatest mean accumulation occurred in the pH 8 treatment
with averaging 24.75 % by mass, compared to 21.8 % by mass
in unbuffered controls (Fig. 2; one-way ANOVA—df = 3, F =
2.79, p =0.0767). The FAME profile shows the dominant fatty
acids to be 14:0, 16:0, 16:1, and 18:1 (Fig. 3). Also present in
lower abundance is C20:5n3. Figure 3 shows acyl hydrocarbon
content (measured as FAME) as a percentage of lyophilized
tissue mass. Accumulation of unsaturated, saturated, and mono/
polyunsaturated lipid is similar across treatments, and no sig-
nificant differences were found (Table 1).
Invading organisms (experiments 1 and 2)
Densities of invading organisms were evaluated for day 30 of
the first experiment. Ciliates appeared consistently throughout
all treatments, while diatoms appeared in only two aquaria,
and thus, only the former were included in analysis. The pH
levels effects growth of ciliates (one-way ANOVA—df = 3, F
= 2.91, p =0.0004). A subsequent Tukey’s test (Hsu 1996)
shows that ciliate abundance was lower at pH 7–9 than at
pH 6 (Table 2).
15 20 25 30 salina by weight 2 weeks subsequent to pH alterations. All cultures began
growth without pH control and pH was buffered to pH 6, 7, 8, and 9 in
select treatments. Control treatments exhibited pH levels between pH 8.6
and 9.25
J Appl Phycol (2014) 26:1431–1437 1435

Table 2 Mean densities ± SE of invading organisms at different levels of

pH

pH Phytoplankton Zooplankton
(diatoms mL−1) (ciliates mL−1)

Exp. 1 6 0 569,200 ± 161,890

7 2,800 ± 2,800 12,900 ± 5,620
8 0 1,160 ± 860
9 270 ± 270 67,900 ± 65,810
Exp. 2 Control 127,300 ± 29,570 0
7 36,500 ± 13,770 0
8 43,900 ± 6,510 0
9 47,400 ± 7,200 0

Fig. 3 FAME profile of lipid extracts from control and each pH treatment
group represented as % of total FAME
Invading organism densities for the second experiment
were also analyzed. These cultures contained exclusively
diatoms (Table 2). pH significantly effected diatom density
(one-way ANOVA—df = 3, F = 6.27, p =0.0170). Tukey’s
test shows a significant lower diatom density in treatments
buffered to pH 7, 8, and 9 compared to the unbuffered control.
Discussion
Our research suggests that a pH of 8 might be ideal to maximize
growth, reach high cell densities, and maximize lipid
accumulation while minimizing abundance of undesired organ-
isms. This is one of few experiments that separate the effects of
CO2 addition, a required component of photosynthesis, from
the effects of pH changes. Spolaore et al. (2006) utilized buffers
to explore the effect of pH on Nannochloropsis oculata growth
rates. However, they do not consider lipid accumulation and
their experiment is conducted in a laboratory setting, making
invading organisms less of an issue. Rocha et al. (2003) utilized
a buffer in their research, but they did not explore the effects of
different pH levels; instead, they were able to show that Tris/
HCl buffer (40 mM), keeping a constant pH 8, had a positive
effect on the growth rate of Nannochloropsis gaditana.
Our observed optimum pH range for N. salina growth was
pH 8–9. We found that N. salina was not able to grow at pH 10,

Table 1 Percent of total FAME distribution between control versus treatments

Control pH 7 pH 8 pH 9

C12:0 0.20 ± 0.000 0.24 ± 0.000 0.23 ± 0.000 0.21 ± 0.000

C14:0 3.08 ± 0.002 3.56 ± 0.002 3.22 ± 0.001 3.20 ± 0.001
C15:0 0.37 ± 0.001 0.45 ± 0.000 0.40 ± 0.000 0.37 ± 0.000
C16:0 37.59 ± 0.010 36.88 ± 0.006 37.54 ± 0.006 37.16 ± 0.013
C16:1 43.38 ± 0.010 44.88 ± 0.006 43.72 ± 0.004 43.79 ± 0.006
C17:0 0.31 ± 0.001 0.34 ± 0.000 0.29 ± 0.000 0.31 ± 0.000
C18:0 1.95 ± 0.001 1.69 ± 0.002 1.82 ± 0.001 1.90 ± 0.001
C18:1n9c 8.88 ± 0.005 7.56 ± 0.005 8.30 ± 0.004 9.01 ± 0.005
C18:2n6c 0.47 ± 0.001 0.47 ± 0.001 0.48 ± 0.000 0.47 ± 0.000
C18:3n6 0.48 ± 0.001 0.37 ± 0.000 0.54 ± 0.001 0.38 ± 0.002
C20:3n6 0.32 ± 0.001 0.35 ± 0.001 0.38 ± 0.000 0.34 ± 0.000
C20:4n6 0.97 ± 0.001 1.00 ± 0.003 0.96 ± 0.001 0.96 ± 0.002
C20:5n3 1.99 ± 0.005 2.18 ± 0.005 2.13 ± 0.005 1.90 ± 0.003
Sum of saturated 43.51 43.18 43.50 43.15
Sum of monounsaturated 52.26 52.45 52.02 52.80
Sum of polyunsaturated 4.23 4.37 4.48 4.05
Sum of unsaturated 56.49 56.82 56.50 56.85
Sum of polyunsaturated/sum of unsaturated 0.10 0.10 0.10 0.09
Sum of monounsaturated/sum of polyunsaturated 12.35 11.99 11.62 13.02
1436
while still growing well at pH 7. This led to our regression
analysis predicting pH 7.7 as the optimum pH, closer to pH 7/8
than 9/10. Our results are similar to findings by Spolaore et al.
(2006) who also did not alter inorganic carbon supply, with an
optimum pH of 8.4 for N. oculata. Most other studies in which
pH manipulation occurs through CO2 supply typically find
lower pH optima (7–7.8) for Nannochloropsis spp (Abu-Rezq
et al. 1999; Brown et al. 1993; Sukenik et al. 1993). Growth
rates for previous experiments employing a similar setup than
used in this study (Bartley et al. 2013), where algae were grown
unbuffered at salinities of 34 PSU, were less than growth rates
for this experiment at treatments buffered to pH 8 or 9 (0.13 vs.
0.19, respectively). And while these growth rates are only
relative values obtained in a model system not aimed at max-
imizing growth rates in general, this suggests that the buffers for
pH 8 and 9 did not negatively impact growth of N. salina.
However, since we used different buffers for different pH
values, we cannot exclude the possibility that some of the other
buffers were toxic to the algae. However, we are able to argue
that Nannochloropsis cultures are able to tolerate high pH
values (8 and 9). This is independent of findings that algae
can benefit from carbon addition, when CO2 is used to manip-
ulate pH, and thus may show higher growth rates at lower pH.
We were able to find only one previous study that explored
the effects of pH on lipid accumulation. Moheimani (2013)
found pH 7 and 7.5 ideal for lipid accumulation in Tetraselmis
suecica and Chlorella sp. While we found no significant effect
of pH change on lipid accumulation, the treatment with a pH
change to 8 exhibited the greatest overall accumulation (aver-
aging 24.75 % by mass). Another study (Griffiths and Harrison
2009) reports average lipid contents of 27 % of dry weight
(nutrient replete) and 46 % of dry weight (nitrogen deficient).
Rodolfi et al. (2008) found that lipid content (% biomass) for
different Nannochloropsis spp to be 24.4–35.7 %. This indi-
cates that pH might not be an important stress factor that
triggers increased lipid accumulation. Previous research
(Bartley et al. 2013) indicated that other stressors like salinity
(increase from 22 to 34 PSU when reaching stationary phase)
can cause a significant increase in lipid content (35 % by
weight, compared to 17 % with no salinity increase). pH also
had no effect on the fatty-acyl profile observed for each treat-
ment. In general, we found a low abundance of C20:5n3 in N.
salina, which is the ω-3 fatty acid eicosapentaenoic acid meth-
yl ester, an essential fatty acid and known health supplement.
In the experiment described here, invading organisms were
limited to ciliates and diatoms. We found that invading ciliates
are highest at pH 6 and lower at high pH values. This finding
is probably specific to the ciliate species invading the aquaria.
Other research has indicated that freshwater ciliate Urotricha
castalia shows positive growth above pH 6.5 (Weisse and
Stadler 2006). Other research has suspected that high pH
values (up to pH 11) during daytime in Pleurochrysis carterae
cultures limit algae contamination due to restriction of
J Appl Phycol (2014) 26:1431–1437
available inorganic carbon (Moheimani and Borowitzka
2011). Diatoms did not appear to be influenced by pH, rather
than the addition of buffers themselves. To date, we have no
explanation for this finding. Generally, invading organisms
are difficult to evaluate and will vary by geographical location
and season. Thus, much more research on invading organisms
is needed. Unfortunately, we did not consider bacteria and
viruses in our study. Another essential component of invaders
(SevrinReyssac et al. 1996), especially when considering
alternative water sources like wastewater (Sigala and Unc
2012) for algae cultivation, should be more closely observed
in the future. Invading organisms occurred at low density and
are unlikely to have significantly altered lipid measurements
in this experiment.
In conclusion, we find that pH of around 8 seems most
beneficial for maximum growth rate and lipid accumulation of
N . salina and to minimize invading organisms. Although
adding buffers will not be cost effective or realistic at a large
scale, we were able to demonstrate that higher pH values per
se do not slow Nannochloropsis production. Thus, the addi-
tion of CO2 at large scales is mostly valuable for providing an
inorganic carbon source for algae.
Acknowledgments We are grateful for the valuable work from the
following undergraduate students: Herman Campos, Levi Chavez, Renee
Pardee, Herberto Chaparro, and Zach Brecheisen. Darren James provided
valuable help with statistical analyses for this research. This work is
supported by the U.S. Department of Energy under contract DE-
EE0003046 awarded to the National Alliance for Advanced Biofuels
and Bioproducts. This is a New Mexico Agricultural Experiment Station
publication, supported by state funds and the U.S. Hatch Act.
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