Algal Research 16 (2016) 141149

Contents lists available at ScienceDirect

Algal Research
journal homepage: www.elsevier.com/locate/algal

Effect of temperature and nitrogen concentration on lipid productivity

and fatty acid composition in three Chlorella strains
Vince rdg a,b, Wendy A. Stirk b,, Pter Blint a, Adeyemi O. Aremu b, Ambrose Okem b, Csaba Lovsz c,
Zoltn Molnr a, Johannes van Staden b
a
b
c

Institute of Plant Biology, Faculty of Agricultural and Food Sciences, University of West Hungary, 9200 Mosonmagyarvr, Hungary
Research Centre for Plant Growth and Development, School of Life Sciences, University of KwaZulu-Natal, Pietermaritzburg, P/Bag X01, Scottsville 3209, South Africa
Central Agricultural Ofce, Food and Feed Safety Directorate, Feed Investigation National Reference Laboratory, Remny Street 42, 1114 Budapest, Hungary

a r t i c l e

i n f o

Article history:
Received 21 July 2015
Received in revised form 26 February 2016
Accepted 1 March 2016
Available online xxxx
Keywords:
Carbohydrates
Fatty acids
Lipids
Nitrogen-deprivation
Proteins
Temperature

a b s t r a c t
The effects of temperature and nitrogen (N) on lipid productivity and fatty acid composition in three Chlorella
strains grown at 20 C, 25 C and 30 C in modied Tamiya media with low (7 and 21 mg L1 N) and moderate
(70 mg L1 N) N were investigated. Temperature and N inuenced biomass accumulation with the largest
biomass accumulation (1697, 1732 and 1809 mg DW L1 for the three strains) at higher temperatures and N concentrations. Proteins decreased and lipids increased over time with N-deprivation. Strain, temperature and N
concentration inuenced lipid productivity with the highest productivity in cultures grown in 3% N at higher
temperatures (68, 70 and 90 mg lipid L1 day1 for the three strains). The fatty acid methyl ester (FAME) prole
was similar in the three strains with C16:0 N C18:1n9c N unidentied FAMEs N C18:2n6c N C18:3n3 N C18:0
comprising 90% of the total FAME. C18:1n9c and C16:0 showed the largest variation in response to culture
conditions in Chlorella sp. MACC-438 (0.226.8% and 2.316.2% respectively) and C. minutissima MACC-452
(0.226.7% and 3.023.3% respectively); unidentied FAMEs (1.19.7%) and C18:2n6c (0.911.2%) were the
most variable in Chlorella sp. MACC-728. Lower temperatures resulted in higher % FAME in Chlorella sp. MACC438 (68.9%) and C. minutissima MACC-452 (91.7%). Nitrogen concentration had more inuence on the specic
FAME content compared to temperature. Thus, temperature tolerance is important when selecting strains to ensure high lipid productivity while the specic strains response to N-deprivation is important to ensure quality of
the biofuel feedstock.
2016 Elsevier B.V. All rights reserved.

1. Introduction
Owing to limited fossil fuel reserves, alternative sources of
renewable energy are being investigated. Oleaginous microalgae are
able to accumulate high amounts of reserve lipids in the form of
triacylglycerides (TAGs) when cultured in growth limiting conditions.
TAGs are a suitable substrate for biodiesel production as well as a source
of various long chain fatty acids such as C20:4 (arachidonic acid), C20:5
(eicosapentaenoic acid) and C22:6n3 (docosahexaenoic acid). These
are valuable bioactive compounds for human and animal use [1,2].
Microalgal species, especially those grown in outdoor cultivation
systems need a wide tolerance to environmental conditions. Geographical location and climatic conditions, especially temperature and solar

Abbreviations: FAME, Fatty acid methyl ester; HCA, Hierarchical Cluster Analysis;
MACC, Mosonmagyarvr Algal Culture Collection; MUFA, Monounsaturated fatty acid;
PUFA, Polyunsaturated fatty acid; SFA, Saturated fatty acid; TAGs, Triacylglycerides.
Corresponding author.
E-mail address: stirk@ukzn.ac.za (W.A. Stirk).

http://dx.doi.org/10.1016/j.algal.2016.03.001
2211-9264/ 2016 Elsevier B.V. All rights reserved.

radiation are the main environmental factors affecting lipid productivity

[3,4].
Microalgae strains suitable for commercialization should have a fast
growth rate and high lipid content. Promising strains have been identied from the genera Chlorella, Nannochloropsis, Neochloris and
Scenedesmus [3,5]. Most commonly, nitrogen (N) deprivation is applied
as the growth limiting factor as it causes a signicant increase in the
lipid content of many microalgae, is cost effective and is one of the
more easily adjustable factors in both open and closed systems [6]. A
survey of the available literature calculated the average lipid content
in a variety of microalgal cultures grown in N-sufcient conditions as
23% dry weight (DW), which increased in N-limiting conditions to
41% DW [7]. However, fast growth rates (achieved in optimal growth
conditions) and high lipid content (stimulated by growth limiting
conditions) are mutually exclusive with high lipid yield often accelerating with the onset of the stationary growth phase in batch cultures [6,8].
While many studies have reported on ways to optimize microalgal
biomass and lipid productivity, there are fewer reports on the quality
of the lipids produced [reviewed in 6,7]. Commercialization of biodiesel
has led to the formulation of specic biodiesel standards and guidelines

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V. rdg et al. / Algal Research 16 (2016) 141149

with the properties of biodiesel directly determined by the fatty acid

composition of the microalgal feedstock [6,9]. The two most important
factors are i) the length of the carbon chain with the long chain C16C18 TAGs being the best substrate for biodiesel production and ii) the
number of double bonds which affect other biodiesel properties such
as cetane number (ignition quality), cold-ow properties, viscosity
and oxidative stability [9]. To meet the specic technical biodiesel
standards and to achieve the best balance between these properties,
the most desirable TAGs are those containing the monounsaturated
fatty acids (MUFAs) C18:1 (oleic acid) and C16:1 (palmitoleic acid).
High concentrations of saturated fatty acids (SFAs) negatively affect
the cold-ow properties and the polyunsaturated fatty acid (PUFA)
C18:3 (-linolenic acid) content is required to be below 12% as higher
concentrations result in poor oxidative stability [9]. Thus the fatty acid
composition is important in determining the quality of the microalgal
feedstock for biodiesel production.
While the fatty acid composition in microalgae can be used as a
taxonomic biomarker at the generic level, e.g., separating a true Chlorella clade from the Parachlorella clade in indigenous Chlorella strains from
Malaysia [10], environmental variables can also alter the fatty acid
composition of a species [6,9]. Physical cultivation parameters such as
temperature and light intensity as well as media characteristics such
as the source and concentration of N, salinity and pH inuence the
fatty acid composition and hence both the quantity and quality of lipids
produced [11]. For example, when the arachidonic acid- producing
Parietochloris incisa (Trebouxiophyceae) was grown in high light conditions to facilitate rapid growth, there was a low C20:4 content, while Ndeprivation increased the C20:4 content in proportion to the total fatty
acid content, allowing the cultures to be manipulated to achieve a
higher C20:4 content [2].
Lipid production of specic strains in different culture conditions is
required to predict overall lipid productivity in outdoor systems [4].
By investigating the relationship between fatty acid composition and
environmental factors in microalgae, suitable strains can be selected to
take into account the effect of uncontrollable environmental changes
such as seasonal trends. As temperature is one of the main environmental factors, which inuence fatty acid proles in microalgae [12], the aim
of the present study was to investigate the effects of temperature and N
concentrations in three fast growing, oleaginous Chlorella strains.
Growth, protein, carbohydrate, lipid content, and fatty acid composition
were quantied, and lipid productivity calculated. These results should
provide insight on lipid productivity and fatty acid composition in
microalgae in response to variable ambient temperatures.
2. Materials and methods
2.1. Microalgal cultures and growth conditions
Three fast growing axenic Chlorella strains namely Chlorella sp.
MACC-438, Chlorella minutissima MACC-452 and Chlorella sp. MACC728 isolated from soil samples collected in Brazil, were selected from
the Mosonmagyarvr Algal Culture Collection (MACC), Hungary.
Culture suspensions were initiated from agaragar stock cultures and
grown in modied Tamiya medium [13,14] where the nitrogen concentration in the media was reduced from 700 mg L1 N (100% N) to
70 mg L 1 N (10% N). This nitrogen concentration was selected for
the present study as previous experiments using the same culture apparatus and C. minutissima MACC-452 showed that the highest lipid
productivity was obtained with 10% N [14]. Culture conditions were
25 2 C, 14:10 h light:dark photoperiod and illumination from
below at 130 mol m2 s 1. Cultures were aerated with 20 L h 1
1.5% CO2-enriched sterile humidied air during the light periods.
After 7 days, cultures of each strain were inoculated into asks containing 250 mL modied Tamiya medium (10% N) to give an algal starting
density of 10 mg L1 DW. After 7 days, cultures were re-inoculated in the
same way to obtain sufcient inoculum for the experiment. For the

experiment, the Tamiya medium was modied to give two low nitrogen
concentrations 7 and 21 mg L1 N (1% and 3% N respectively of the total
N content in Tamiya media) and a moderate nitrogen concentration
70 mg L1 N (10% N). The asks were inoculated to give an algal starting
density of 10 mg L1. These cultures were grown at three temperatures,
namely 20 C, 25 C and 30 C. For each algal strain and treatment, 18
and 10 asks were harvested on day 3 and day 6 respectively. Samples
were taken from three randomly selected asks for biomass quantication. Thereafter, the biomass from all the asks was combined to give a
single sample for each strain and for each sampling time (days 3 and
6) to ensure sufcient biomass for the biochemical analysis. Samples
were lyophilized and stored at 70 C until analysis.
2.2. Dry weight quantication
Samples (520 mL) were ltered and dried as previously described [14]
and the density of the suspension cultures calculated as mg DW L1. As
all cultures had the same initial biomass (10 mg L1 DW), increase in biomass accumulation was used as a measure of growth. There were three
replicates per treatment. Three-way analysis of variance (ANOVA)
comparing the effect and interaction of temperature, N concentrations
and time of harvest was performed on the biomass data. Statistical computations were done using SPSS for Windows (SPSS, version 10.0 Chicago,
USA).
2.3. Estimation of protein content
The N content of the microalgae samples (250 mg DW) was
determined using a Kjeldahl method and the protein content calculated
from the N content as previously described [14]. Results were expressed
as g protein 100 g1 DW (%).
2.4. Carbohydrate determination
The carbohydrate content of the samples was measured using a
method based on the MSZ 6830/26 Hungarian Standard (Determination
of feed nutritive value: Determination of sugar content). Microalgal
samples (150500 mg DW) were extracted in 50 mL hot (50 C) deionized water after which 5 mL HCl (37% w/w) was added and the asks
heated to 100 C for 45 min. During this extraction, the asks were gently shaken on a horizontal shaker. The extracts were cooled and claried
with the addition of 510 mL Carrez I and Carrez II solutions (see above
MSZ 6830/26 Standard Method) and deionized water. After 15 min, the
solutions were ltered on lter paper. The glucose content of the ltrate
was determined by the Luff-Schoorl method with titration (see above
MSZ 6830/26 Standard Method). Various amounts of ltrate (5
25 mL) made up to volume with deionized water and a blank prepared
with 25 mL deionized water were prepared and 2 mL NaOH (20% w/w)
and 25 mL Luff-Schoorl reagent solution added. The solutions were
heated to reux and reuxed for an additional 10 min. The reagent
mixture was cooled with cold water and 10 mL potassium iodide solution (30% w/w) added, followed by 25 mL 3 M sulfuric acid. The mixture
was titrated with 0.1 M sodium thiosulfate solution with the addition of
starch solution as an indicator. Based on the titration, the carbohydrate
content in the microalgal samples was calculated.
2.5. Lipid determination and productivity
Lipids were quantied using two parallel samples (150 mg DW) that
were hydrolyzed with 3 M HCl at 95100 C and then extracted using
methanol, hexane and diethyl ether as previously described [14]. The
resulting extract was dried and weighed as a measure of lipid yield
that was expressed as g lipid 100 g 1 DW (%). Lipid productivity
on day 3 and day 6 was calculated using the DW and lipid content as
previously described [14].

V. rdg et al. / Algal Research 16 (2016) 141149

143

[15]. Individual fatty acid methyl ester (FAME) peaks were identied according to their retention times in comparison to the retention times of
known FAMEs in a reference solution (Supelco 37 Component FAME
Mix, Sigma-Aldrich Corp.). All medium and long chain FAMEs (C8-24)
present in the samples were identied, while short chain (bC8) and
very long chain (N C24) FAMES were not identied and grouped as
unidentied fatty acids. Using a standard calibration curve, FAME
content was calculated as g FAME 100 g1 lipid (% FAME in lipid) [15].
Two sets of dendrograms were generated using Hierarchical Cluster
Analysis (HCA) on the FAME data for each microalgal strain to determine, i) FAMEs that showed the most variation in concentration in
response to the culture conditions; and ii) which of the tested culture
conditions resulted in the largest variation in the FAME composition.
This was an unsupervised technique that involved classication and
measurement of similarity between samples to be clustered. The HCA
technique was applied to the data sets using Ward's method with
Euclidean distance to calculate the sample interpoint distance.
3. Results
3.1. Biomass accumulation
There was a signicant increase in biomass from day 3 to day 6 in
all the cultures. Both temperature and N concentration also had a
signicant effect on biomass with the higher temperatures (25 C and
30 C) and moderate 10% N concentration having the highest biomass
accumulation on day 6 (16971809 mg DW L1), while the cultures
grown at 20 C in 1% N had the lowest biomass accumulation (393
583 mg DW L1; Fig. 1). Three-way ANOVA revealed signicant differences between temperature, N and time in culture in the three Chlorella
strains (Table 1).
3.2. Macromolecule content

Fig. 1. Biomass accumulation in three Chlorella strains grown in various nitrogen

concentration and temperature combinations. Cultures were harvested on day 3 and
day 6. Data is presented as mean SE (n = 3).

2.6. Fatty acid analysis

After determining the lipid content, the dried residue was
methylated, an internal standard (ethyl tridecanoate) added and the
samples analyzed by gas chromatography as previously described

Protein content decreased over time with N concentration, strain

and temperature having an effect. For Chlorella sp. MACC-438 and
Chlorella sp. MACC-728 grown in low (1%) N medium, the protein content decreased from day 0 (2226% DW; indicated as a dotted line in
Fig. 2) so that by day 3, the proteins were at their lowest (1023%
DW) and remained so up to day 6 (1012% DW). Although following a
similar trend, the protein content had a slower decrease with the 3% N
treatment, showing little change from day 0 to day 3 (2129% DW)
but decreasing from day 3 to day 6 (1416% DW). In the moderate
(10%) N treatments, the protein content initially increased from day 0
to day 3 (4760% DW) and then decreased from day 3 to day 6 (35
47% DW), although the concentrations remained above the day 0 protein content (Fig. 2A and G). Although C. minutissima MACC-452 followed a similar trend as the other strains when grown at 30 C, in the lower
temperatures (20 C and 25 C), the protein content in the 1% N cultures
only decreased from day 3 (1823% DW) to day 6 (612% DW), while
the protein content initially increased in the cultures grown in 3% N
and then decreased from day 3 (3747% DW) to day 6 (16% DW;
Fig. 2D).

Table 1
Three-way analysis of variance of the effect of harvest time (day), temperature and nitrogen concentrations on biomass accumulation in three Chlorella strains.
MACC-438

MACC-452

MACC-728

Source of variation

F value

P value

F value

P value

F value

P value

Day
Nitrogen
Temperature
Day temperature
Day nitrogen
Temperature nitrogen
Day temperature nitrogen

2158.52
1248.69
92.318
11.876
336.361
49.728
7.347

0.000
0.000
0.000
0.000
0.000
0.000
0.000

1511.93
907.013
166.271
5.837
163.442
24.091
4.201

0.000
0.000
0.000
0.006
0.000
0.000
0.007

771.346
821.564
52.287
1.656
170.54
5.497
1.785

0.000
0.000
0.000
0.205
0.000
0.001
0.153

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V. rdg et al. / Algal Research 16 (2016) 141149

Fig. 2. Protein, carbohydrate and lipid content of three Chlorella strains grown in various nitrogen concentration and temperature combinations. Cultures were harvested on day 3 and day
6. The dashed line indicates the protein, carbohydrate and lipid content on day 0. Biomass from 18 asks (day 3) and 10 asks (day 6) were combined prior to analysis to ensure sufcient
biomass for two parallel samples.

Changes in the carbohydrate content were not as large as for the

protein and lipid contents with each strain having a different response
to N levels and temperature. In the low (1% and 3%) N media, there
was generally no change or a decrease in the carbohydrate content
from day 0 (37% DW) to day 3 in Chlorella sp. MACC-438 (1839%
DW) and C. minutissima MACC-452 (1443% DW). There was no discernible trend with some treatments showing an increase and others
a decrease in carbohydrate content from day 3 to day 6 (2140% DW;
Fig. 2B and E). In contrast, the carbohydrate content increased from
day 0 (28% DW) to day 3 in Chlorella sp. MACC-728 when grown in
1% N and 3% N (3240% DW) at all temperatures and then generally
decreased from day 3 to day 6 (2131% DW) to levels similar to day 0
(Fig. 2H). In all three strains grown in 10% N media, there was an
initial decrease in carbohydrate content from day 0 to day 3 (7
20% DW) with the largest decrease at the lowest temperature
(20 C). However, from day 3 to day 6, the carbohydrate concentration increased (2129% DW) although it remained less than at day
0, with the largest increase in the 25 C and 30 C treatments
(Fig. 2B, E and H).
Lipid content increased over time from day 0 (922% DW) to day 6
(3747% DW) in Chlorella sp. MACC-438 and Chlorella sp. MACC-728
when grown in low 1% N and 3% N media regardless of temperature.
The lipid content remained low when grown in moderate (10%) N
media with little change or decreasing over the 6 days (1118% DW;
Fig. 2C and I). As with the protein content, C. minutissima MACC-452
had a different response with generally lower lipid content than the
other two strains. In C. minutissima MACC-452, the lipid content generally decreased or remained unchanged from day 0 (19% DW) to day 3
(727% DW) in most of the nitrogen and temperature treatments.
From day 3 to day 6, there was an increase in the lipid content when
grown in the lower (1% and 3%) N media so that by day 6 (2235%
DW), the lipid content was higher than at day 0. The lipid content
remained low in the 10% N treatments (813% DW; Fig. 2F).

3.3. Lipid productivity

Chlorella sp. MACC-728 generally had the highest lipid productivity
(up to 96 mg L 1 day1) of the three strains investigated (Fig. 3C),
and C. minutissima MACC-452 the lowest productivity (up to 70 mg L1day1; Fig. 3B) owing to its lower lipid content (Fig. 2F). The highest
lipid productivity was recorded when the three strains were grown
in 3% N. Temperature had little effect on the productivity of Chlorella
sp. MACC-438, with similar rates measured on day 6 for cultures
grown at the three temperatures (3648 mg L1 day1 at 20 C; 65
68 mg L1 day1 at 25 C; 2832 mg L1 day1 at 30 C; Fig. 3A). In
contrast, the highest productivity was measured in C. minutissima
MACC-452 (5470 mg L 1 day 1) and Chlorella sp. MACC-728 (90
96 mg L1 day1) grown at 30 C in 3% N, and the lowest productivity
was recorded in the cultures grown at 20 C (2335 mg L1 day 1 and
3755 mg L1 day1 respectively; Fig. 3B and C). Time also inuenced
lipid productivity with Chlorella sp. MACC-438 and C. minutissima
MACC-452 having higher productivity on day 6 compared to day 3
(Fig. 3A and B), while lipid productivity was higher on day 3 compared
to day 6 in Chlorella sp. MACC-728 (Fig. 3C).

3.4. FAME composition

There was a decrease in the proportion of FAME in the lipid content
from day 0 (5367%) to day 3 in most treatments, with the largest
decrease occurring in the 10% N cultures (622%; Fig. 4). The exceptions
were C. minutissima MACC-452 grown in 1% N at 30 C (67%; Fig. 4B),
and Chlorella sp. MACC-728 grown in 1% N at 25 C (67%) and 30 C
(52%; Fig. 4C), where there was no decrease in the % FAME. This was
followed by an increase in the proportion of FAME in the lipid content
from day 3 to day 6, with a higher proportion FAME in the cultures
grown in low (1% N and 3%) N media (5091%), and little increase in

V. rdg et al. / Algal Research 16 (2016) 141149

Fig. 3. Lipid productivity of three Chlorella strains grown in various nitrogen concentration
and temperature combinations. Cultures were harvested on day 3 and day 6, where
biomass from 18 asks (day 3) and 10 asks (day 6) were combined to ensure sufcient
biomass for analysis.

most cultures grown in 10% N (430%; Fig. 4). The exceptions were
C. minutissima MACC-452 grown in 10% N at 25 C (41%), and Chlorella
sp. MACC-728 grown in 10% N at 30 C (61%), where there was a large
increase in % FAME from day 3 to day 6 (Fig. 4B and C).
Temperature also inuenced the proportion of FAME with the
highest FAME content at 20 C in Chlorella sp. MACC-438 (69%) and
C. minutissima MACC-452 (92%), while temperature had little effect on
Chlorella sp. MACC-728 (Fig. 4).
In total, between 24 and 27 FAMEs were detected in the samples
as well as some unidentied short (b C8) and very long chain (N C24)
FAMEs (Supplementary Table A).When the results from all the
treatments were combined, the three strains had a similar FAME prole
with seven predominant FAMEs (C16:0 N C18:1n9c N Unidentied
FAMEs N C18:2n6c N C18:3n3 N C18:0 [stearic acid]). These contributed
approximately 90% to the total FAME content (Table 2). In some cases,
specic long chain fatty acids e.g. C22:0 (behenic acid), C24:0
(lignoceric acid) and C24:1n9 (nervonic acid) were only detected in

145

Fig. 4. The proportion of FAME in the lipid content of three Chlorella strains grown in
various nitrogen concentration and temperature combinations. Cultures were harvested
on day 3 and day 6. The dashed line indicates the % FAME on day 0.

cultures harvested on day 6. This was probably due to an increase in

the fatty acid content over time, such that these FAMEs were above
the limit of detection by day 6 (Supplementary Table A).
The composition of the FAME content also changed in response to
culture conditions. In all instances, there were lower SFA and MUFA
and higher PUFA and unidentied FA components in the cultures
grown in 10% N compared to the lower N treatments in the three strains
(Fig. 5). Some medium chain fatty acids (e.g., C8:0 (caprylic acid), C11:0
(undecylic acid)) and long chain fatty acids (e.g., C20:2 (eicosadienoic
acid)) were also only detected in the cultures grown in 10% N. The
number of long chain FAMEs detected also increased with increasing
temperature, with C18:2n6t (linolelaidic acid), C20:3n6 (eicosatrienoic
acid) and C22:6n3 not detected in cultures grown at 20 C, but were
present in some cultures grown at 25 C and 30 C (Supplementary
Table A).
HCA indicated which FAMEs showed the largest variation in concentration due to the various culture conditions. In Chlorella sp. MACC-438
and C. minutissima MACC-452, the FAMEs were separated into 5 clusters
with C18:1n9c and C16:0 (cluster 5) showing the most variation,
followed by C18:2n6c, unidentied FAMES (cluster 4), C18:3n3 (cluster

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V. rdg et al. / Algal Research 16 (2016) 141149

Table 2
Average content (%) of the predominant fatty acids in the three Chlorella strains grown in a range of nitrogen concentrations (1%, 3% and 10% N) and temperatures (20 C, 25 C and 30 C)
when harvested on day 3 and day 6. Results are presented as mean SE (n = 18).
FAME
Strain

C16:0

C18:1n9c

Unid FA

C18:2n6c

C18:3n3

C18:0

Total

MACC-438
MACC-452
MACC-728

24.0 1.3
22.1 1.0
26.1 1.5

21.1 3.3
21.1 3.2
21.4 2.8

19.9 2.9
18.8 2.8
18.3 3.6

14.5 0.7
13.6 0.6
13.6 0.6

9.0 0.8
10.8 1.2
9.0 0.6

4.5 0.7
3.1 0.5
5.0 0.7

93.2
89.5
93.4

Unid FA = unidentied fatty acids of short chain (bC8) and very long chain (NC24).

4 or 3) and C18:0 (cluster 3) and C20:2 and C17:1 (heptadecenoic acid;

cluster 2; Supplementary Fig. A). Chlorella sp. MACC-728 differed in that
the largest variation in concentration was in the unidentied FAME content (Cluster 6) as well as large variation in the C18:2n6c content (cluster 5) followed by C18:3n3 and C18:0 (cluster 4); C18:1n9c and C16:0
(cluster 3) and C20:2 and C17:1 (cluster 2) (Supplementary Fig. A). In
addition, HCA indicated that the largest changes in the FAME proles
were due to N concentration rather than temperature. Chlorella sp.
MACC-438 and C. minutissima MACC-452 responded in a similar manner with the cultures grown in 1% N and 3% N showing the largest

variation in their FAME prole, while Chlorella sp. MACC-728 grown in

10% N had the highest variation in their FAME prole (Supplementary
Fig. B) due to the large changes in the unidentied FA content (Fig. 5).
4. Discussion
Growth rate and yield are inuenced by environmental factors with
microalgae having a minimum, optimum and maximum requirement
for each factor with acclimation strategies to adjust to this variation. In
the present study, temperature had a signicant effect on the biomass

Fig. 5. Changes in the fatty acid proles with respect to the saturation state in three Chlorella strains grown in various nitrogen concentration and temperature combinations. Cultures were
harvested on day 3 and day 6. Unid FAs are those unidentied short chain (bC8) and very long chain (NC24) FAMEs not authenticated with GCMS standards.

V. rdg et al. / Algal Research 16 (2016) 141149

accumulation with the highest rates achieved at 30 C and growth

decreasing with lower temperatures. Similarly, optimum temperatures
were 20 C for Nannochloropsis oculata and 30 C for Chlorella vulgaris
with both sub-optimum and super-optimum temperatures having a
negative effect on the growth rate [11]. Light and temperature were
the most important factors in determining biomass productivity in a
long-term experiment, where Scenedesmus obliquus was grown in
outdoor photobioreactors with N and CO2 maintained at a constant
level [4].
Nitrogen is rapidly utilized by growing microalgal cultures. For
example, rdg et al. [14] showed that when a Chlorella strain was
grown using the same growth apparatus as in the present study, N
was below the limit of detection by day 3 in the media when the initial
N concentration was 1% N (7 mg L1 N); initial concentrations of 10% N
(70 mg L1 N) decreased to less than 2% N (14 mg L1 N) in the media
by day 3 and to less than 1% N (5 mg L1 N) by day 6. Despite this Ndeprivation, the cultures continued to increase in biomass [14]. Similarly, in the present study, the three Chlorella strains continued to increase
in biomass despite N-deprivation although even small adjustments in
the initial N concentration (from 1% N to 3% N) had a signicant effect
on biomass accumulation (Fig. 1).
Macromolecules such as proteins, carbohydrates, lipids and nucleic
acids are derived from a carbon substrate and can account for up to
85% of the cell DW [16]. During exponential growth, carbon allocation
is diverted to cell growth and reproduction, while under stressful
conditions, such as N-deprivation, there is a switch in carbon allocation
to storage reserves [17]. Thus, the production of proteins is favored during optimum growth conditions with limited carbohydrate and lipid
production, while during periods of stress, protein production decreases
and carbohydrate and lipid production increase [1]. Compounds such as
chlorophyll that make up the intracellular N pool can be utilized as an
alternative N source to sustain limited growth over a short period of
time [8]. However, degradation of chlorophyll and other proteins
lower photosynthetic and growth rates compared to those achieved in
N-sufcient cultures [8]. Nitrogen levels had a signicant effect on the
protein content in the three Chlorella strains used in the present experiment with a positive correlation between protein and N levels (Fig. 2A,
D and G). This larger decrease in the protein content in response to Ndeprivation in the lower N concentrations may be due to the degradation of proteins to provide an N source to sustain growth over the
short experimental time used in the present study. Conversely, lipid
content increased in the N-deprived cultures (Fig. 2C, F and I). Nitrogen
levels did have some effect on the carbohydrate levels in the present
study, with concentrations generally higher in the N-deprived cultures
(Fig. 2B, E and H). These trends in macromolecule content are similar
to those reported for other microalgae. For example, the protein content
decreased and the lipid content increased in Chlorella protothecoides
when grown under N-deprivation, while the carbohydrate content
was the least affected [16]. Reduced N conditions did not signicantly
affect the growth rate of C. vulgaris but caused a threefold increase in
the lipid content [11]. In Scenedesmus acutus, the protein content
decreased, and both the lipid and carbohydrate content increased,
under N-deprivation [18].
Although varying from species to species, cultures grown at high
temperatures generally have a faster growth rate, but with decreased
protein content and increased lipid and carbohydrate content [12]. In
the present study, while the fastest growth rate was recorded at the
highest temperature tested (Fig. 1), temperature had little effect on
the protein, carbohydrate and lipid content on day 6 in the three
Chlorella strains (Fig. 2). Similarly, investigation of three Botryococcus
braunii strains revealed that variation in total lipid content was strainspecic rather than temperature dependent [19]. In contrast, proteins
and lipids decreased with increasing temperature in four tropical
microalgae species, while there was no apparent trend in the carbohydrate content with only small uctuations (3%) over the tested temperature range [12].

147

The results of the present study conrm that these Chlorella strains
are oleaginous, producing lipids in response to N-deprivation with
adjustments in the initial N concentration having a greater effect than
temperature on the carbon allocation in these strains. However,
responses were strain-specic with C. minutissima MACC-452 generally
having the lowest lipid content but the largest increase in carbohydrates
from day 3 to day 6 (Fig. 2). Similarly, temperature also had no effect on
lipid content in the freshwater diatom Fragilaria capucina, while N and
Si levels signicantly affected lipid content [20]. In contrast, both
suboptimum temperatures and reduced N concentrations led to an
increase in lipid content in N. oculata and C. vulgaris [11].
Based on data from the literature, the average daily lipid productivity in microalgae strains grown in N-replete media was calculated as
50 mg L1 day1 [9]. Similar rates of lipid productivity were obtained
on day 6 in the present study for the cultures grown at higher temperatures and 3% N concentration (Fig. 3). Both temperature and N had a
signicant effect on lipid productivity with the lowest N (1% N) and
lowest temperature (20 C) having a negative effect on lipid productivity due to lower biomass accumulation (Fig. 1) rather than lipid content
(Fig. 2C, F and I). Higher temperatures (30 C) had a positive effect with
lipid productivity increasing to over 60 mg L1 day1 when grown in
3% N due to faster biomass accumulation at optimum temperatures
for growth (Fig. 1). However, this response was strain-specic with
C. minutissima MACC-452 having the lowest lipid productivity
(Fig. 3B) due to a lower lipid content (Fig. 2F). Similarly, lipid productivity decreased in 31 Chlorella strains when grown in sub-optimum and
super-optimum temperatures [21] and increased in C. vulgaris when
grown in 375 mg L1 N compared to 750 mg L1 N [11].
Apart from adjusting carbon allocation to favor lipid accumulation,
microalgae also modify their lipid metabolism in response to changing
environmental conditions. Under optimal conditions, synthesis of
glycerolipids, which are major constituents of membranes, is favored
and accounts for 520% DW [6]. There is a switch in lipid metabolism
in response to unfavorable conditions towards synthesis of neutral
lipids, mainly TAGs, which are deposited in lipid bodies in the cytoplasm. TAG accumulation is via de novo biosynthesis of fatty acids in
the chloroplast and by recycling of membrane lipids, which account
for up to 30% TAG content and results in the partial collapse of the membrane system [22]. TAGs do not have a structural function but serve as
carbon and energy storage forms during unfavorable conditions. Once
favorable conditions are restored, the acyl groups from the TAGS are
used to synthesize new membranes and metabolites [22]. TAGs may
also have an active role in stress responses with production of TAGs
preventing excess accumulation of electrons from the photosynthetic
electron transport chain, which can lead to photoinhibition as well as
maintaining membrane integrity and uidity [6]. In a recent study, enzymes and genes involved in the biosynthesis, catabolism and degradation of fatty acids and TAGs were identied in Dunaliella purva. Some
differentially expressed genes such as the transcription factor gene
wri1 were revealed, shedding light on the regulating mechanisms of
TAG production induced by nitrogen limitation [23]. As expected, a
change in lipid metabolism to favor TAG production occurred in the
three Chlorella strains used in the present study, with the proportion
of fatty acids increasing in the total lipid content from day 3 to day 6.
This response was strain-specic, with the largest shift in metabolism
generally occurring in the most N-deprived cultures (1% N) grown in
the lowest temperature (20 C) in Chlorella sp. MACC-428 and
C. minutissima MACC-452. By comparison, in Chlorella sp. MACC-728,
the largest increase in the % FAME occurred from day 3 to day 6 in the
cultures grown at the highest temperatures.
SFA and MUFAs are the predominant fatty acids present in microalgae
[3]. In general, the major fatty acids in green microalgae (Chlorophyceae)
are the SFAs C16:0 and C18:0, the MUFA C18:1, while the major PUFAs
are C18:2 and C18:3. In the present study, the FAME proles were similar
in the three strains, with the predominant fatty acids being
C16:0 N C18:1n9c N unidentied FAMEs N C18:2n6c N C18:3n3 N C18:0,

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V. rdg et al. / Algal Research 16 (2016) 141149

which accounted for over 90% of the total FAME content by day 6
(Table 2; Supplementary Table A). Unlike vascular plants, microalgae
have a greater variation in medium-chain fatty acids (C10C14) and a
higher proportion of very long chain fatty acids (N C24) [8], with 45
medium chain fatty acids (C10C15), 1718 long chain fatty acids
(C16C24) and a high proportion of unidentied short (bC8) and very
long chain (N C24) fatty acids detected in the Chlorella strains analyzed
in the present study (Supplementary Table A).
Lipid accumulation due to nutrient deprivation tends to result in a
FAME prole with higher amounts of SFA and MUFAs and lower PUFA
content [24], as was the case in the present study, where the SFA and
MUFA content was the highest in the cultures grown in 1% N and the
PUFAs and unidentied FAs were the highest in cultures grown in 10%
N (Fig. 5). The changes in specic fatty acids in response to Ndeprivation appear to be strain specic. In the present study, the
MUFA (C18:1n9c) and the SFA (C16:0) showed the most variation in
concentration in Chlorella sp. MACC-438 and C. minutissima MACC452, while the unidentied FAs and PUFA (C18:2n6c) were the most
variable FAMEs in Chlorella sp. MACC-728 (Supplementary Fig. A).
These changes in specic fatty acids are similar to those reported for
C. vulgaris where the concentration of C16:0, which contributed approximately 60% of the overall lipid fraction, was not signicantly affected by
N concentration or temperature. However, higher temperatures
increased the oleic acid (C18:1) content [11].
Temperature is also an important factor in determining the fatty acid
composition in microalgae [6,20]. It is speculated that microalgae modify their fatty acid composition as a strategy to acclimatize to the
changing temperatures, as fatty acids are essential in maintaining the
integrity and uidity of the cell membrane phosolipid layer with uidity
depending on the degree of fatty acid unsaturation [6,12]. Limited data
suggests that the trend appears for increasing fatty acid unsaturation
with decreasing temperature, and increasing saturated fatty acids with
increasing temperature [6,12], although there are exceptions. In the
present study, while temperature inuenced the % FAME with a higher
FAME content at the lower temperatures in Chlorella sp. MACC-438 and
C. minutissima MACC 452, temperature did not have much effect on the
specic fatty acid proles, apart from a few long chain fatty acids that
only occurred in cultures grown at the higher temperatures. In contrast,
the N concentration had more inuence on the specic FAME prole.
These changes were strain-specic, with the most variation due to low
N concentrations in Chlorella sp. MACC-438 and C. minutissima MACC452 while there was more variation in Chlorella sp. MACC-728 grown
in 10% N (Supplementary Fig. B).
5. Conclusions
Microalgal species grown in outdoor cultivation systems need
a wide tolerance to environmental conditions as they grow over a
range of seasons and are exposed to uctuating ambient weather conditions. Climatic conditions, especially temperature and solar radiation
are the main factors affecting lipid productivity in outdoor cultivation
systems [3]. In the present study, biomass accumulation, macromolecule content and fatty acid proles were quantied in three Chlorella
strains grown in a combination of low to moderate N concentrations
and temperatures. Strain-specic responses were observed with variation in the FAME composition being similar in Chlorella sp. MACC-438
and C. minutissima MACC-452 with C18:1n9c and C16:0 being the
most variable FAMEs. In Chlorella sp. MACC-728, the unidentied
FAMEs and C18:2n6c were the most variable FAMEs. Generally,
temperature had a signicant effect on biomass accumulation and
lipid productivity, with Chlorella sp. MACC-438 and C. minutisisma
MACC-452 having the highest productivity and FAME content (%
FAME) at lower temperatures, while lipid productivity and FAME
content in Chlorella sp. MACC-728 were similar regardless of temperature. N concentration had a greater inuence than temperature on
lipid accumulation and FAME proles, with increased SFAs and MUFAs

at lower N concentrations. Thus, temperature tolerance should be

considered when selecting strains for cultivation in outdoor ponds to
ensure high lipid productivity over a range to temperatures, while the
strain-specic response to N-deprivation is important to ensure the
quality of the biofuel feedstock. For example, while Chlorella sp.
MACC-728 had the best temperature tolerance and the highest productivity over the temperature range tested, the large variation in the concentration of the short and very long chain fatty acids (unidentied
FAMEs) and the polyunsaturated C18:2n6c would result in a lower
quality biodiesel feedstock.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.algal.2016.03.001.

Acknowledgments
The University of KwaZulu-Natal, National Research Foundation
(South Africa) and TAMOP-4.2.2.A-11/1/KONV-2012-0003 (Microalgal
Biotechnology in Sustainable Agriculture) are gratefully acknowledged
for their nancial support. A.O.A. thanks the Claude Leon Foundation,
Cape Town, South Africa for the nancial support.

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