Algal Research 11 (2015) 227–233

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Algal Research

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

Baltic Sea microalgae transform cement flue gas into valuable biomass
M. Olofsson, E. Lindehoff, B. Frick, F. Svensson, C. Legrand ⁎
Center for Ecology and Evolution in Microbial model Systems (EEMiS), Department of Biology and Environmental Science (BoM), Linnæus University, Kalmar 39182, Sweden

a r t i c l e i n f o a b s t r a c t

Article history: We show high feasibility of using cement industrial flue gas as CO2 source for microalgal cultivation. The toxicity
Received 24 April 2015 of cement flue gas (12–15% CO2) on algal biomass production and composition (lipids, proteins, carbohydrates)
Received in revised form 22 June 2015 was tested using monocultures (Tetraselmis sp., green algae, Skeletonema marinoi, diatom) and natural brackish
Accepted 1 July 2015
communities. The performance of a natural microalgal community dominated by spring diatoms was compared
Available online 9 July 2015
to a highly productive diatom monoculture S. marinoi fed with flue gas or air–CO2 mixture. Flue gas was not toxic
Keywords:
to any of the microalgae tested. Instead we show high quality of microalgal biomass (lipids 20–30% DW, proteins
Microalgae 20–28% DW, carbohydrates 15–30% DW) and high production when cultivated with flue gas addition compared
Baltic Sea to CO2–air. Brackish Baltic Sea microalgal communities performed equally or better in terms of biomass quality
Flue gas and production than documented monocultures of diatom and green algae, often used in algal research and de-
Biomass composition velopment. Hence, we conclude that microalgae should be included in biological solutions to transform waste
Natural communities into renewable resources in coastal waters.
Brackish © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction
1.1. Flue gas as CO2 source for microalgae
The cement industry is responsible for approximately 4–5% of global
CO2 emissions [1–3]. Using mass cultivation of microalgae is considered
environmentally safe and sustainable for the abatement of CO2 from in-
dustrial flue gas [4–8]. Recent studies questioned the contribution of
algae to global CO2 removal since algae fix CO2 from flue gas but do
not offer permanent storage nor are energy efficient [9,10]. However,
microalgae biomass can deliver products (biofuels, industrial material
etc.) that may replace an equivalent amount of fossil fuels, hence facili-
tating the sustainability of microalgae-based product development [9].
By 2030 the EC proposes that emissions from sectors covered by emis-
sion trade scheme (ETS) will be 43% lower than in 2005 [11]. This pro-
posal, combined with political climate change targets and market
forces can provide economic incentive for future company investments
in new technology.
Flue gas generally contains 3–15% CO2 (v/v) depending on fuel feed-
stock and type of operation [12] and thus can be used as a source of CO2
for microalgae cultivation. Microalgae show a good growth potential in
CO2 concentrations up to 10–20% regardless of the source, e.g. pure CO2
Abbreviations: Chla, chlorophyll a; DW, dry weight; EC, European Commission; FG,
flue gas; KAC, Kalmar Algae Collection; NC, natural community; PBR, photobioreactor;
PTFE, polytetrafluoroethylene; Sm, Skeletonema marinoi; SYKE, Finnish Environment
Institute; TC, total carbohydrates; TL, total lipids; TP, total proteins; v, volume; w,
weight.
⁎ Corresponding author.
E-mail address: catherine.legrand@lnu.se (C. Legrand).
[13,14] and industrial flue gas [15–17,6,18,8]. Industrial flue gas con-
tains over 100 substances, of which several are potentially toxic to
microalgae (e.g. SOx, NOx, HF, heavy metals) [19]. Numerous studies
have weighed opportunities and limitations of microalgal cultivation
and based on predictions have showed the potential of a process
where industrial waste CO2 is converted to bioproducts through algae
[20–24]. Empirical studies on the tolerance of microalgae to industrial
flue gas are increasing steadily but rarely include various taxonomic
groups of microalgae, and trials with natural or multispecies communi-
ties are noticeably lacking.
1.2. Importance of diversity and production for microalgae cultivation
Outdoor mass cultivation of microalgae has generally focused on
highly productive monoclonal cultures for biomass production or
targeting specific chemicals. Few studies have been using multispecies
cultures or natural assemblages of microalgae to improve production
yields. Productivity and stability of natural terrestrial ecosystems have
been positively linked to diversity and species richness [25–28] but
may be applicable to marine habitats [29]. Productivity of agro systems
is considered to benefit from intercropping through the establishment
of stable and sustainable ecosystems within crop farmlands [30]. Both
observational [31] and experimental studies [32–34] indicate that this
applies also for microbial communities, including microalgae. The posi-
tive relationship between diversity and productivity may be explained
by 1) the complementary effect, where resource utilization is higher
in a more diverse community [35,29,36] and 2) the selection effect,
where one highly productive species, is favored by certain environmen-
tal conditions over other species in a diverse community [37]. The two

http://dx.doi.org/10.1016/j.algal.2015.07.001
2211-9264/© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
228 M. Olofsson et al. / Algal Research 11 (2015) 227–233

mechanisms may not be complete contrasts but should both be Table 1

accounted for, while estimating the effect of diversity on productivity Composition of the conditioned flue gas collected from Cementa AB, Degerhamn,
Sweden for this study. Values are valid from April 2013 to May 2014, metal levels
[26,37]. The complementary effect in diverse microalgae communities were measured in September 2012.
could lead to a more stable and resilient system less prone to invasive Source: environmental report HeidelbergCement, Degerhamn 2013.
species and zooplankton grazing pressure [38]. The same mechanism
Temperature 150–200 °C
of decreased pest susceptibility was shown for plant crops [39]. Diverse
CO2 12–15%
communities seem to be more productive and resilient in natural vari- O2 0–21%
able environments where changing abiotic factors affect productivity. H2O 0–15%
Additionally, the spectrum of valuable chemicals in terms of lipids, NOx b800 mg/Nm3
SO2 b50 mg/Nm3
proteins, carbohydrates and pigments produced will most likely be
CO 0–1000 mg/Nm3
more diverse in a diverse community. Smith et al. [40] suggested that NH3 b50 mg/Nm3
multispecies communities of microalgae in open pond cultivation sys- HCl b10 mg/Nm3
tems could accumulate more solar energy as lipids due to a more effi- HF 0–0.01 mg Nm−3
cient utilization of light from different functional groups of microalgae, Dust particles b10 mg/Nm3
Metalsa b0.5 mg/Nm3
in contrast to closed systems with monocultures. Positive effects of spe-
Mercury (Hg) b0.03 mg/Nm3
cies richness (level of two, three and four species) were found on both Cadmium (Cd) + titanium (Ti) b0.05 mg/Nm3
algal biovolume and lipid content compared to monocultures [41]. a
Metals: Sb + As + Pb + Cr + Co + Cu + Mn + Ni + V.
These effects were attributed to complementarity rather than selection
effects. Stockenreiter et al. [42] found the relationship of functional
group richness more strongly linked to lipid content than mere species 2.3. Microalgal stock cultures
richness, suggesting that a more efficient light utilization within func-
tionally diverse communities contributed to the higher lipid content. The green algae Tetraselmis (strain KAC21) and the diatom S. marinoi
Extrapolating these findings from natural and artificial ecosystems to (strain SMTV1) were grown in filtered Baltic seawater (salinity 7,
industrial mass cultivation of microalgae leads to a combination of un- filtered 0.2 μm) enriched with original Guillard's f/2 medium and
certainties. Nonetheless, the use of natural community as inoculum in f/2 + Si respectively [43]. Strains were obtained from the Kalmar Algal
large-scale production system could increase the stability, resilience Collection (KAC) and the Finnish Environment Institute (SYKE).
and productivity of the system. Cultures were grown in 10 L glass flasks, gently bubbled with air at tem-
Composition of the flue gas from the cement industry varies with the perature 18 °C (Tetraselmis) and 15 °C (S. marinoi), and at irradiance
origin of the raw substrate and the combustion process. For the first 300–500 μmol photons s−1 m−2. Irradiance was measured with a digi-
time the potential use of flue gas from Cementa AB, Degerhamn, tal scalar irradiance meter (Biospherical Instruments Inc.). A natural
Öland, SE for microalgal biomass production was evaluated using microalgal community was sampled during the spring bloom of 2013
monocultures and natural communities from the Baltic Sea. in the SW Baltic Sea (PRODIVERSA cruise, station 8, 18 April, longitude
This study aims to test the toxicity of cement flue gas on biomass 17.33342 latitude 56.2559). Diatoms dominated the community, pri-
production of a monoculture (Tetraselmis sp., green algae), and to com- marily of the genus Chaetoceros spp. dinoflagellates, cryptophytes,
pare biomass composition (lipids, proteins, carbohydrates) and produc- euglenophytes and chlorophytes (green algae) were also present in
tion of a natural microalgal community dominated by spring diatoms to lower abundance.
one highly productive diatom monoculture during treatments of flue
gas or air–CO2 mixture. The reference monoculture was Skeletonema 2.4. Experimental design
marinoi (strain SMTV1), a rapidly growing diatom common in the
spring bloom community in the Baltic Sea. 2.4.1. Can microalgae use the CO2 in flue gas and how is the quality of the
biomass affected?
2. Material and methods Tetraselmis was inoculated (5000 cells mL− 1) in six cylinders in
photobioreactor 1 (PBR1, Fig. 1, Table 2) filled with f/2 Baltic seawater
2.1. Study site medium. Three replicates were supplied with air (control) and three
with cement flue gas daily for 40–120 s at a flow rate of 5 L min−1.
Cementa AB Degerhamn, Öland, southeast Sweden, manufactures Growth performance of Tetraselmis was monitored over 10 days. Sam-
300,000 tons of cement annually and releases approximately ples were taken daily in each replicate cylinder for cell density and pH
260,000 tons of CO2 through their flue gas. The major components of (days 1–10), and dry weight (DW) from day 3 to 10. Endpoint lipids
the flue gas emissions, besides CO2, are SO2, NOx and dust (Table 1). and inorganic nutrient levels were also measured at day 10. Cells were
The Cementa HeidelbergCement group is working to reduce the CO2 fixed with Lugol's solution prior to counting. pH was measured with a
emissions by 10% by the year 2020 at the plant in Degerhamn, and
will strive to achieve 0% by 2050. The possibility of lowering their car-
bon footprint by using mass cultivation of microalgae to capture the
CO2 rich flue gas is currently being evaluated in an academia–industry
collaboration. The valorization of the produced algal biomass to gener-
ate bulk chemicals, such as lipids, carbohydrates and proteins for con-
version to bioenergy or high value products is also assessed.

2.2. Flue gas and CO2

The cement flue gas was collected from the monitoring sampling
point in one of the flue stacks at the Cementa AB Degerhamn factory
by using a high-pressure compressor and filling the flue gas into gas
cylinders at 150–200 bar. The composition of the flue gas varied
12–15% during the experiments (Table 1). Industrial grade CO2–air Fig. 1. Dry weight (DW) of Tetraselmis sp. during the experiment excluding the initial
mixture (13.5% CO2) was obtained from AGA Gas AB. two-day lag-phase (n = 2).
 M. Olofsson et al. / Algal Research 11 (2015) 227–233
Table 2
Technical specifications and set-up of the two photobioreactors (PBR) used in the study.
PBR1 PBR2
Dimension (H × d) mm 1000 × 100 531.6 × 117.3
Cylinders 6 12
Material Acrylic Polystyrene
Culture volume L 5.8 4.2
Irradiancea μmol m−2 s−1 340–550 300–500
Light supply Metal-halide lamps (250 W Osram)
Light:dark 16:8 16:8
Temperature °C 18 15
Air feed (0.2 μm filtered) Perforated pipes Air stone
Air release 20 to 150 mm Air release 0 to 20 mm
from bottom of cylinder from bottom of cylinder
FG/CO2 feed Bottom up through Bottom up through airstone
perforated plug
Daily FG/CO2 distribution Single pulse, 40–120 s Multi pulses (3×), 60 s
Gas flow rate 5 L min−1 1 L min−1
a
Measured with a digital scalar irradiance meter (Biospherical Instruments Inc.).
compact inoLab Level 1 pH meter. Dry weight was determined by fil-
tering 25 mL onto prewashed (10 mL 0.5 M ammonium formate),
dried and preweighed 45 mm glass fiber filters (Whatman GF/C). Max-
imum specific growth rate was derived from the steepest slope by plot-
ting the natural logarithm of the cell density using at least 3 successive
data points (days 1–6). Productivity (mg DW L−1 d−1) was determined
using the natural logarithm of DW values during the same time interval.
Tetraselmis samples (50 mL) for analysis of total lipids were centrifuged
for 20 min at 11,900 ×g, at 4 °C (Beckman, Avanti J-25). The supernatant
was discarded and the pellet frozen in 50 mL Falcon tubes.
2.4.2. How does the production and chemical composition differ between
a monoculture and a natural microalgal community under flue gas
treatment?
S. marinoi (Sm) and the natural community (NC) were inoculated
(3 μg Chla L− 1) in 3 × 2 × 2 replicate cylinders in photobioreactor 2
(PBR2, Table 2.) filled with f/2 Baltic seawater medium. Triplicates of
Sm and NC were repeatedly (3 times during the light period; 08.00,
12.00 and 16.00) sparged with cement flue gas alternatively with indus-
trial grade CO2–air mixture for 1 min at a flow of 1 L min−1. Upon injec-
tion of flue gas or industrial grade CO2 pH decreased in the PB2 bottles
by 1 to 1.5 units. Recovery of pH to initial values occurred gradually
over 3–4 h before the next injection.
Growth performance was monitored over 10 days. Samples were
taken daily in each replicate cylinder for Chla concentration, cell abun-
dance and community composition and pH, and DW from days 3 to
10. Endpoint chemical composition (lipids, proteins, carbohydrates)
and inorganic nutrient levels were also measured at day 10. Cells were
fixed with Lugol's solution prior to counting. pH was measured daily
with a HI98128 hand pH-meter (Hanna instruments). Dry weight was
determined by filtering 25–100 mL onto prewashed (Milli-Q), dried
and preweighed 45 mm glass fiber filters (Whatman GF/C). Growth
rate was derived from the slope in the growth phase by plotting the
natural logarithm of the Chla concentration over days 1–5. Productivity
(mg DW L−1 d−1) was calculated using endpoint values of DW over the
experimental period (10 days). The theoretical optimum productivity
(mg DW L−1 d−1) was calculated using the initial biomass (DW) and
the growth rate at maximum yield (day 4). Biomass for analysis of
chemical composition was collected by filtration of 1200 mL from
each replicate onto a 3 μm SS Millipore filter and transferred to 50 mL
Falcon tubes. The Falcon tubes were centrifuged at 2850 ×g (Hettich
Universal 16R) and the supernatant was discarded. The pellet was
frozen and then vacuum dried using a Labconco bulktray drier and a
Scanvac Coolsafe until constant weight. At constant weight, the biomass
was divided for analysis of total protein (10 mg) and carbohydrates
(10 mg) and the remaining biomass of 96 ± 0.6 mg was used for total
lipid analysis.
229
2.5. Microalgal biomass and composition
Cell density of monoculture Tetraselmis and Sm was measured using
a 0.1 mL Palmer Maloney counting chamber. Cell abundance and compo-
sition of NC were determined using sedimentation chambers [44]. At
least 300 cells were counted in each replicate using an inverted light
microscope (Zeiss PrimoVert). Cells were identified to the genus or spe-
cies level when possible. Cell biovolume was calculated using the
method of Olenina et al. [45] and multiplied with cell numbers to obtain
the total biovolume (μm3 L−1). Chlorophyll a (Chla) was analyzed
fluorometrically (Turner Trilogy fluorometer) on ethanol extracts ac-
cording to Jespersen & Christoffersen [46]. Depending on concentration,
5–20 mL of microalgal suspension was filtered onto 25 mm Pall AE/E fil-
ters prior to extraction in ethanol. Filters for DW were immediately
washed after sampling to remove excess salt with 10 mL 0.5 M
ammonium-formate and dried in 80 °C (Tetraselmis) or washed with
10 mL MilliQ and dried in 100 °C (Sm and NC) until constant weight. Fil-
ters were weighed on a Mettler Toledo, College-B B154 scale. Dry weight
was calculated by subtracting the weights of the dried filters with bio-
mass and the dried prewashed filters.
2.6. Product analyses
2.6.1. Total lipids
Lipid extractions were performed using a modified Bligh & Dyer
method [47]. The lipids were extracted from the algal pellet with
Chloroform:MeOH 1:2 (v/v). In order to break cell walls samples were
sonicated using a Sonics Vibra-cell (VCX 130) for 5 min at 100%
for Tetraselmis and 2 min at 50% for Sm and NC. For Sm and NC the
chloroform:MeOH 1:2 extraction procedure was repeated three times
by collecting the supernatant in new falcon tubes after centrifugation
(10 min, 2850 ×g, Hettich Universal 16R). Water and chloroform were
then added to the recovered supernatant in the final proportions
of chloroform:MeOH:H2O 2:2:1 and the solution was vortexed until
homogenous. The solution was then centrifuged (10 min, 2850 ×g,
Hettich Universal 16R). The aqueous-methanol layer was removed
and the remaining chloroform/lipid layer was transferred into pre-
weighed glass sampling tubes and dried until constant weight (Mettler
Toledo, College-B B154 scale). The empty pre-weighed values were
subtracted from the weighed sample values. For the Sm and NC sam-
ples the collected supernatant was filtered through a 25 mm syringe
filter w/0.2 μm PTFE membrane into pre-weighed glass vials. To re-
move any traces of water a few drops of MeOH were added and
then the samples were evaporated by using a filtered (0.2 μm) stream
of compressed air. When the samples appeared dry and the chloro-
form had completely evaporated the samples were further dried in
an oven at 60 °C until constant weight.
2.6.2. Protein analysis
5 mg vacuum-dried biomass was resuspended in 2 mL 1 M NaOH
and incubated in a waterbath at 95 °C for 60 min. Samples were then
centrifuged (10 min, 2850 ×g, Hettich Universal 16R) and 100 μL of
the supernatant was transferred to glass tubes. A ready-made assay,
Bio-Rad DC Protein assay kit II, using the Folin-Phenol protein quantifi-
cation method [48] was used to prepare the samples to be read in a
spectrophotometer. The sample values were compared to a standard
curve with the range of 0.2–1.0 mg mL− 1 made from the bovine
serum albumin standard included in the assay.
2.6.3. Carbohydrate analysis
Carbohydrate extraction was carried out using the phenol-sulfuric
acid method described by DuBois et al. [49]. The vacuum-dried biomass
was dissolved in 5 mL 1 M H2SO4 and incubated in 90 °C for 60 min.
After incubation samples were cooled down to room temperature and
centrifuged (10 min, 2850 ×g, Hettich Universal 16R), 100 μL of the
supernatant was added to a glass vial and was let to react with 1 mL
230 M. Olofsson et al. / Algal Research 11 (2015) 227–233

Table 3 Addition of FG did not affect the total lipid levels in Tetraselmis sp. re-
Cell density, growth rate, biomass and productivity of Tetraselmis sp. fed with air and flue gardless of treatment (air: 10.9 ± 5.8%, FG: 10.1 ± 6.8% of DW). No sig-
gas (FG). Slope, confidence interval (CI) and coefficient of determination (R2) of simple re-
gressions of the natural logarithm of cell density were calculated over the experimental
nificant differences were found among the treatments (one-way
period (days). Statistical comparison of slopes for cell density between treatments of Air ANOVA, F = 0.433, p = 0.661). The experiment was repeated using ref-
and FG is represented by p-value. Biomass and productivity are the mean of duplicate erence cultures bubbled with CO2, in which the lipid levels were similar
values. n.a. not applicable. as for the air and FG treatments (data not shown).
Ln cells mL−1 Growth rate d−1 CI R2 Day p-Value
3.2. Production and chemical composition of monoculture and a natural
Air 0.83 0.67–0.98 0.95 2–6 0.0283
FG 1.04 0.91–1.18 0.98 1–5 microalgal community fed with flue gas

Biomass Productivity mg L−1 d−1 Range
Air 34.7 31.3–38.0 n.a.
FG 57.3 50.7–64.0 n.a.
of Phenol (5% w/v) and 3 mL of H2SO4 (72 wt.%). A glucose standard
curve using Sigma D-(+)-glucose with the range of 0.2–1.0 mg mL−1
was prepared. Samples were incubated in a waterbath at 90 °C for
5 min. Absorbance was measured using a VWR UV-1600 PC spectropho-
tometer at 490 nm and concentrations were determined by comparing
to the standard curve.
2.7. Statistics
Statistical analyses (2-way ANOVAs, paired t-test) followed by
Tukey's post-hoc test were performed using Graph Pad prism (version
6.0d for Mac OS X, GraphPad Software, La Jolla California USA). The
level of significance was determined to p b 0.05. Multiplicity adjusted
p values, accounting for multiple comparisons, are reported. In the ex-
periment with Tetraselmis, biofouling occurred in one replicate per
treatment, hence simple regression based on the natural logarithm
during linear growth of the exponential phase was performed from
two replicates. The slopes of the regressions were then compared
statistically between the flue gas and air treatment using Graph Pad
prism.
3. Results
3.1. Utilization of CO2 in flue gas and quality of algal biomass
Productivity was significantly higher in NC (~6.5 mg L−1 d−1) com-
3–9 n.a. pared to Sm (~4.5 mg L−1 d−1) regardless of treatment (2-way ANOVA,
3–6 n.a. community: F = 7.351, p = 0.027, treatment: F = 0.057, p = 0.818)
(Table 4). Yield in DW after 10 days was similar for Sm and NC regard-
less of treatment (2-way ANOVA, community: F = 0.183, p = 0.680,
treatment: F = 0.057, p = 0.818). On the other hand, growth rates
based on Chla were higher in Sm monoculture (~ 1 d−1) compared
to the NC (~ 0.8 d− 1) (2-way ANOVA, community: F = 57.860,
p b 0.0001, treatment: F = 0.028, p = 0.871, Tukey's post-hoc test)
(Table 4, Fig. 2). Cell density was twice as high in Sm cultures compared
to NC (2-way ANOVA, community: F = 64.100, p b 0.0001, treatment:
F = 0.062, p = 0.810). Hence, biovolumes were marginally higher in
Sm (biovolumes, 2-way ANOVA, community: F = 6.892, p = 0.030,
treatment: F = 0.361, p = 0.564) (Table 4). The ratio between
biovolume and cell density showed larger cells in the NC (3.0:1) com-
pared to Sm (1.4:1). This was confirmed by the species composition in
NC, dominated by diatoms (90% of the total biovolume) in both treat-
ments, mostly Chaetoceros wighamii (67% of the total biovolume in
both treatments) (Fig. 3). All together the genera Chaetoceros and
Tabellaria consist of cells 2–10 times larger than Skeletonema cells.
Smaller cells (the diatom Cylindrotheca and Skeletonema, the genus
Euglena and flagellates) only made up for 10% of the biomass. No signif-
icant difference was found between treatments (paired t-test, p N 0.99).
Total lipid (TL) ranged 16–28% DW in Sm and 24–27% DW in NC
with no significant difference (Fig. 4a). Total proteins (TP) content of
Sm and NC were similar ranging 21–28% DW (Fig. 4b). Total carbohy-
drates (TC) were higher in Sm (30% DW) compared to NC (20% DW)
with a significant difference for FG treatment (2-way ANOVA, commu-
nity: F = 21.67, p = 0.01, Tukey's post-hoc test) (Fig. 4c).

The FG composition used is shown in Table 1. The main compounds 4. Discussion

of the FG were CO, NOx and SOx, in addition to CO2. In addition the FG
also contained NH3 and various metals that can be used for algal growth,
while the presence of dust particles, mercury and cadmium can have a
negative effect on algal metabolism. Experiment performed in batch
cultures demonstrated that FG can be used as the CO2 source for
Tetraselmis increasing growth performance (Fig. 1). The growth of
Tetraselmis sp. was significantly higher for FG (1.04 d−1) compared to
air control (0.83 d−1) (F = 5.815, p = 0.028, Table 3). Maximum yield
(200 mg L−1) was attained on day 6 with FG compared to day 9 in air
control cultures (Fig. 1). Consequently, the highest productivity was
achieved in FG treatment (57.3 ± 6.7 mg L−1 d−1) compared to air con-
trol (34.7 ± 3.3 mg L−1 d− 1), corresponding to a 1.6-fold increase
(Table 3).
4.1. Potential for using flue gas for algal cultivation
Marine and brackish microalgal production provides a sustainable
mitigation biofiltration method for the removal of greenhouse gases
such as CO2 and various pollutants, nutrients and metals. Due to the
composition of the flue gas released at the Cementa AB plant in
Degerhamn, SE we needed to test the tolerance limits and biomass pro-
ductivity of selected microalgal strains and natural communities.
The results reported here demonstrate that flue gas from the
Degerhamn plant can be used to produce algae in brackish waters
in the Baltic Sea region. Productivity of the Baltic Sea microalgae dou-
bled with flue gas indicating a good source of CO2 and no toxicity to

Table 4
Dry weight, productivity, maximum growth rate, cell density, biovolume and theoretical maximum production ± SD for Skeletonema marinoi (Sm) and a natural microalgae community
(NC) fed with CO2 and flue gas (FG). Different letters indicate significant differences for a given parameter.

Treatment Dry weight Productivity Growth rate Cell density Biovolume Theoretical maximum production
(mg L−1) (mg L−1 d−1) (d−1) (109 cells L−1) (mm3 L−1) (mg L−1 d−1)

Day 10 0–10 1–5 10 10 2–5

Sm CO2 77 ± 0.02 4.27 ± 1.70a 1.05 ± 0.04a 3.35 ± 0.88a 462 ± 122a 306 ± 32
Sm FG 81 ± 0.01 4.73 ± 0.61a 1.03 ± 0.04a 3.33 ± 0.37a 460 ± 52a 291 ± 29
NC CO2 77 ± 0.01 6.47 ± 0.99b 0.87 ± 0.12b 1.16 ± 0.05b 377 ± 23b 71 ± 25
NC FG 75 ± 0.01 6.33 ± 1.29b 0.88 ± 0.02b 1.04 ± 0.12b 329 ± 43b 70 ± 4
 M. Olofsson et al. / Algal Research 11 (2015) 227–233
Fig. 2. Natural logarithm of chlorophyll a (Chla) values for a) Skeletonema marinoi (Sm) and b) the natural microalgae community (NC) during the experiment. Trend line marks the days
used for calculation of growth rate (n = 3 ± SD).
microalgae. The productivity of microalgae depends heavily on the
conditioning of the raw flue gas due to the substantial levels of toxic
compounds [19]. We demonstrate that the conditioning of flue gas at
Cementa AB, Degerhamn, removing NOx, SOx, dioxins, and particles is
satisfactory for microalgal cultivation. Another challenge when using
flue gas is the decreased pH due to CO2 and other compounds. The use
of brackish seawater in our study provides a natural buffer capacity
compared to freshwater and this is particularly relevant on an island
where freshwater valuable resources are scarce.
Productivity of microalgae is related to the availability of CO2 in
the growth environment and the 12–15% CO2 in the cement flue gas
in this study fits the requirements for growth and the CO2 tolerance
of brackish microalgae. Green algae and diatoms dominate the natural
microalgal community in the Baltic Sea during spring and early sum-
mer [50,51]. Both in marine and freshwater, these two algal groups
have a broad CO2 tolerance (14–40%) while optimal CO2 for growth
is 10–15% [52,53].
Productivity performance of brackish microalgae monocultures and
natural communities in this study was in the lower range compared to
reported values [54,55]. Worth mentioning is that the primary intention
of the present study was not to optimize productivity but provide a
comparison of microalgae growth with flue gas. Weis et al. [34] demon-
strated that under homogenous conditions a monoculture would per-
form better than a diverse community. Our results confirm this as
maximum growth rate, hence theoretical maximum productivity,
achieved in the monocultures exceeded the ones of natural communi-
ties four times. Increased species richness can be linked to increased re-
source use, nutrient recycling and productivity. With increased diversity
the cultures can be more adaptable and resilient in a spatial heteroge-
neous environment [29], which could explain the higher productivity
of the natural community at maximum yield through complementary
effects [26,41]. Since flue gas had no stress effect on the microalgal
100
80
% Biovolume
60
40
20
0
FG
2
O
C
Fig. 3. The contribution (%) of algal taxonomic groups (flagellates) and species to the total biovolume of natural microalgal communities fed with CO2 and flue gas (FG).
231
community composition, this finding is encouraging for the use of
natural communities in view of a stable production in large-scale culti-
vation systems.
When evaluating the potential of using flue gas for algal cultivation
and product development, it is essential to determine the quality of
the microalgal biomass in terms of lipids, proteins and carbohydrates.
In this study, lipid, protein and carbohydrate contents in green algae
and diatoms were not affected by the cement flue gas (12–15% CO2)
compared to standard cultivation methods, using aeration or pure
CO2. A moderate increase in CO2 (5–20%) can stimulate the accumula-
tion of lipids [56], while higher CO2 concentrations (N 20%) can inhibit
growth and decrease lipid content [57,7]. Similar results have been re-
ported for green algae (e.g. Tetraselmis suecica, Chlorella sp.) grown
with untreated flue gas from coal fire power plant or industrial CO2
[58]. Furthermore, the diatom Chaetoceros muelleri showed highest
yield and lipid accumulation at 10% CO2 [59]. The fatty acid composition
in total lipids of Chlorella sorokiniana changed drastically when cultured
with flue gas (~10% CO2) instead of pure CO2 [60], suggesting that bio-
mass quality should be investigated beyond bulk measurements. The
large variability of reported values of biomass quality between algal
groups, species and isolates renders it difficult to identify trends in re-
spect to flue gas effect. In addition, there is a predominant focus on bio-
mass oil content for algal biofuel research, hence neglecting other
valuable products.
4.2. Valuable biomass from Baltic Sea microalgae communities
Most of the research in biological solutions using algal biomass is
based on the use of monocultures that ensures repeatability, which is
paramount in food industry, pharmaceuticals, cosmetics, and biofuel
production. Increasing productivity, yield and valuable product content
often relies on screening for the optimal strains that should be resilient
Flagellates <10 µm
Flagellates 10-20 µm
Euglena spp.
Tabellaria spp.
Cylindrotheca spp.
Chaetoceros spp.
C. wighamii
Skeletonema spp.
232 M. Olofsson et al. / Algal Research 11 (2015) 227–233
Fig. 4. Chemical composition (% DW) of Skeletonema marinoi (Sm) and natural microalgal
communities (NC) fed with CO2 and cement flue gas (FG); a) total lipids (TL), b) total pro-
teins (TP) and c) total carbohydrates (TC) (mean, n = 3 ± SD), *indicates a significant dif-
ference between treatments p b 0.05. Two replicates and a dummy value were used to
calculate average values in CO2-TP and FG-TC.
to different culture and environmental conditions, contamination and
predation. Ironically, algal monocultures often provide high quality
food for predators and pathogens, and are susceptible to contamination
by other microbes. Our results show that natural brackish microalgal
communities dominated by diatoms are of comparable quality in bio-
chemical composition as other Baltic Sea diatom monocultures. The
natural community in this study, dominated by C. wighamii contained
high levels of lipids (16–28%) similar to other cold water species;
S. marinoi (5–25%, this study), Skeletonema costatum (6–16%) and
C. wighamii (3–27%) [61]. Protein levels (20–30%, this study) were also
in the range of reported values for other Skeletonema strains [62,63].
Carbohydrates are the main organic compounds produced by algae
photosynthesis and accumulate when cells enter stationary growth
phase. Our results showed a higher amount of carbohydrates in
S. marinoi monocultures compared to S. costatum (5–7%, [62]) or the
natural community (20%, this study). This reflected that the monocul-
tures entered stationary phase earlier than the natural community,
due to higher growth rate. However, there was no accumulation of
lipids in S. costatum, as a consequence of this stress, as reported in the
literature [64,16]. Comparable results to ours are reported in Fig. 3
from Bertozzini et al. [63], regardless of nitrogen stress, carbohydrates
were also stored rather than lipids in S. marinoi. Thus, in our study,
since nutrients were still available in stationary phase it is likely that
light was limiting growth (self shading).
The brackish Baltic Sea algal community performed at the same level
as documented species used in algal cultivation systems in terms of bio-
chemical composition. The combination of a high quality biomass and
higher productivity at maximum yield emphasizes the potential of
using natural communities in algal production.
It remains unclear how flue gas or high CO2 concentrations influence
other nutritional values, such as fatty acids, vitamins and pigments.
Toxic components in algal biomass, possibly originating from flue gas
or recycled nutrients from waste streams, need to be carefully screened
in the valorization process.
5. Conclusion
The cement flue gas was not toxic to the microalgae tested, and
proved to be a suitable CO2 source with increased productivity relative
to air, and comparable quality of biomass composition between flue
gas and CO2. The natural brackish microalgal communities dominated
by diatoms show high quality of biomass, and higher productivity than
other Baltic Sea diatom monocultures at maximum yield. Hence, we
demonstrate the potential of using naturally occurring microalgal
communities in outdoor large scale algal cultivation systems. It re-
mains to be proven if this applies to other algal communities such as
green algae, cyanobacteria, and flagellates along the seasonal succes-
sion. For a successful and sustainable large-scale algal biomass produc-
tion, locally (Baltic Sea) adapted species are sought for, as they are
able to cope not only with environmental conditions (temperature,
eutrophication, large pH variation), but also with the constraints
coupled to the cultivation process (CO2 fluctuations, contamination
and pathogens). Therefore, using the natural succession over the
year with seasonally adapted species could aid in the stability and sus-
tainability of algal cultivation. Hence, microalgae should be included in
biological solutions to transform waste into renewable resources in
coastal waters.
Acknowledgements
We would like to thank Natalie Henriksson, Jesper Björk Rengbrandt,
and Emmelie Nilsson for laboratory assistance, Lars Malmer for crafting
PBR1, Anke Kremp (Finnish Environmental Institute) for providing
S. marinoi strain, Carina Bunse (Lnu) for NC sampling, Claes Kollberg
and his staff at Cementa AB, Degerhamn, Caroline Littlefield Karlsson
for proof reading the manuscript, and Stefan Sandelin (Cementa
HeidelbergCement, Sweden), Leif Norlander (SMA Mineral), Max
Larsson (ÅF Group), Kjärstin Hagman Boström (Lnu), and Erik
Wennerhag (The Swedish Agency for Economic and Regional Growth,
Tillväxtverket) for the support during this project. Funding was
provided by the Stiftelsen för kunskaps- och kompetensutveckling
(KK- Stiftelsen, project 20120194), the European Regional Develop-
ment Fund (project 170405), The Regional Council in Kalmar county,
the Swedish Research Council FORMAS (Ecochange project 2009-149),
Linnæus University, Faculty of Health and Life Science (grant to CL),
Åforsk Forskningstiftelsen (project 11-385), and industry partners
Cementa HeidelbergCement and SMA Mineral.
References
[1] E. Worrell, L. Price, N. Martin, C. Hendriks, L.O. Meida, Carbon dioxide emissions
from the global cement industry, Annu. Rev. Energy Environ. 26 (2001) 303–329.
 M. Olofsson et al. / Algal Research 11 (2015) 227–233
[2] B. Metz, O. Davidson, P. Bosch, R. Dave, L. Meyer, Climate change 2007—mitigation of
climate change, Intergovernmental Panel on Climate Change, Geneva, Working
Group III, 2007.
[3] T.A. Boden, G. Marland, R.J. Andres, Global, regional, and national fossil-fuel CO2
emissions, Carbon Dioxide Information Analysis Center, Oak Ridge National Labora-
tory, U.S. Department of Energy, Oak Ridge, TN, 2011.
[4] M. Huntley, D. Redalje, CO2 mitigation and renewable oil from photosynthetic mi-
crobes: a new appraisal, Mitig. Adapt. Strateg. Glob. Chang. 12 (4) (2007) 573–608.
[5] B. Wang, Y.Q. Li, N. Wu, C.Q. Lan, CO2 bio-mitigation using microalgae, Appl.
Microbiol. Biotechnol. 79 (5) (2008) 707–718.
[6] I. Douskova, J. Doucha, K. Livansky, J. Machat, P. Novak, D. Umysova, V. Zachleder, M.
Vitova, Simultaneous flue gas bioremediation and reduction of microalgal biomass
production costs, Appl. Microbiol. Biotechnol. 82 (1) (2009) 179–185.
[7] C. Yoo, S.Y. Jun, J.Y. Lee, C.Y. Ahn, H.M. Oh, Selection of microalgae for lipid produc-
tion under high levels carbon dioxide, Bioresour. Technol. 101 (2010) S71–S74.
[8] K. Kumar, C.N. Dasgupta, B. Nayak, P. Lindblad, D. Das, Development of suitable
photobioreactors for CO2 sequestration addressing global warming using green
algae and cyanobacteria, Bioresour. Technol. 102 (8) (2011) 4945–4953.
[9] F.G.A. Fernandez, C.V. Gonzalez-Lopez, J.M.F. Sevilla, E.M. Grima, Conversion of CO2
into biomass by microalgae: how realistic a contribution may it be to significant
CO2 removal? Appl. Microbiol. Biotechnol. 96 (3) (2012) 577–586.
[10] S. Grierson, V. Strezov, J. Bengtsson, Life cycle assessment of a microalgae biomass
cultivation, bio-oil extraction and pyrolysis processing regime, Algal Res. 2 (3)
(2013) 299–311.
[11] EU, The EU Emissions Trading System (EU ETS) Factsheet, 2013. (doi: 102834/55480).
[12] M. Packer, Algal capture of carbon dioxide; biomass generation as a tool for green-
house gas mitigation with reference to New Zealand energy strategy and policy,
Energ Policy 37 (9) (2009) 3428–3437.
[13] E. Ono, J.L. Cuello, Selection of optimal microalgae species for CO2 sequestration. Sec-
ond National Conference on Carbon Sequestration, Proceeding on the 2nd Annual
Conference on Carbon Sequestration, USA, 2003.
[14] Y.L. Jiang, W. Zhang, J.F. Wang, Y. Chen, S.H. Shen, T.Z. Liu, Utilization of simulated
flue gas for cultivation of Scenedesmus dimorphus, Bioresour. Technol. 128 (2013)
359–364.
[15] A. Hamasaki, N. Shioji, Y. Ikuta, Y. Hukuda, T. Makita, K. Hirayama, H. Matuzaki, T.
Tukamoto, S. Sasaki, Carbon-dioxide fixation by microalgal photosynthesis using ac-
tual flue-gas from a power-plant, Appl. Biochem. Biotechnol. 45–6 (1994) 799–809.
[16] L.M. Brown, Uptake of carbon dioxide from flue gas by microalgae, Energy Convers.
Manag. 37 (6–8) (1996) 1363–1367.
[17] J. Doucha, F. Straka, K. Livansky, Utilization of flue gas for cultivation of microalgae
(Chlorella spp.) in an outdoor open thin-layer photobioreactor, J. Appl. Phycol. 17
(5) (2005) 403–412.
[18] C.G. Borkenstein, J. Knoblechner, H. Fruhwirth, M. Schagerl, Cultivation of Chlorella
emersonii with flue gas derived from a cement plant, J. Appl. Phycol. 23 (1) (2011)
131–135.
[19] S. Van Den Hende, H. Vervaeren, H. Boon, Flue gas compounds and microalgae: (bio-)
chemical interactions leading to biotechnological opportunities, Biotechnol. Adv. 30
(2012) 1405–1424.
[20] J.R. Benemann, W.J. Oswald, Systems and economic analysis of microalgae ponds for
conversion of CO2 to biomass, Final Report. US DOE-NETL No: DOE/PC/93204-T5,
Prepared for the Energy Technology Center, Pittsburgh, USA, 1996.
[21] J.R. Benemann, CO2 mitigation with microalgae systems, Energy Convers. Manag. 38
(1997) S475–S479.
[22] K.L. Kadam, Environmental implications of power generation via coal-microalgae
cofiring, Energy 27 (10) (2002) 905–922.
[23] R. Sayre, Algal biofuels, a systems approach, In Vitro Dev. Biol. Anim. 46 (2010)
S67–S68.
[24] P.J. McGinn, K.E. Dickinson, S. Bhatti, J.C. Frigon, S.R. Guiot, S.J.B. O'Leary, Integration
of microalgae cultivation with industrial waste remediation for biofuel and
bioenergy production: opportunities and limitations, Photosynth. Res. 109 (1–3)
(2011) 231–247.
[25] D. Tilman, P.B. Reich, J. Knops, D. Wedin, T. Mielke, C. Lehman, Diversity and produc-
tivity in a long-term grassland experiment, Science 294 (5543) (2001) 843–845.
[26] M. Loreau, A. Hector, Partitioning selection and complementarity in biodiversity
experiments, Nature 412 (6842) (2001) 72–76.
[27] C.Y. Li, X.H. He, S.S. Zhu, H.P. Zhou, Y.Y. Wang, Y. Li, J. Yang, J.X. Fan, J.C. Yang, G.B.
Wang, Y.F. Long, J.Y. Xu, Y.S. Tang, G.H. Zhao, J.R. Yang, L. Liu, Y. Sun, Y. Xie, H.N.
Wang, Y.Y. Zhu, Crop diversity for yield increase, PLoS ONE 4 (11) (2009).
[28] Y. Zhang, H.Y.H. Chen, P.B. Reich, Forest productivity increases with evenness, spe-
cies richness and trait variation: a global meta-analysis, J. Ecol. 100 (3) (2012)
742–749.
[29] B. Worm, E.B. Barbier, N. Beaumont, J.E. Duffy, C. Folke, B.S. Halpern, J.B.C. Jackson,
H.K. Lotze, F. Micheli, S.R. Palumbi, E. Sala, K.A. Seloke, J.J. Stachiwicz, R. Ward, Im-
pacts of biodiversity loss on ocean ecosystem services, Nature 314 (2006) 787–790.
[30] D. Tilman, J. Hill, C. Lehman, Carbon-negative biofuels from low-input high-diversity
grassland biomass, Science 314 (5805) (2006) 1598–1600.
[31] D.R. Ptacnik, A.G. Solimini, T. Andersen, T. Tamminen, P. Brettum, L. Lepisto, E.
Willen, S. Rekolainen, Diversity predicts stability and resource use efficiency in
natural phytoplankton communities, Proc. Natl. Acad. Sci. U. S. A. 105 (13) (2008)
5134–5138.
[32] J. McGrady-Steed, P.M. Harris, P.J. Morin, Biodiversity regulates ecosystem predict-
ability, Nature 390 (6656) (1997) 162–165.
[33] T. Bell, J.A. Newman, B.W. Silverman, S.L. Turner, A.K. Lilley, The contribution of
species richness and composition to bacterial services, Nature 436 (7054) (2005)
1157–1160.
233
[34] J.J. Weis, D.S. Madrigal, B.J. Cardinale, Effects of algal diversity on the production of
biomass in homogeneous and heterogeneous nutrient environments: a microcosm
experiment, PLoS ONE 3 (7) (2008).
[35] B.J. Cardinale, D.S. Srivastava, J.E. Duffy, J.P. Wright, A.L. Downing, M. Sankaran, C.
Jouseau, Effects of biodiversity on the functioning of trophic groups and ecosystems,
Nature 443 (7114) (2006) 989–992.
[36] K. Gross, B.J. Cardinale, Does species richness drive community production or vice
versa? Reconciling historical and contemporary paradigms in competitive commu-
nities, Am. Nat. 170 (2) (2007) 207–220.
[37] J.W. Fox, Interpreting the ‘selection effect’ of biodiversity on ecosystem function,
Ecol. Lett. 8 (8) (2005) 846–856.
[38] A.A. Corcoran, W.J. Boeing, Biodiversity increases the productivity and stability of
phytoplankton communities, PLoS ONE 7 (11) (2012).
[39] S. Satpathy, D. Mishra, Use of intercrops and antifeedants for management of egg-
plant shoot and fruit borer Leucinodes orbonalis (Lepidoptera: Pyralidae), Int. J.
Trop. Insect Sci. 31 (1–2) (2011) 52–58.
[40] V.H. Smith, B.S.M. Sturm, F.J. de Noyelles, S.A. Billings, The ecology of algal biodiesel
production, Trends Ecol. Evol. 25 (5) (2010) 301–309.
[41] M. Stockenreiter, A.K. Graber, F. Haupt, H. Stibor, The effect of species diversity
on lipid production by micro-algal communities, J. Appl. Phycol. 24 (1) (2012)
45–54.
[42] M. Stockenreiter, F. Haupt, A.K. Graber, J. Seppala, K. Spilling, T. Tamminen, H. Stibor,
Functional group richness: implications of biodiversity for light use and lipid yield in
microalgae, J. Phycol. 49 (5) (2013) 838–847.
[43] R.L. Guillard, Culture of phytoplankton for feeding marine invertebrates, in: W.
Smith, M. Chanley (Eds.), Culture of Marine Invertebrate Animals, Springer, USA
1975, pp. 29–60.
[44] H. Utermöhl, Zur Vervollkommnung der quantitativen Phytoplankton-Methodik,
Mitt. Int. Verein. Limnol. 9 (1958) 1–38.
[45] I. Olenina, S. Hajdu, L. Edler, A. Andersson, N. Wasmund, S. Busch, J. Göbel, S. Gromisz,
S. Huseby, M. Huttunen, A. Jaanus, P. Kokkonen, I. Ledaine, E. Niemkiewicz,
Biovolumes and size-classes of phytoplankton in the Baltic Sea. HELCOM, Balt. Sea
Environ. Proc. (No. 166) (2006).
[46] A.M. Jespersen, K. Christofferson, Measurements of chlorophyll—a from phytoplank-
ton using ethanol as extraction solvent, Arch. Hydrobiol. 109 (3) (1987) 445–454.
[47] E.G. Bligh, W.J. Dyer, A rapid method of total lipid extraction and purification, Can. J.
Biochem. Physiol. 37 (8) (1959) 911–917.
[48] O.H. Lowry, N.J. Rosenbrough, A.L. Farr, R.J. Randall, Protein measurements with folin
phenol reagent, J. Biol. Chem. 193 (1951) 265–275.
[49] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Colorimetric method for
determination of sugars and related substances, Anal. Chem. 28 (1956) 350–356.
[50] S. Suikkanen, S. Pulina, J. Engstrom-Ost, M. Lehtiniemi, S. Lehtinen, A. Brutemark,
Climate change and eutrophication induced shifts in northern summer plankton
communities, PLoS ONE 8 (6) (2013).
[51] C. Legrand, E. Fridolfsson, M. Bertos-Fortis, E. Lindehoff, P. Larsson, J. Pinhassi, A.
Andersson, Interannual variability of phyto-bacterioplankton biomass and produc-
tion in coastal and offshore waters of the Baltic Sea, AMBIO (2015), http://dx.doi.
org/10.1007/s13280-015-0662-8.
[52] Y. Ishida, N. Hiragushi, H. Kitaguchi, A. Mitsutani, S. Nagai, M. Yoshimura, A highly
CO2-tolerant diatom, Thalassiosira weissflogii H1, enriched from coastal sea, and its
fatty acid composition, Fish. Sci. 66 (2000) 655–659.
[53] F.M. Salih, Microalgae tolerance to high concentrations of carbon dioxide: a review,
J. Environ. Prot. 2 (2011) 648–654.
[54] M.R. Tredici, Photobiology of microalgae mass cultures: understanding the tools for
the next green revolution, Biofuels 1 (1) (2010) 143–162.
[55] M. Olofsson, T. Lamela, E. Nilsson, J.P. Bergé, V. del Pino, P. Uronen, C. Legrand, Com-
bined effects of nitrogen concentration and seasonal changes on the production of
lipids in Nannochloropsis oculata, Mar. Drugs 12 (4) (2014) 1891–1910.
[56] C. Yoo, G.G. Choi, S.C. Kim, H.M. Oh, Ettlia sp. YC001 showing high growth rate and
lipid content under high CO2, Bioresour. Technol. 127 (2013) 482–488.
[57] S.Y. Chiu, C.Y. Kao, M.T. Tsai, S.C. Ong, C.H. Chen, C.S. Lin, Lipid accumulation and CO2
utilization of Nannochloropsis oculata in response to CO2 aeration, Bioresour.
Technol. 100 (2) (2009) 833–838.
[58] N.R. Moheimani, Inorganic carbon and pH effect on growth and lipid productivity
of Tetraselmis suecica and Chlorella sp. (Chlorophyta) grown outdoors in bag
photobioreactors, J. Appl. Phycol. 25 (2013) 387–398.
[59] X.W. Wang, J.R. Liang, C.S. Luo, C.P. Chen, Y.H. Gao, Biomass, total lipid production,
and fatty acid composition of the marine diatom Chaetoceros muelleri in response
to different CO2 levels, Bioresour. Technol. 161 (2014) 124–130.
[60] K. Kumar, D. Banerjee, D. Das, Carbon dioxide sequestration from industrial flue gas
by Chlorella sorokiniana, Bioresour. Technol. 152 (2014) 225–233.
[61] D. Schwenk, J. Seppala, K. Spilling, A. Virkki, T. Tamminen, K.M. Oksman-Caldentey,
H. Rischer, Lipid content in 19 brackish and marine microalgae: influence of growth
phase, salinity and temperature, Aquat. Ecol. 47 (4) (2013) 415–424.
[62] M.R. Brown, S.W. Jeffrey, The amino acid and gross composition of marine diatoms
potentially useful for mariculture, J. Appl. Phycol. 7 (6) (1995) 521–527.
[63] E. Bertozzini, L. Galluzzi, F. Ricci, A. Penna, M. Magnani, Neutral lipid content and
biomass production in Skeletonema marinoi (Bacillariophyceae) culture in response
to nitrate limitation, Appl. Biochem. Biotechnol. 170 (7) (2013) 1624–1636.
[64] G.A. Dunstan, J.K. Volkman, S.M. Barrett, C.D. Garland, Changes in the lipid-
composition and maximization of the polyunsaturated fatty-acid content of 3
microalgae grown in mass-culture, J. Appl. Phycol. 5 (1) (1993) 71–83.