spilling 2011
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Inducing autoflocculation in the diatom Phaeodactylum
tricornutum through CO2 regulation
Kristian Spilling & Jukka Seppälä & Timo Tamminen
Received: 29 June 2010 /Revised and accepted: 14 October 2010 /Published online: 6 November 2010# Springer Science+Business Media B.V. 2010
Abstract The effect of pH on flocculation was studied
using the diatom Phaeodactylum tricornutum and the greenalgae Scenedesmus cf. obliquus as surrogate species. There
was a distinct, species-specific threshold of pH where
flocculation started. P. tricornutum started to flocculate at
pH 10.5 and S. cf. obliquus at pH 11.3. Above this
threshold, settling rates up to 360 cm h−1 were observed for
P. tricornutum and the concentrating factor was up to
60-fold. The combined effect of pH, turbulence, and cell
density on flocculation of P. tricornutum was additionally
studied in a factorial 53-design experiment. pH was the
most important factor affecting flocculation, but at the pH
threshold (pH 10.5), the concentrating factor was increased
by increasing cell density and turbulence. Algae increases
the pH during photosynthesis, and the P. tricornutum and S.
cf. obliquus cultures increased the pH to a maximum of
10.8 and 9.5, respectively, after discontinuing the CO2
supply. For P. tricornutum, this was above the flocculation
threshold, and rapid settling of this species due to increased
pH was observed in a matter of hours after the CO2 supply
was turned off. This could be used as a simple, low-cost,
initial dewatering step for this species.
Keywords pH . Turbulence . Harvesting . Algal culturing .
Particle encounter rate
Introduction
Harvesting and dewatering of algal cultures is a significant
cost of algal production (Gudin and Thepenier 1986).
Low-cost solutions to the problem of dewatering must be
developed before algae can be considered as a raw material
for low-cost commodities such as biofuel. In particular, the
first steps of the harvesting process should be very simple
and have minimal energy requirement.
Aggregation due to addition of polymers (flocculation)
or electrolytes (coagulation) has been seen as a potential,
first step in algal harvesting. The most common method has
been to add a flocculating (or coagulating) agent (Molina
Grima et al. 2003), which makes the algae aggregate and
settle. For example, multivalent salts (McGarry 1970) and
chitosan (Divakaran and Pillai 2002) have frequently been
used. This approach works well on a small scale and/or when
the end-product is of high value. However, at larger scales,
this approach would require substantial amounts of the
flocculation agent, and albeit being relatively inexpensive, it
would impose an extra cost, not well-suited for production of
low-value, raw materials. In addition, the flocculating agents
may have to be removed afterwards.
There are ways to induce flocculation without the use of
a flocculating agent, and high pH is one example that has
been reported (Sukenik and Shelef 1984; Ayoub et al. 1986;
Yahi et al. 1994; Nurdogan and Oswald 1995). Flocculation
at high pH takes place mainly due to precipitation of
CaCO3, Mg(OH)2 (Vråle 1978; Ay ou b e t a l. 1986;
Semerjian and Ayoub 2003), and calcium phosphate
(Sukenik and Shelef 1984). However, the pH threshold
for flocculation seems to vary with the algal species being
cultured (Yahi et al. 1994; Blanchemain and Grizeau 1999),
K. Spilling (*) : J. Seppälä : T. Tamminen
Marine Research Centre, Finnish Environment Institute,
Erik Palménin aukio 1, P.O. Box 140, 00251 Helsinki, Finland
e-mail: [email protected]
K. Spilling
Tvärminne Zoological Station, University of Helsinki,
10900 Hanko, Finland
J Appl Phycol (2011) 23:959 – 966
DOI 10.1007/s10811-010-9616-5
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and for some algae, the effect of pH on flocculation is
minimal (Knuckey et al. 2006). Consequently, there must
be some inherent properties of specific algae that alter their
adhesion.
For those species that flocculate at elevated pH, this is an
interesting option for harvesting because algae themselves
increase the pH when photosynthesizing (Falkowski and
Raven 1997). CO2 is a weak acid when dissolved in water,and during photosynthesis, CO2 is fixed into organic carbon,
thereby increasing the pH (algal uptake of nutrients, e.g.,
nitrate, also alter the pH, but to a much smaller extent). When
growing algae in dense cultures, effective gas exchange is
vital, and input of CO2 is needed to provide inorganic carbon
and to regulate the pH. Potentially, simply discontinuing the
CO2 supply could be a first step in the harvesting procedure,
making the algae increase the pH until flocculation starts
(Sukenik and Shelef 1984). The concentrated biomass could
then be taken out for further dewatering.
There is of course a limit to how much the pH can
increase due to photosynthetic activity because the algaewill slow down photosynthesis and eventually stop
altogether once their upper pH tolerance is reached
(Falkowski and Raven 1997; Spilling 2007). For most algal
species, growth slows down significantly at pH>9.0, but
there are examples of species that can drive the pH up well
above 10 (Goldman et al. 1982; Møgelhøy et al. 2006),
which potentially could be high enough for inducing
flocculation (Blanchemain and Grizeau 1999). Although
the potential of pH regulation has been suggested to be a
way of harvesting algae through autoflocculation (Sukenik
and Shelef 1984; Benemann and Oswald 1996), it has
received very little attention during the past decade.
The flocculation of the diatom Phaeodactylum tricornutum
and the green alga S . cf. obliquus at high pH was studied.
The reason behind choosing these species was that adhesive
proteins in P. tricornutum have been identified (Dugdale et
al. 2006), and diatoms in general seem to flocculate at high
pH (Knuckey et al. 2006). In contrast, previous studies have
suggested that S. obliquus does not flocculate very easily at
high pH (Lavoie and de la Noüe 1986). Our main objective
was to observe how pH would affect flocculation in the two
species grown under the same conditions and additionally, if
photosynthesis could drive the pH high enough to induce
autoflocculation. Furthermore, we also documented the
effect of particle encounter rate through different cell
concentration and turbulence regimes for P. tricornutum.
Materials and methods
Non-axenic monocultures of P . tricornutum (fusiform
growth form) and S . cf. obliquus were used. The P.
tricornutum culture originated from Tvärminne Zoological
Station (strain Tv 335), while the S. cf. obliquus culture
originated from isolation of a single cell picked from a
natural plankton community (taken from a rock pool at the
outer archipelago, SW coast of Finland, summer 2008). Both
cultures were grown at 18°C, salinity of 6 psu, and 16 h
light/8 h dark cycle and ∼200 μ mol photons m−2 s−1
irradiance. Cultures were bubbled with prefiltered air
(0.2 μ m) in order to keep cells in suspension and stabilizethe pH. The pH was approximately pH 8.2 during culturing.
The culture was grown semi-continuously in T2 medium,
which is modified f/2 medium (Guillard 1975), where the
molar nutrient ratios, N/P/Si, are adjusted to 16:1:8. Sub-
samples of the parent cultures were used for all measurements
and experiments. Adjusting the pH in subsamples and
experimental setups was done by adding NaOH.
Dry weight (DW) of the algal culture was determined by
filtering onto dry, pre-weighed GF/F filters (Whatman); the
filters were dried overnight (>12 h) at 60°C and weighed
again, and the DW was calculated by subtracting the filter
weight.Chlorophyll a (Chl a) fluorescence was used as an
approximation for biomass during experiments, and it was
measured using a fluorometer (Varian Cary Eclipse) with
430 nm excitation and 680 nm emission light. In cases with
very high biomass, the samples were diluted up to 1,000
times, depending on the amount of biomass, in order to be
in the linear range of the fluorescence signal to biomass
unit. Minimum fluorescence ( F 0) was measured on dark
acclimated cells (5 min). For measurement of the
maximal fluorescence ( F m ), 10 μ l of 2 mM DCMU
(3-(3,4-dichlorophenyl)-1,1-dimethylurea) was added
mL−1 of sample, and the sample was left for 5 min in
light before determination of fluorescence using the method
described above. As an index of the health of the cells,
photochemical efficiency in PS II was calculated according
to the equation: F m À F 0ð Þ= F m ¼ F v= F m where F 0, F m , and
F v is minimum, maximum, and variable fluorescence,
respectively.
Measuring the effect of pH The pH was measured using a
p H meter. T he e ffec t o f p H o n flo cc ulatio n was
examined in small (14 mL) polycarbonate tubes. The
cultures were kept in 300-mL Erlenmeyer flasks, and the
pH was gradually increased by addition of NaOH.
Subsamples of 12 mL were taken out and filled in
replicate tubes for each pH step. The cultures in the
tubes were subsequently left for 1 h to settle. After the
settling time, the uppermost 3 mL was collected. The F v/
F m was determined from this sample and for the
completely mixed culture before pH had been adjusted.
The percentage removal from the uppermost water layer
was calculated using the equation: P r ¼ A0 À Af ð Þ Â
100= A0 where P r is the percentage removal, A0 the initial
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In order to determine the longer-term concentrating
factor, we filled a glass cylinder (50 cm, Ø =5 cm) with P.
tricornutum culture. The pH was increased to 10.5, by
adding NaOH, and left for 48 h. After the settling period,
the thickness of the settled material was compared with that
of the clear water phase above, and samples from the
bottom part were taken out and compared with the original
dry weight. Settling rate was calculated by measuring the
time and distance of sinking of the largest particles (∼1 mm
diameter) that could easily be identified.
20 40 60 80 100 120
10
20
30
40
20 40 60 80 100 120
10
20
30
40
20 40 60 80 100 120 140
10
20
30
40
20 40 60 80 100 120 140
10
20
30
40
20 40 60 80 100 120
10
20
30
40
a
b
c
d
e
T u r b u l e n c e
( r o t a t i o n s
h - 1 )
Biomass concentration (mg DW L-1
)
Fig. 2 The concentrating factor
of P . tricornutum at different pH
(10.0 (a), 10.1 (b), 10.3 (c), 10.5
(d), and 10.8 (e), biomass
concentration ( x-axis) and
turbulence regimes ( y-axis). The
different treatments were
followed by 1 h settling, and the
concentrating factor was
determined from the bottom of the experimental units. The dots
in (a) represent the sampling
combinations, which were equal
for all plots (a – e), and the dotted
lines in (d) represent the
sampling transects presented in
Fig. 3. The negative correlation
of biomass concentration and
concentrating factor at pH 10.8
(e) is probably due to a
methodological error; please see
the text for discussion
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Results
The effects of pH on flocculation
The P. tricornutum culture started to flocculate and formed
large aggregates when pH reached 10.5, and the culture
subsequently started to settle (Fig. 1). The same took place
in the S. cf. obliquus culture, but the threshold for
flocculation was at pH 11.3. The clearance rates above the
pH threshold were for the two cultures ∼85% and ∼70%,
respectively, after 1 h settling time (Fig. 1a ).
The P. tricornutum culture became visibly lighter brown
in color at pH>11, but the photochemical efficiency
( F v/ F m ) was relatively stable at 0.35 – 0.45 up to pH 11.5
(Fig. 1b), indicating healthy cells, 1 h after the increase in
pH. However, at pH 12.1, there was a marked drop in the F v/ F m to <0.1 for this species. For S. cf. obliquus, the
photochemical efficiency was on average 38% higher than
for P. tricornutum at pH<11 and did not decrease notably
even at pH 12.1 (Fig. 2).
After the increase in pH, a clear phase at the top of the
culture was created, and the settling rate of the borderline
between the visibly colored culture and the clear water phase
on top was 22 cm h−1 at pH 12. However, large aggregates
were settling considerably faster than this. The largest
aggregates (Ø ∼1 mm) settled at rates of 180 – 360 cm h−1.
Experiment
The experimental results supported the existence of a
threshold at pH 10.5 for P. tricornutum where aggregates
started to form, which consequently increased the settling
rate (Fig. 2). At pH 10.5, the concentrating factor increased
up to 11-fold while at pH 10.8, a 12-fold increase in
biomass at the bottom of the experimental unit was
recorded, which was the theoretical maximal increase with
the setup used.
Flocculation and settling speed increased with increasing
turbulence and cell concentration at pH 10.5, but at pH
below or above this, there was little or no effect of neither
turbulence nor cell concentration. At pH<10.4, there was
very little flocculation overall, while at pH 10.8 the major
part of the biomass settled (>sixfold increase) regardless of
turbulence or cell concentration, with the exception at the
lowest biomass concentration.
The effect of turbulence was not linear at pH 10.5
(Fig. 3). At the high end of turbulence range, the settling
was approaching the maximal asymptote. For cell concen-
tration, in contrast, non-linearity was not as obvious.
However, using a rise-to-maximum equation (see Fig. 3)
gave a better fit to the data ( R2=0.98) compared with linear
regression ( R2=0.91).
Potential of cultures to increase pH
In the separate batch culture test, the P. tricornutum and S.
cf obliquus cultures increased the pH to 10.8 and 9.5,
respectively, when left without air bubbling. Using a
biomass concentration of 1.1 g DW L−1, P. tricornutum
increased the pH from ∼7 to 10.8 within 5 h (Fig. 4), which
was well above the pH threshold for flocculation (Fig. 1a ).
Time (h)
0 1 2 3 4 5
p H
7
8
9
10
11
Fig. 4 Increase in pH over time in a culture of P . tricornutum. The
biomass concentration of the culture was 1.1 g DW L−1. The cultures
were in 250 mL TC flasks, and cells were kept in suspension with
magnetic stirring (∼60 rpm). The dotted horizontal line denotes the
pH 10.5 flocculation threshold (Fig. 1). Three replicates were used,
and the solid line represents the regression fit of the equation F ð X Þ ¼ Y 0 þ a» 1 À exp À ÀbX ð Þð Þ. S et tl in g a t t he e nd p oi nt
(pH 10.8) is presented in Fig. 5
Turbulence (rotations h-1
)
0 20 40
C o n c e n t r a t i o n f a c t o r
0
5
10
Biomass concentration (mg DW L-1
)
0 50 100 150
Turbulence
Biomass concentration
Fig. 3 Effect of cell concentration and turbulence on sedimentation of
algal material at pH 10.5; the sampling transects is presented in
Fig. 2d. The solid line represents the regression fit using the equation: F ð X Þ ¼ Y 0 þ a» 1 À exp ÀbX ð Þð Þ. Note the two different x-axis
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The cells formed aggregates and started to settle rapidly after
the stirring was discontinued. After 30 min, the biomass in the
bottom on the TC flasks was concentrated by a factor 8.6±0.3
(SD) and this increased to 10.7±1.8 (SD) after 12 h (Fig. 5).By comparison, the same culture at pH 7.6 yielded
concentration factors of 1.4± 0.02 (SD) and 3.5± 0.2 (SD)
after 30 min and 12 h settling time, respectively. Using a
longer settling chamber (50 cm glass cylinder) and settling
period of 48 h, the concentrating factor was 60-fold.
Discussion
There was a clear difference in the effect of pH on the
two cultures. P. tricornutum flocculated at a much lower
pH than S. cf. obliquus. The two main forces at work
during the initial coagulation phase are van der Waals
attractive forces and electrostatic repulsive forces. The
surface charge of most suspended particles (including
algae) is negative, and the repulsive forces between like-
charged particles must be overcome before flocs can start
to form. For this, the surface chemistry of the involved
particles is critical, and it is not surprising that different
algae are different in this respect. It is outside the scope of
this paper to make any analysis of the underlying
chemistry, but the flocculation of P. tricornutum at a
relatively low pH could be due to adhesive proteins at the
cell surface (Dugdale et al. 2006). The adhesiveness of the
surface can to a large degree be altered by the algae
themselves (when healthy), and increasing adhesiveness is
an active strategy for some species in order to, e.g., attach
to surfaces (Hoagland et al. 1993). Additional factors that
could alter the flocculation properties are algal excretion
of dissolved organic matter that could have autoflocculat-
ing properties (Aluwihare and Repeta 1999) and
co-occurring bacteria that could produce bioflocculants
(Deng et al. 2003; Lee et al. 2009).
The algal species that only flocculate at very high pH
(e.g., >11) will most likely not be able to increase the pH
above the threshold for flocculation, which was the case of S.
cf. obliquus in this study. The potential of using pH in theharvesting procedure must be evaluated for the particular
algal species that is grown. However, this study clearly
shows that P. tricornutum had the ability to drive the pH far
enough to induce autoflocculation. Thus, regulation of pH,
through decreasing the flow of CO2, could be used as a first
low-cost step in the harvesting procedure. An alternative way
of regulating the pH could be addition NaOH or by bubbling
with N2 gas (Cohen and Kirchmann 2004), but this would
come with additional cost.
Another aspect of pH that is highly species-specific is
the effect on the physiology of the algal cells, which was
also the case for P. tricornutum and S. cf. obliquus. The
photochemical efficiency ( F v/ F m ) can be used as a proxy
for stress (Falkowski and Raven 1997), and the results
indicate that P. tricornutum was not negatively affected by
short-term exposure (1 h) to pH<11.5. S. cf. obliquus was
even more tolerant to high pH (Fig. 1b). Other flocculation
studies have also concluded that short-term of exposure to
high pH has relatively little effect on algal cells, if the pH is
lowered again after flocculation (Blanchemain and Grizeau
1999; Knuckey et al. 2006). For example, the fatty acid
profile of Skeletonema costatum did not change during 1 h
incubation at pH 10.2 compared with cells harvested by
centrifugation; however, after longer exposure to high pH,
the cells started to lyse (Blanchemain and Grizeau 1999).
At the pH threshold where aggregates started to form, both
turbulence and cell concentration had a clear effect on the
flocculation and settling of P. tricornutum. At pH below 10.5,
there was very little effect of turbulence and cell concentra-
tion, although cell encounter rate is well-established as a key
factor in aggregate formation. The reason for the lack of effect
was most likely a low probability of particles aggregating after
contact at pH< 10.5. Consequently, the experimental time (1 h
Settling time
30 min 12 h
B i o m a s s ( g L - 1 )
0
5
10
pH 7.6
pH 10.8
Fig. 5 Settling of P .
tricornutum at different pH and
settling time. The cultures were in
250 mL TC flasks and samples of
2 mL were taken from thebottom:
30 min and 12 h after stirring was
stopped. The dotted horizontal
line denotes the initial biomass
concentration. The difference in
pH was obtained with different gas exchange prior to settling,
e.g., the high pH culture
(pH 10.8) was obtained after 5 h
incubation in light without any
bubbling (Fig. 4). Error
bars=standard deviation (n=3)
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treatment and 1 h settling time) was too short to produce any
effect. At pH above 10.5, the response surface was a relatively
high and flat plateau, meaning that most of the cells were
sinking to the bottom within the 1 h settling time, regardless
of turbulence or cell concentration.
The cell concentration used in these experiments was
relatively low due to the cultivating environment we used,
and the results suggest that a higher cell concentrationwould be better not only for biomass yield, but also from a
harvesting point of view. The increase in flocculation with
increasing cell concentration was not linear, however, and
the optimal cell concentration should be weighed against
other production processes such as growth rate. In Fig. 2e,
the results suggest that, at pH 10.8, the flocculation
decreased with increasing cell concentration, which is
somewhat counterintuitive. Flocculation and settling took
place at this pH (Fig. 1), and the observed decreased
flocculation with increasing cell concentration seen in
Fig. 2e is probably a methodological artifact. Chl a
fluorescence was used as an approximation for biomass,and the larger aggregates formed at high biomass at
pH 10.8 would lead to higher packaging effect (Falkowski
and Raven 1997), leading to decreasing difference in
fluorescence signal before and after flocculation. This
conclusion is also supported by the flocculation results
with a much higher biomass (Fig. 4), which indicates that
pH can be used to make the cells flocculate also at high cell
concentrations.
Determining the kinetic energy in a turbulent flow is
notoriously difficult, and this is the reason why we used
number of rotations per hour as the proxy for turbulence.
Although this is not a quantifiable unit (kinetic energy will
vary for example with size of experimental unit, amount of
headspace, etc.), it reveals some general characteristics of
the effect turbulence has on flocculation. Turbulence
increased flocculation at the pH threshold, but, as with cell
concentration, the relationship between flocculation and
turbulence was not linear. The results showed that turbu-
lence had a positive effect on flocculation at pH 10.5, but
increasing turbulence substantially would probably not
increase the concentrating factor much, as the maximum
turbulence used in this study gave close to the maximum
effect on flocculation. This information could be useful if
the algae are able to increase the pH close to the
flocculation threshold. In that case, some turbulence will
likely increase the concentrating factor, but this should be
weighed against the energy needed for creating turbulence.
In conclusion, high pH did make P. tricornutum and S.
cf. obliquus flocculate, and P. tricornutum was able to
increase pH above the threshold for flocculation through
the photosynthetic uptake of CO2. This could be used as an
autoflocculation mechanism, which can be induced by
turning off the CO2 supply to the algal culture. The pH
was the single most important factor, but cell concentration
and turbulence also had an effect on flocculation. Cell
concentration should be as high as possible before harvest-
ing. Turbulence will also increase aggregate formation and
settling and had the highest effect close to the pH threshold
of flocculation.
Acknowledgments This study was funded through the FinnishAcademy program Sustainable Energy (SusEn) and through the
Nordic Energy Research program N-INNER. We would also like to
thank DSc (Tech) Perttu Koskinen (Neste Oil Corporation) for
comments on the manuscript.
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