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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