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Harvesting of the marine microalga Conticribraweissflogii (Bacillariophyceae) by cationic polymericflocculants

Ricardo Bessler K€onig a, Rafael Sales b, Fabio Roselet a,Paulo Cesar Abreu b,*

a Post-graduate Course on Aquaculture, Institute of Oceanography, Federal University of Rio Grande-FURG,

Av. Italia, Km 08, Rio Grande, RS 96201-900, Brazilb Laboratory of Phytoplankton and Microorganisms, Institute of Oceanography,

Federal University of Rio Grande e FURG, Brazil

a r t i c l e i n f o

Article history:

Received 4 October 2013

Received in revised form

26 March 2014

Accepted 3 June 2014

Available online

Keywords:

Marine

Microalga

Conticribra weissflogii

Thalassiosira weissflogii

Flocculation

FLOPAM

* Corresponding author. Tel.: þ55 53 3233 65E-mail addresses: docpca@furg.br, copa@

http://dx.doi.org/10.1016/j.biombioe.2014.06.0961-9534/© 2014 Elsevier Ltd. All rights rese

a b s t r a c t

The harvesting of microalgae is currently one of the bottlenecks hindering the commercial

production of microalgae-based biofuels and products. The objective of this study was to

determine the best flocculant and its optimum concentration in order to harvest the ma-

rine microalga Conticribra weissflogii (previously Thalassiosira weissflogii) for further use in

the production of biofuels or bioelements. Experiments were conducted with cultures in

the logarithmic and stationary growth phases. The low-charge FLOPAM® FO 4240 SH was

the most effective at concentrations of 2 and 4 mg m�3 in the LOG phase cultures, with

flocculation efficiencies >90%. Smaller flocculation efficiencies were observed for cells in

the stationary growth phase, most likely due to the production of dissolved organic carbon

by the microalga. The highest microalgae density generated higher flocculation rates,

whereas the pH and salinity negatively impacted flocculant efficiency.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Microalgae have shown significant advantages in the pro-

duction of biodiesel [1] compared to oilseed crops because

they can be grown in areas that are unsuitable for agriculture,

use nitrogen and phosphorus from domestic or industrial

sewage, and can even be grown in marine or brackish waters

that are unsuitable for human consumption or agricultural

uses. Moreover, these autotrophic microorganisms are

important sources of valuable bioproducts, such as pigments,

09.mikrus.com.br (P.C. Abre001rved.

vitamins, amino acids, and many others, with important uses

in the pharmaceutical and food industries [2].

However, the lack of an efficient harvesting method for

these microorganisms is a major obstacle to their large-scale

production. Microalgae are small in size, ranging from 2 to

200 mm in diameter, and are diluted in culture medium

(0.5e2.0 g m�3), requiring the manipulation of large volumes

to a minimum profitability, since the removal of microalgae

from the liquid medium significantly increases the costs of

production of biodiesel or other bioproducts [3].

u).

Table 1 e Charge density and cationicity of flopamflocculants. Information furnished by the manufacturer.

Flocculant Charge density Cationicity (mol%)

FO 4140 SH Very low 5

FO 4240 SH Low 20

FO 4490 SH Medium 40

FO 4700 SH High 70

FO 4990 SH Very high 100

b i om a s s a n d b i o e n e r g y 6 8 ( 2 0 1 4 ) 1e62

Currently, the most common methods employed in the

separation of microalgae are centrifugation and filtration, but

these are high-energy methods and may cause damage to the

cells. Flocculation, on the other hand, has been indicated as a

more economical and practical solution to concentrate pro-

duced microalgae biomass [4]. Flocculating agents act by

neutralizing the negative charges of the microalgae cell walls,

promoting their coagulation and further sedimentation.

Flocculants can be inorganic or organic, natural or synthetic.

Inorganic flocculants, such as aluminum and iron sulfate, are

effective, but used in high doses can result in contamination

of the produced biomass [5]. Natural organic flocculants, such

as chitosan, are biodegradable and are often used in small

doses [6]; however, they are more expensive than synthetic

polymers [7].

Themain aggregationmechanism of polymeric flocculants

is the establishment of links between the polymer and the cell

walls, known as “polymer bridging”. The neutralization of

electric charges, including the effect of “electrostatic pack-

aging”, is another process that promotes the flocculation of

particles [4,8]. The most important features of polymeric

flocculants are their molecular weight and charge density.

Conventionally, flocculants are classified as very low, low,

medium, high, or very high molecular weight and are also

characterized based on their ionic nature as cationic, anionic

or non-ionic [7]. These polymers have proven to be efficient

and economically viable for the flocculation of the diatom

Chaetoceros calcitrans [8]. However, there is a need for studies to

evaluate the efficiency of flocculants applied to other micro-

algae species with greater commercial interest and especially

to marine species due to the problems caused by saltwater

during the flocculation process.

Previous research has indicated that the marine diatom

Conticribra weissflogii (Grunow) K. Stachura-Suchoples & DM

Williams, previously known as Thalassiosira weissflogii, is a

rapid growing species with high rates of carbon fixation and

high levels of lipid production [9]. Thus, the large-scale culti-

vation of this microalga could contribute not only to the pro-

duction of biodiesel but also to a decrease in the carbon

dioxide concentration in the atmosphere.

The main objective of this study was to evaluate the use of

five polyacrylamide flocculants with different charge den-

sities to harvest the biomass of C. weissflogii.More specifically,

we wanted to determine i) the most efficient cationic floccu-

lant and its minimum concentration for removal of C. weiss-

flogii; ii) the best growth phase in which to use the flocculant;

and iii) the minimum density of microalgae that would pre-

sent the best response to the flocculant.

2. Materials and methods

2.1. Flocculants

Five commercial flocculants (Flopam®, SNF Floerger, France)

were evaluated. The flocculants were all cationic polymers

with a high molecular weight but displayed different charge

densities ranging from very low to very high and cationicity

from 5 to 100mol/% (Table 1). The flocculantswere supplied as

powders and were prepared according to the manufacturer's

instructions. Each flocculant solution (5000 ppm) was vigor-

ously homogenizedwith amagnetic stirrer until the flocculant

was completely dissolved.

2.2. Microalga culture

The strain of C. weissflogii used in all experiments was ob-

tained from the Microalgae Collection of the Laboratory of

Phytoplankton and Marine Microorganisms of the Institute of

Oceanography from the Federal University of Rio Grande

(FURG), registered under the code THAL WEIS-1.

For the cultures, 0.03 m3 of saltwater was filtered (5 mm)

and chlorinated. After 24 h, de-chlorination was carried out

using ascorbic acid. With this treated water, f/2 medium was

prepared [10], and the cells were cultured to the exponential

(Log) and stationary growth phases for the experiments. Car-

boys with the cultures were kept inside a greenhouse covered

with transparent LDPE UV stabilized film with 90% light

transmission. The air temperature was partially controlled

with the use of a thermostat-controlled fan. The cultures were

stirred by continuous air injection, which also furnished CO2

for the cells [11]. These culture conditions generated differ-

ences in temperature between the growth phases. Salinity

differences, with higher values in the stationary growth

phase, were mainly produced by aeration and water evapo-

ration, while pH variability was caused by the CO2 uptake by

the microalgae.

2.3. Abiotic parameters

The temperature (±0.1 �C), salinity (±0.01 PSU) and pH (±0.01unit) were measured during the experiments with a YSI 556

MPS Handheld Multiparameter (Yellow Springs Instruments,

OH, USA).

2.4. Flocculation efficiency

The efficiencies of the flocculants were determined by

considering the decrease in cell number in the water column

in a period before and after the addition of the flocculant. For

this, the initial and final microalgae cell densities were

determined by cell counting using a hemacytometer and an

optical microscope with a final magnification of 400�.

The flocculation efficiency was calculated according to the

formula:

Ef ¼��

Ci � Cf

�Ci

��100

where Ci ¼ the initial concentration and Cf ¼ the concentra-

tion after 15 min [7].

b i om a s s a n d b i o e n e r g y 6 8 ( 2 0 1 4 ) 1e6 3

2.5. Experimental design

2.5.1. Experiment 1The efficiencies of five flocculants (Table 1) were tested for the

removal of C. weissflogii. The experiments were performed in

triplicate, and the final concentration of the flocculant was set

at 1 ppm. The flocculant solutions were added to beakers

containing 300 cc of C. weissflogii culture. This experiment was

repeated with the culture in the Log and stationary growth

phases. The homogenization of the flocculant was performed

by vigorous agitation using a glass rod (approximately 30 s).

After that, the cultures were left for 15 min to settle. The

flocculation efficiencies were determined as described above.

In this experiment, the initial density of C. weissflogii was

6.1 � 105 cm�3. The culture conditions were as follows: 1) Log

phase cultivation: pH 9.1, salinity, 35.6 and temperature

24.9 �C; 2) Stationary phase cultivation: pH 8.7, salinity 30.7

and temperature 28.3 �C.

2.5.2. Experiment 2The best flocculant determined in the previous experiment

was used to determine the minimum concentration to be

added. For this experiment, several dilutions were made from

a 2500 ppm solution of the flocculant FO 4240 SH, which was

tested in eight concentrations (in triplicate); namely, 0.2, 0.4,

0.6, 0.8, 1.0, 1.5, 2.0 and 4.0 ppm. Each concentration of floc-

culant was added to a beaker containing 300 cc of C. weissflogii

culture in the Log phase. The flocculation efficiency was

evaluated in the same way described above.

In this experiment, the conditions of cultivation of C.

weissflogii were pH 9.9, salinity 29.9, and temperature 24.9 �C,and the initial density of cells was 6.1 � 105 cm�3.

2.5.3. Experiment 3After the determination of the optimal concentration of the

flocculant FO 4240 SH, another experiment was performed to

Fig. 1 e Comparison of the efficiency of five distinct

flocculant agents on the harvesting of microalgae C.

weissflogii cultures in the LOG (grey) and stationary (black)

growth phases. The initial cell density was 61 £ 104 cells

cm¡3, and the concentration of all flocculants was

1 mg m¡3. The bars represent the mean value (with

standard deviation) of three replicates.

test its efficiency with different cell densities. Four cell di-

lutions were tested from a culture of C. weissflogii, with the

following treatments (in triplicate): 1.20, 2.45 3.55 and

4.25 � 105 cm�3. For the dilution of the culture media, filtered

seawater was used. The concentration of the flocculant used

was 4 ppm. The flocculant solution (4.0 ppm) was added to

beakers containing 300 cc of a Log phase culture of C. weiss-

flogii at different densities. The flocculation efficiency was

evaluated by the same method described above.

The cultures presented the following conditions: pH: 8.9,

9.4, 9.8 and 10.0; salinity: 33.1, 34.0, 34.6 and 35.3 and tem-

perature: 24.6, 25.4, 25.8 and 26.4 �C.

2.6. Statistical analysis

The data normality and homoscedasticity were verified for

each data set using the ShapiroeWilk and Bartlett's tests. The

flocculation efficiencies were compared by one-way ANOVA

(a¼ 0.05) followed by Tukey'smultiple comparisons ad hoc test

[12]. Statistical analyses were performed using the software

STATISTICA (StatSoft Inc., Tulsa, OK, USA).

3. Results

3.1. Experiment 1

The flocculation efficiencies for the different flocculants and

different growth phases are shown in Fig. 1. There were sta-

tistically significant differences (P < 0.05) in the efficiency of

flocculation between the growth phases and for each treat-

ment. The highest efficiencies were registered for the floccu-

lants FO 4140 SH and FO 4240 SH, which removed more than

90% of the cells in the Log phase but did not show the same

efficiency for cells in the stationary phase. In general, the cells

in the Log phase resulted in higher (85%) flocculation effi-

ciencies in comparison to the cells in the stationary phase,

which resulted in amaximal removal efficiency of 63%. Higher

charge densities resulted in lower efficiencies in the Log phase,

but no significant effect was observed in the stationary phase.

3.2. Experiment 2

Statistical analysis indicated that the treatments with floc-

culant concentrations of 2 and 4 mgm�3 were bigger (P < 0.05)

than the other treatments (Fig. 2). A positive relationship was

observed between the flocculant concentration and efficiency.

3.3. Experiment 3

Fig. 3 shows the results of the flocculation efficiency assays at

different cell densities. The best removal results were ob-

tained for treatments with 3.55 and 4.25 � 105 cm�3, although

no significant differences among treatments were observed.

4. Discussion

The use of salt and brackish water is, perhaps, the greatest

advantage of the large-scale culture of marine microalgae for

Fig. 3 e Comparison of the flocculant Flopam FO 4240 SH

(4.0 mg m¡3) in the harvesting of the microalgae C.

weissflogii at different cell densities. The bars represent the

mean value (with standard deviation) of three replicates.

b i om a s s a n d b i o e n e r g y 6 8 ( 2 0 1 4 ) 1e64

biofuel and bioelement production because it releases fresh-

water for more critical purposes, such as food production and

drinkingwater. However, similar to all freshwatermicroalgae,

the dewatering and concentration of the produced biomass is

also a problem for marine microalgae, representing a signifi-

cant portion of the production costs [3,13].

The flocculation ofmicroalgae has a good cost-benefit ratio

because it is a low energy consumption process and does not

damage the cells. Recently, several studies have evaluated the

potential of flocculation for the concentration of microalgae.

However, studies on the flocculation ofmarinemicroalgae are

not as abundant as those on freshwater species [4], most likely

because flocculants are not as efficient in marine water as

they are in freshwater. For instance, Eldridge et al. [14] tested

several types of organic and inorganic flocculants (metals,

hydroxides and polyacrylamides) for the concentration of five

marine microalgae species. The best results were obtained

with the use ofmetals (Al and Fe) as coagulants, while cationic

polyacrylamides were less effective. Moreover, most floccu-

lants, when used inmarinewater, need to be applied in higher

doses than used in freshwater [4,15].

Regarding cationic polymeric flocculants, previous studies

have demonstrated the inhibition of flocculation mainly due

to the high ionic strength of saltwater [15,16]. According to

Bilanovic et al. [16], the configurations and dimensions of

polymers change according to the ionic strength. At high ionic

strengths, as in saltwater, cationic polymers shrink and fail to

establish bridges among the microalgae cells. Moreover,

polymers with higher charge densities tend to strongly adsorb

to the cell surface, hampering the bridging [17].

In this study, a clear difference was observed in the floc-

culant efficiency as a function of charge density. There was a

decrease in the cell removal capacity with increasing charge

density of the flocculants. According to Bolto&Gregory [7], the

higher the charge density, the harder it is to stretch the

polymer chain and, consequently, the more difficult it is to

establish cross connections among the cells.

Fig. 2 e Comparison of the flocculation efficiency of the

microalgae C. weissflogii with different concentrations of

the flocculant Flopam FO 4240 SH (0.2e4.0 mg m¡3). The

bars represent the mean value (with standard deviation) of

three replicates.

Another problem that can complicate the flocculation of

microalgae cells in salt and freshwater is the production of

soluble extracellular-polymeric-substances (EPS). In fact,

recent studies have demonstrated that the negative effect of

EPS on the flocculation process can be greater than that

caused by the increased ionic strength in saltwater [18,19]. EPS

are a mixture of polymers with high molecular weights.

However, polysaccharides and proteins are the major ele-

ments found in EPS. Polysaccharides present negatively

charged carboxyl groups that interact with cationic floccu-

lants [18,20]. According to Sheng et al. [21], the EPS produced

bymicroalgae can be either bound to the cells or soluble in the

watermedium.When bound to the cells, the EPS helps to form

a net-like structure that maintains cell aggregates, especially

when divalent cations (Ca2þ and Mg2þ) are employed as co-

agulants. On the other hand, soluble EPS in the culture me-

dium interacts with cationic flocculants, neutralizing them.

Our results showed that the flocculation efficiencies of all

employed flocculants were smaller for cultures in the sta-

tionary growth phase. This fact could be related to the pro-

duction of soluble EPS by the diatom C. weissflogii. Borges et al.

[9] showed that this species presented the highest production

rates of dissolved organic matter (6.67 mg C mg Chla�1 h�1)

during the stationary and senescent growth phases.

The presence of EPS in the culturemedia in the experiment

may also have contributed to the higher flocculant concen-

tration needed to achieve greater efficiency when comparing

with experiments 1 (1 ppm e 90% efficiency) and 2 (4 ppm e

90% efficiency) with the same cell density. Thus, to overcome

the EPS effect, it was necessary to increase the dosage of

flocculant in the second experiment (from 1 to 4 mg m�3) in

order to obtain the same efficiency as in the Log phase of

experiment 1. However, a similar dose (3e4 ppm) of cationic

polyelectrolyte flocculant was used by Uduman et al. [4] and

Garzon-Sanabria et al. [19] to same flocculation efficiency.

The cell size, surface area and density in the culture can

also influence the process of polymer bridging because the

increased surface area and decreased cell density will require

a greater concentration of flocculant to obtain the same effi-

ciency [13,22]. Experiment 3 showed that the highest

b i om a s s a n d b i o e n e r g y 6 8 ( 2 0 1 4 ) 1e6 5

efficiencies occurred under conditions of increased cell den-

sity. This difference most likely occurred due to the smaller

distances between the cells in the more concentrated cul-

tures, stimulating the formation of flocs that sedimented

faster. Moreover, at high cell densities, the cell wall material is

less electrically charged on its surface, and due to the smaller

distances among cells, the cell collision rate increases,

contributing to greater flocculation efficiency [16].

The abiotic data of the third experiment showed a pH

gradient among the treatments, with the highest value (10.0)

in the cultures with higher cell densities. These differences

were most likely generated by the higher CO2 consumption in

the denser cultures. The pH of an aqueous medium can affect

the degree of ionization, charge density and extension of the

polymer as well as the surface charge density of the micro-

algae and even the flocculation process as a whole [4]. A

higher pH makes flocculation more effective, especially with

cationic polyelectrolytes [17]. This is because at high pH, there

is a reduction in the electrostatic repulsion between the water

molecules, leading to a greater probability of forming bridges

between the polymers. Vandamme et al. [18] tested the effect

of dissolved magnesium and calcium as catalysts in the floc-

culation of Chlorella vulgaris at three different pH levels (10.5,

11.0 and 12.0) and observed that pH can affect the zeta po-

tential of the medium, causing flocculation to be more or less

efficient. Chen et al. [23] observed that pH values between 7

and 9 do not alter the zeta potential of themedium, but higher

or smaller pH values can inhibit the action of flocculants,

decreasing the flocculation efficiency.

5. Conclusions

According to the results of this study, the flocculant FO 4240

SH was the most efficient in concentrating the C. weissflogii

cells. Differences in the results for the Log and stationary

phase cultures were observed, most likely due to the negative

effect of soluble EPS. Furthermore, it was found that the

flocculant operates more efficiently at concentrations greater

than 1 mg m�3, while higher concentrations were required

probably due to the presence of higher concentrations of

dissolved organic matter.

Finally, it was found that the cell density is also of great

importance for the efficiency of flocculants, with improved

effects observed at densities greater than 35.5 � 104 cm�3.

Acknowledgments

The authors would like to thank the important contributions

of Reviewers and Editor to the final version of this text. We are

grateful to Mr. Jo~ao Brito (SNF e FLOEGER) who furnished the

flocculants used in this study. This study had financial sup-

port of Brazil's Ministry of Science and Technology - MCT,

through the Council for Scientific and Technological Devel-

opment - CNPq (Proc. no. 574737/2008-1) and Ministry of

Fisheries and Aquaculture eMPA (Proc. no. 0350.009678/2008-

1) and Studies and Project Funding – FINEP (Proc. no.

04.11.0234.00). F. Roselet was funded by a Ph.D. grant from

Coordenaç~ao de Aperfeiçoamento de Pessoal de Nível Superiore CAPES. P.C. Abreu is research fellow of the CNPq.

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