1

Optimal conditions of different flocculation methods for harvesting

Scenedesmus sp. cultivated in an open-pond system

Lu Chen1, Cunwen Wang

1*, Weiguo Wang

1, Jiang Wei

2*

1 Key Laboratory for Green Chemical Process of Ministry of Education, Wuhan

Institute of Technology, Xiongchu road 693, Wuhan 430073, China

2Alfa Laval Nakskov A/S, Stavangervej 10 , DK-4900 Nakskov, Denmark

*Corresponding author:

Cunwen Wang: Key Laboratory for Green Chemical Process of Ministry of Education,

Wuhan Institute of Technology, Xiongchu road 693, Wuhan 430073, P.R.China

Email: wangcw118@hotmail.com Tel./fax: +8627 87195639

Jiang Wei: Alfa Laval Nakskov A/S, Stavangervej 10 , DK-4900 Nakskov, Denmark

Email: jiang.wei@alfalaval.com Tel./fax: +45 54971771

2

Abstract

The effects of flocculation efficiency on harvesting Scenedesmus sp. cultivated in an

open-pond system were investigated by changing culture medium pH value, flocculants

(FeCl3, Al2(SO4)3, Alum, Ca(OH)2, chitosan, polyacrylamide), different dosages and

sedimental times. Meanwhile, the relation between initial biomass concentration and the

flocculant dosage needed was also investigated. The results from this work indicated

that the flocculation efficiency achieved 97.4% after 10 minutes sedimentation when the

pH was adjusted to be 11.5, without adding flocculants. FeCl3 and chitosan showed a

good flocculation efficiency at 0.15 g/l and 0.08 g/l respectively without pH adjustment.

The flocculation efficiency increased from 49.74% to 90.63% when the final medium

pH was adjusted to 6 after adding 0.1 g/l Alum. An increment from 68.18% to 92.84%

was observed after adding 0.1 g/l Al2(SO4)3 followed by pH adjustment. Finally, the

most suitable flocculation method is discussed in this paper.

Keywords: flocculation; Scenedesmus sp.; open-pond cultivation; inorganic flocculants;

organic flocculants

1. Introduction

As the depletion of fossil fuels, governments and research institutions all over the world

are making a great effort to search for new fuels. Biodiesel is a non-toxic, biodegradable

and renewable fuel that makes no net carbon dioxide or sulfur contribution to the

atmosphere and emits less gaseous pollutants than conventional diesel fuels(Hu et al.,

2008; Hu et al., 2006; Kim et al., 2011). Now biodiesel production from vegetable oils

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is a proven technology and is widely available in the world. However, plantation oil

crops, waste vegetable oil and animal fat are only available in limited amounts (Ahmann

and Dorgan, 2007; Lee et al., 2009; Wu et al., 2012). Moreover, using common food

crops such as maize, sugarcane, soybean or oilseed rape for biodiesel will entail a

decrease in food production(Schlesinger et al., 2012). Microalgae, as a potential source,

have been attracting the worldwide attention as it has a high areal productivity, a

relatively high lipid oil and protein content compared with traditional crops (Chisti,

2007; Halim et al., 2011; Mata et al., 2010). It has been reported that the average yield

of biodiesel produced by microalgae is nearly 10-20 times higher than that from

oleaginous seeds and/or vegetable oils(Chisti, 2007; Tickell and Tickell, 2000).

Nonetheless, due to the small size (3-30 µm), low concentration (0.5-5 g/L) of

microalgae and the stable suspended state in the culture medium due to their negative

surface charge, the separation and recovery of microalgae from culture medium have

been seen as a critical step in the microalgae biomass production process, which

accounts for about 20%-30% of the total production cost (Gudin and Thepenier, 1986;

Wu et al., 2012). Thus, it’s necessary for developing an efficient and low cost

downstream process to harvest the microalgae cells from culture medium as well as to

preserve their viability and bioactivity prior to use in the appropriate fields(Harith et al.,

2009).

Until now, several methods have been applied to harvest microalgae(Chen et al.,

2011): centrifugation(Heasman et al., 2000; Price et al., 1978), foam

fractionation(Csordas and Wang, 2004; Lockwood et al., 1997)，filtration(Turker et al.,

2003)，flocculation(Avinmelech Yoram et al., 1982) and gravity sedimentation. Most

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existing commercial systems choose centrifugation, the traditional method, for

harvesting microalgae, but there exists a non-negligible problem that it consumes a

great deal of electric power. And high-speed centrifugation usually ruptures the cells

which leads to the contents inside cells flow into the medium(Divakaran and

Sivasankara Pillai, 2002). Some microalgae can be harvested using filtration, but

membranes will rapidly fouled by extracellular organic matter if filtrating the medium

directly(Babel and Takizawa, 2010). Therefore, considering the feasibility in terms of

economy and technology, flocculation can be an effective and convenient method to

harvest microalgae from large quantities of microalgae cultures rapidly(Wu et al., 2012).

Flocculation is the coalescence of separating suspended microalgal cells into large but

loose particles. Through the interaction between the flocculant and the surface charge of

microalgal cell, cells aggregate into large flocs and then settle out of the suspension

subsequently(Knuckey et al., 2006). Lots of chemicals have been investigated as the

flocculants for many types of microalgae.

As it’s reported, Scenedesmus sp. was considered as one of the most promising

microalgae for biodiesel because it has relatively high lipid productivity and it is

relatively easy to be cultivated(Jena et al., 2012). Until now, many researchers have

discussed the method for harvesting Scenedesmus sp.. Different methods were

investigated to reduce the membrane fouling for harvesting Scenedesmus sp. with

polyvinylidene fluoride (PVDF) microfiltration membrane(Chen et al., 2012). It shows

a potential of industrial micro-organisms harvesting by membrane. Consecutive

treatment with CaCl2 and FeCl3, and a bioflocculant were used to be the flocculants to

harvest Scenedesmus sp. with a high density(Kim et al., 2011). However, the production

process of bioflocculant is complex and the cost is relative expensive. The pH increase

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of culture medium could induce the flocculation of microalgae. The flocculation

efficiency of several freshwater microalgae (Chlorella vulgaris, Scenedesmus sp.,

Chlorococcum sp.) and marine microalgae (Nannochloropsis oculata, Phaeodactylum

tricomutum) have been discussed by increasing the pH value of culture medium(Halim

et al., 2011). The various flocculation potential of different microalgae depends on their

different properties, such as the cell wall compositions, the extent and type of excretions,

physiological conditions, age and other factors (Avinmelech Yoram et al., 1982).

Therefore, the optimal flocculation method for the microalgae should be chosen

according to their own situation.

In this study, the flocculation efficiency of different types of flocculants on harvesting

Scenedesmus sp. cultivated in an open-pond cultivation system was investigated. The

effects of pH, sedimental time and flocculant dosage and pH adjustment after adding

flocculants on flocculation efficiency were also discussed.

2. Materials and methods

2.1 Microalgae and culture condition

The microalgae used in this study were Scenedesmus sp. which was provided by Algae

Innovation Center of Denmark. It was cultivated using Bold’s Basal medium (BBM) in

an open-pond cultivation system. Cells were harvested at late logarithmic growth phase

and stored under darkness at 4°C for subsequent use in the flocculation experiments.

2.2 Flocculation experiments

2.2.1 Effect of pH

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The effect of pH value on flocculation efficiency was carried out by adjusting the pH of

1 L culture medium ranging from pH 7.5 to pH 12.5 using 5M sodium hydroxide and

1N hydrochloric acid. The medium was mixed rapidly (800rpm) until the required pH

value was achieved and then slowly (250rpm) for 1 minute using magnetic bar stirrer.

After sedimentation under gravity for different sedimentation times, an aliquot of

medium was withdrawn for measuring the optical density at the height of two-thirds

from the bottom. The optical density (OD) of the aliquot was measured by UV-

spectrophotometer (Hach Lange DR5000) at a wavelength of 665 nm to evaluate the

flocculation efficiency. The flocculation efficiency was calculated using the following

equation:

Flocculation efficiency (%) = (1-B/A)*100

Where A is the optical density of the initial culture medium at 665 nm and B is the

optical density of the sample at 665 nm.

2.2.2 Effect of different flocculants with different dosages

Six flocculants (chitosan, polyacrylamide (PAM), Alum, Al2(SO4)3, Ca(OH)2 and

FeCl3), which were purchased from Sigma (Denmark), were used for harvesting

Secendesmus sp. from culture medium. All of them were common chemicals that have

been proved to be efficient flocculants to many types of microalgae and widely used on

many flocculation processes (Bajza and Hitrec, 2004; Harith et al., 2009; Schlesinger et

al., 2012). Several dosages of these flocculants were added to 1L culture medium and

mixed rapidly (800rpm) for 1 minute and then slowly (250rpm) for 1 minute using

magnetic bar stirrer. Thereafter, an aliquot of medium was taken for measuring the

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flocculation efficiency at the height of two-thirds from the bottom after sedimentation

under gravity for different sedimentation times.

2.2.3 Effect of flocculation with pH adjustment

The effect of medium pH after adding flocculants on flocculation efficiency was carried

out by adjusting the pH using 1M sodium hydroxide and 1N hydrochloric acid. The

base or acid were added to the medium at high mixing rate (800rpm) provided by

agitation using magnetic bar stirrer. After the required pH value was reached, the

medium was agitated at 250 rpm for 1 minute to achieve homogeneity in pH in whole

medium solution. After 10 minutes sedimentation, an aliquot of medium was taken for

measuring the flocculation efficiency at the height of two-thirds from the bottom.

2.2.4 Effect of flocculant dosage with different algal concentrations

Four initial algal biomass concentrations (0.23 g/l, 0.41 g/l, 0.53 g/l and 0.66 g/l) were

investigated to test the effect of flocculant dosage with different algal concentrations.

The experimental method was the same as previously mentioned in 2.2.2. The

sedimentation time was 10 minutes.

2.3 Determination of cell growth

A calibration curve of known OD values and corresponding dry weights was calculated

by measuring the dry cell weight (DCW) of microalgal culture. The dry weight was

determined gravimetrically after centrifugation at 4000 rpm for 15 minutes and then

drying the algal cells at 60 ◦C in the oven until constant weight was reached. There was

a direct correlation between OD665 and dry weight expressed by a function:

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Dry weight (g/l) =0.7323*OD665 (R2=0.9983)

The relationship between DCW and OD665 was described by a power regression with a

R2 close to 1 within an OD range from 0.15-1.8. Based on this relation, all the OD

values were converted to biomass (g/L). The results presented in this paper are based on

the average of the three replicates.

3. Results and discussion

3.1 Autoflocculation by pH adjustment

Figure 1. Effect of pH adjustment on flocculation efficiency of Scenedesmus sp. at 0.54

g/l

Figure 1 shows the effect of pH ranging from 7.5 to 12.5 on flocculation efficiency for

harvesting Scenedesmus sp. at an algal biomass concentration of 0.54 g/l. The original

pH value of culture medium was 10.3. Around this pH, 50% algae cells were settled

down after 120 minutes. The efficiency increased to 97.4% after only 10 minutes

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sedimentation when the pH was adjusted up to 11.5. In addition, after 120 minutes

sedimentation, the flocculation efficiencies could keep higher than 96% when the pH

was adjusted to 11.5 or 12.5. Therefore, the results demonstrated that effective

flocculation for harvesting Scenedesmus sp. could be attained by pH increase. Various

groups have realized that microalgae could be flocculated by high pH value. They

proposed that the reason for autoflocculation could be due to the metal cations in the

medium such as calcium and magnesium ions that could form hydroxide precipitates

with a positive superficial charge as pH increased. These positively charged precipitates

would absorb the negatively charged algal cells, causing the compression of the

electrical double-layer and then the cells become destabilized and hence to flocculate

(Lavoie and de la Noüe, 1987; Schlesinger et al., 2012; Semerjian and Ayoub, 2003).

However, the algal cells in the medium at pH 12.5 were dead partially after 24 h and the

color turned to be yellow. Hence, the flocculation efficiency could be the highest at the

shortest time when the pH was adjusted to 11.5.

3.2 Effect of different types and dosages of flocculants on flocculation efficiency

As the flocculant dosage will influence both the extent and the rate of flocculation

reaction, it has been recognized as a critical parameter in all flocculation processes.

Therefore, preliminary experiments were undertaken to determine the optimum

flocculant dosage and sedimental time on the flocculation of algal cells. Six flocculants

were chosen to be discussed in this study. Among them FeCl3, Alum, Al2(SO4)3 and

Ca(OH)2 belong to inorganic flocculants, Chitosan is organic cationic polymer which

only dissolved in dilute acid and PAM is an polymer flocculant with high molecular

weight. The reason for adding cationic flocculants is the positive charge carried by them

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could neutralize the negative charged microalgal cells. All of them have been used as

efficient flocculants on many algae, such as Chlorella, Thalassiosira pseudonana.

Figure 2. Effect of different dosages of six flocculants, (a) FeCl3, (b) Al2(SO4)3, (c)

Alum, (d) Chitosan, (e) PAM, (f) Ca(OH)2, at different sedimental time on flocculation

efficiency. The biomass concentration of Scenedesmus sp. culture medium was 0.54 g/l.

Figure 2 shows the flocculation efficiencies of harvesting Scenedesmus sp. culture

medium by different types of flocculants. As Figure 2 shown, FeCl3, Al2(SO4)3, Alum

and Chitosan could lead to a high flocculation efficiency over 95% at a relatively short

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time while the flocculation efficiencies of Ca(OH)2 and PAM were lower especially

PAM. The flocculation efficiency of FeCl3 sharply increased from 53.31% to 97.32%

when the dosage increased from 0.1 g/l to 0.15 g/l after 2 minutes sedimentation. Figure

2e shows that it has no significant change of flocculation efficiency with different

dosages of PAM. The mechanism of flocculation by PAM was bridging. The

flocculation efficiency will be affected strongly by the solution properties of the

polymer. The PAM used in this study was a common one whose chain is not expanded

enough for bridging the cells. Modified PAM has better flocculation efficiency than the

common one because of the influence of chain end group(Qian et al., 2004). For

Ca(OH)2, the highest flocculation efficiency was 90% after 120 minutes sedimentation

with adding 0.4 g/l solution to the medium for flocculating the Scenedesmus sp. and the

efficiency did not further increase with the increment of flocculant dosage. Almost

similar results were observed for flocculation using Al2(SO4)3 and Alum. High

flocculation efficiency, 97.88% and 94.93% respectively, were obtained after 10

minutes sedimentation when the dosage of them was 0.3 g/l. However, the flocs

produced from that dosage of Al2(SO4)3 and Alum were not very dense and showed a

tendency to float and the high dosage of flocculants was also harmful to the algal cells.

The culture medium turned to be light white when the Al2(SO4)3 and Alum dosage

reached 0.3 g/l and turned to be orange when the FeCl3 dosage reached 0.2 g/l after

adding the flocculants. This phenomenon might be caused by the excess flocculants.

Part of flocculants reacted with the algal cells, the excess flocculants stayed in the

medium in ionic state. Ferric chloride solution was orange and the Al(OH)3 precipitate

was white. And after 24 hours, most of cells were dead and floated on the surface with

adding high dosages of Al2(SO4)3, Alum and FeCl3. Based on these results, the optimum

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flocculant dosage produces stability flocs and high flocculation efficiency at a shorter

time should be chosen to flocculate the algae according to the conditions of the original

algae culture medium. If the supernatant after flocculation is reused for cultivating the

algae, organic cationic polymer like chitosan will be a suitable choice because it has no

toxic effects and does not contaminate growth medium(Wu et al., 2012). Otherwise, if

the purpose of flocculation process focuses on harvesting the algae economically and

conveniently, the inorganic flocculants such as FeCl3, Al2(SO4)3 and Alum can be

chosen as they are cheaper and easier to get.

3.3 Effect of flocculation efficiency with flocculant and pH adjustment

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Figure 3. Changes in flocculation efficiency at different pH adjustment followed by

addition of different flocculants (a)0.08 g/l chitosan, (b)0,1 g/l Alum, (c)0.1 g/l

Al2(SO4)3 and (d)0.05 g/l PAM. The biomass concentration of Scenedesmus sp. culture

medium was 0.54 g/l.

The changes in flocculation efficiency at different pH adjustments with 1M sodium

hydroxide and 1N hydrochloric acid followed by addition of several flocculants are

given in Figure 3. The importance of pH on flocculation process has been reported by

many researchers. As pH affects the zeta potential of charged particles, it may interfere

with flocculation after adding flocculant. A slight change in flocculation efficiency was

shown in Figure 3a for chitosan at different pH values between 5 and 10. Due to acidic

characteristic of chitosan solution, the pH of the culture medium reduced from10 to7

after the addition the flocculant. The highest flocculation efficiency over 95% was

observed at pH 9 with 0.08 g/l chitosan solution. Chitosan’s molecular structure can be

influenced by pH. The positive charge gradually disappeared and chitosan tended to

form coli structure and precipitate when the pH was alkaline (Rong Huei and Hwa,

1996). The algal cells had the highest negative charge at the pH (neutralization point),

thus the flocculation efficiency was enhanced when the pH increased to that point.

Because of chitosan and the algal cells interact with each other through the electrostatic

interaction. Bridges were formed more than once as the polymer chain had sufficient

length to bind the cells (Harith et al., 2009). As it shown in Figure 3b and 3c, the

flocculation efficiency was higher than 90% with addition of 0.1 g/l Alum and

Al2(SO4)3 followed by adjusting the pH to 6 and 5 respectively. Aluminum salts

released hydrogen ions and consequently lower the medium pH value after adding into

the culture medium. The existing form of Al3+

was affected by pH value. Al(OH)3 was

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the predominant aluminum species around pH 5 and 6. Around this pH, the initially

formed colloidal precipitate was colloidally stable, and it was positively charged. The

flocs stability decreased when the pH increased further because of the soluble anionic

form 4Me(OH) becomes dominant in the solution(Bajza and Hitrec, 2004). It shows

that the flocculation efficiency has no substantially changes from pH 7 to 11 after the

addition of 0.05 g/l PAM in Figure 3d. A sharp increment was shown when the pH

value increased to 12. However, this increment at the high pH was probably caused by

the autoflocculation, which occurs at pH 11.5 or higher.

3. 4 Effect of flocculant dosages with different algal biomass concentrations

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Figure 4. Relation between initial algal biomass concentration and the flocculant dosage

required (line) to achieve high flocculation efficiency (column) (a) FeCl3, (b) Al2(SO4)3,

(c) Alum, (d) Chitosan

Different initial algal biomass concentrations might influence the efficiency during the

flocculation process. Therefore, the relation of algal biomass concentration and

flocculant dosage was investigated and the results are shown in Figure 4. For each

initial algal biomass concentration, different flocculant dosages were compared to get

the optimal one which could result in the highest flocculation efficiency for harvesting

algal cells. A linear relation between the dosage needed and the initial algal biomass

concentration was shown in Figure 4. The dosage needed increased with the increment

of the initial algal biomass concentration. This phenomenon could be explained by the

mechanism of flocculation. The amount of suspended algal cells increased with the

increase of the biomass concentration, thus higher flocculant dosages were needed to

interact with the surface charges of algal cells. As figures shown, when the initial

biomass concentration was 0.66 g/l, the optimal dosage of these four flocculants (FeCl3,

Al2(SO4)3, Alum and Chitosan) was 0.2 g/l, 0.4 g/l , 0.4 g/l and 0.1 g/l respectively.

Compared with Al2(SO4)3 and Alum, the consumption of FeCl3 and chitosan was

smaller. The higher the flocculant dosage is, the higher the residual ions concentration

may be. The residual ions will contaminate the medium and be harmful to the cell

vitality. Therefore, it is better to choose the suitable flocculants according to the

biomass concentration. Furthermore, reducing the amount of flocculant will lower the

cost of flocculate process.

4. Conclusions

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Six flocculants and their suitable conditions were investigated for harvesting the

Scenedesmus sp. cultivated in an open-pond cultivation system. The flocculant needed

to obtain high flocculation efficiency depends on the conditions of algae and further

downstream process. The pH adjustment and nontoxic flocculant like chitosan can be

chosen when the supernatant need to be reused after flocculation. Inorganic flocculants

will be a good choice if there is no strict demand for the rest supernatant or a system

coupled with filtration. A liner relation between the dosage needed and the initial algal

biomass concentration was observed for different flocculants.

Acknowledgements

This work was financed by National Natural Science Foundation of China (Grant

No.20976140). The authors wish to acknowledge Alfa Laval Nakskov A/S for the

support of the work and would like to thank Jørgen Enggaard Boelsmand at Algae

Innovation Center of Denmark for providing algae suspensions and helpful discussions.

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