Aquacultural Engineering 4 (1985) 257-270

Harvesting Marine Microaigae Species by Chitosan Flocculation

J. M o r a l e s , J. d e la N o i i e * a n d G . P i c a r d ? Centre de recherche en nutrition, *D6partement de biologic, tD6partement de sciences et technologic des aliments, Universit6 Laval, Qu6bec, Canada G1K 7P4

ABSTRACT Marine microalgae are still an important larval feeding source. One o f the most promising harvesting techniques o f the algae produced appears to be chemical fiocculation. We report results obtained with chitosan fiocculation o f five marine species o f microalgae o f importance to mariculture

(Skeletonema costatum, Dunaliella tertiolecta, Thalassiosira nordenskoldii, Chlorella sp. and Thalassionema sp.). The algae were grown in the laboratory in 20-liter batch cultures under normal conditions in artificial seawater. Without pH control, a 100% fiocculation efficiency was reached at fairly high chitosan concentrations (above 40 mg liter-I). When the final pH was adjusted to around 7.8-8.0, a 100% flocculation efficiency was obtained with chitosan concentrations o f 40 mg liter -I or more. However, when pH was adjusted to around 7 or less, prior to chitosan addition for S. costatum and Chlorella sp., the concentration o f chitosan required to obtain a 95-100% flocculation efficiency was reduced to 20 mg liter-x for Chlorella and 2 mg liter-1 for S. costatum. The results are discussed in the light o f the currently accepted theories on flocculation.

INTRODUCTION The commercial culture o f marine organisms is an increasingly important industry. However, one o f the bottlenecks still remaining for its complete development is the limited number o f hatcheries and nurseries around the world. In many cases the success of rearing depends upon microalgae that are required for larval feeding. This is particularly true for bivalves (Goldman and Ryther, 1976; Wilson, 1978; Persoone 257 Aquacultural Engineering 0144-8609/85/$03.30- Elsevier Applied Science Publishers Ltd, England, 1985. Printed in Great Britain

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and Claus, 1980), crustaceans (Cook and Murphy, 1969; Mock and Murphy, 1971 ; Bardach et al., 1972; Shigeno, 1975; Hanson and Goodwin, 1977; New, 1982) and larvae of flatfishes (Howell, 1979; Scott and Middleton, 1979; Gatesoupe and Luquet, 1981). The intensive culture of marine phytoplankton is an appealing alternative for nursery operations, but requires a solution to the problem of harvesting the microalgae produced. During the last 20 years, a large number of harvesting methods have been developed (Mohn, 1980). Unfortunately, all these methods still present economical or technical drawbacks (e.g. unfeasibility of scaling up some methods, high energy cost, toxicity problems caused by the chemical products used as flocculants, etc.). However, among all harvesting methods, chemical flocculation is probably one of the most promising. Chemical flocculation techniques were used at the beginning by wastewater investigators to eliminate suspended solids as well as microbes or microalgae from the final effluent (Ives, 1959; Tenney and Stumm, 1965; Tenney et al., 1969; McGarry, 1970; McGarry and Tongkasame, 1971 ; Tilton et al., 1972). A large number of chemical products have been used as flocculants; Table 1 summarizes the most extensively tested. Among them, those with a higher effectiveness are alum and some cationic polyelectrolytes (Tenney and Stumm, 1965; Tenney et al., 1969; McGarry, 1970; McGarry and Tongkasame, 1971; Moraine et al., 1980; Viviers and Van Vuuren, 1981 ). Unfortunately, algal biomass harvested by alum contains a high concentration of aluminium and there are some indications that cationic polyelectrolytes are hazardous for human health (Dodd, 1979). To avoid such problems, chitosan, a natural product, has been used by Indian investigators (Nigam et al., 1980; Venkataraman et al., 1980; Becker and Venkataraman, 1982). Their results have been confirmed more recently by Lavoie and de la NoiSe (1983) in Canada. All the authors worked on freshwater microalgae, except for some preliminary results obtained by Lavoie and de la NoiSe (1983) with Phaeodactylum tricornutum. We have therefore undertaken systematic studies with marine species of importance for aquaculture. We report here the results obtained with five of them using a modification of the Canadian procedure (Lavoie and de la Notie, 1983). The importance of chitosan concentration and pH adjustment strategy for microalgal flocculation will be shown.

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259

TABLE 1 Flocculating Agents Successfullyused for Algae Recovery

Flocculant Concentration (rag liter -1) Reference

Alum Alum Lime FeCI3 Cationic polyelectrolytes Cationic polyamine (Dow C-31) Ca(OH)2 Chitosan Alum Chitosan

75-100 125-170 300-400 30 50-100 3 100 50 70 30

McGarry (1970) Van Vuuren and Van Vuuren (1965) Van Vuuren and Van Vuuren (1965) lves (1959) Tilton et al. (1972) Tenney et al. (1969) Nigam et al. (1980) Nigam et al. (1980) Lavoie et al. (1984) Lavoie et al. (1984)

MATERIAL AND METHODS

Algae
The species used were the following:
S k e l e t o n e m a c o s t a t u m (Thalassiosiraceae, Bacillariales) Dunaliella tertiolecta (Dunaliellaceae, Volvocales) Thalassiosira n o r d e n s k o l d i i (Thalassiosiraceae, Bacillariales) Chlorella sp. (Oocystaceae, Chlorococcales) Thalassionema sp. (Fragilaviaceae, Baccillariales).

Algal batch cultures were grown in 20-liter Pyrex carboys on a medium derived from that proposed by De Pauw et al. (1980), which is itself a modification of that of Walne (1956). Table 2 shows the composition and concentrations of the algal growth medium. Artificial seawater was prepared with 'Instant Ocean' to give a salinity of 31 o ~o. Algal cultures were maintained in a thermostatted room at 20C for 6-7 days. Under these conditions, pH varied without control between 8.7 and 9.9 for all cultures. Light intensity was 30000 lux

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TABLE 2 Algal Culture Medium (Modified after De Pauw et al., 1980) FeSO4.7 H20 MnC12.4 H20 NaH2PO4.2 H20 NaNO3 Na2SiO3 0.278 0-47 5.0 30-0 20.0 g liter -1 g liter -1 g liter -1 (= 1000 mg P liter -1) g liter -1 (= 5000mgN liter -1) g liter -~ (= 1.5 g Si liter -l)

Medium renewal rates: algal stock maintenance, 0.1-1-5% week-1 of stock solution; algal culture maintenance: 0.1-0.2% week-1 of stock solution.

with a photoperiod of 1 4 : 1 0 (L/D). Cell density was measured by spectrophotometry at 678 nm (Stein, 1973) in a 1-cm cuvette for optical density (OD) absorbance. Cell counts of 600-1000 cells were made under a microscope with a Malassez hematocytometer (BordeauxChimic, Bordeaux, France) before each flocculation.

Flocculation procedure
The complete flocculation procedure is shown in Fig. 1. Chitosan efficiency was evaluated and operating conditions, primarily flocculant concentration and pH sensitivity, were determined in l-liter flocculation tests ('jar tests').

Algal culture homogenization (1 min) A d d i t i o n of flocculating agent (chitosan)

~ mixing
pH adjustment (HCI 0.1 N or NaOH 0-1 N) stirring (1 min at lOOrpm and 4 rain at 40 rpm) flocculation

Fig. 1.

Flocculation procedure by chitosan (Modified after Lavoie and de la Nofie, 1983).

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261

The pH was adjusted b y dropwise addition of 0.1 N HC1 or NaOH. Following flocculation, the final OD and pH o f the supernatant were determined. The flocculation efficiency was established b y the difference from the original OD.
Chitosan

Chitosan, obtained by de-acetylation of a ~-N-acetyl-o-glucosamine, mainly extracted from crustacean exoskeleton (Muzzarelli, 1977; Nigam et al., 1980) was provided b y Kypro Co. (Seattle, USA). Chitosan flakes were dissolved in a 1% acetic acid solution to obtain a final chitosan solution o f 0.5% (w/w) at pH 3-5.

RESULTS A. Influence o f chitosan concentration on flocculation efficiency Several chitosan concentrations were tested to determine the best condition for a maximal flocculation efficiency. Algal culture features before and after flocculation are shown in Tables 3 and 4.

TABLE 3 Physico-chemical Features of Algal Cultures Before Flocculation

Species

AIgal eoncentration ( 106 cells, m1-1]

OD (678 nm)

pH

Chlorella sp. Skeletonema costatum Thalassiossira nordenskoldii Thalassionema sp. Dunaliella tertiolecta

80.4 3-0 5.7 3.0 2.2

0.8 0.215 0.34 0-21 0.305

9.50 9.85 9.70 9.70 9.25

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TABLE 4

Flocculation Efficiency for Different Algal Species as a Function of Chitosan Concentration Chitosan concentration (rag liter -I) 30 40 50 60 70 80 20 30 40 45 50 60 10 20 30 40 40 50 60 70 80 90 30 40 50 60 70 80 pH after addition of fiocculant Final OD (6 78 nm) Flocculation efficiency {%) Species

7.7 7-1
-

0-30 0-00 0-00

0.00 0.00 0-00

62.5 100 100

100 100 100

Chorella sp.

8.4 7.9 7.6 7.1 6.9 6.5 9-4 8.8 8-4 7.6 9-0 8.9 8.7 8.4 8.0 7.1 9.3 9.0 8.9 8.6 8.2 7.4

0.30 0.27 0.23 0-00 0.00 0.00 0.18 0.03 0.03 0-00 0.13 0-10 0.10 0-09 0.08 0-01 0.19 O.13 0-15 0.10 0.03 0.00

1-6 11.5 24.6 100 100 100 47.1 91.2 91-2 100 38.1 ) 52.4 52.4 57.4 61.9 95.2 11-6 } 39.5 30.0 53.5 86-0 100

Dunaliella tertiolecta

Thalassiosira nordenskoldii

Thalassionema sp.

Skeletonema costatum

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The chitosan concentration needed to obtain 100% flocculation efficiency was different for each algal culture (Table 4). Several factors related to the flocculation process could explain such differences: cell density, algal size, ageing of cultures, phase of growth, etc. Chitosan concentrations were always above 40rag liter -I and frequently reached 80 and 90 mg liter -a. These values are intermediate between those of Nigam et al. (1980), Venkataraman et al. (1980) and those of Lavoie and de la Notie (1983) obtained with fresh water microalgae. However, they are of the same range as those reported by Lavoie and de la NoiSe (1983) for the marine diatom P h a e o d a c t y l u m
tricornutum.

In these experiments, pH was not adjusted prior to flocculation, but after adding chitosan solution, it was readjusted to 8.0 by dropwise addition of 0.1 N HC1 or NaOH. The addition of acidic chitosan solution reduced pH values but only when pH dropped to near 7.1, before readjustment to 8.0, was a maximal flocculation efficiency obtained. Due to seawater buffer capacity, the quantities of chitosan needed to drop the pH of algal solutions below 7.5 are higher than those mentioned for freshwater media (Lavoie and de la Nofie, 1983). B. Influence of final pH values on flocculation efficiency The importance of pH on flocculation processes was demonstrated by several authors (Ives, 1959; Tenney and Stumm, 1965; Tenney et al., 1969; McGarry and Tongkasame, 1971; Tilton et al., 1972; Venkataraman et al., 1980; Lavoie and de la NoiJe, 1983; Lavoie et al., 1984), but this parameter has never been studied in salt-water media. Since pH values obtained with the same chitosan concentration were different for each algal culture, we chose the smallest quantity of chitosan needed to reach an OD reduction higher than 95% (Table 4). Immediately after adding the flocculant, pH was brought to different final values ranging from 7.4 to 8-2 and higher. We have compared flocculation efficiency for all these final pH values on the basis of optical density fluctuations of the supernatant. Figure 2 shows a strong influence of final pH on flocculation efficiency. For pH values between 7.8 and 8.0 we noticed a slight decrease in flocculation efficiency. For pH below 7-8, we observed a marked

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J. Morales, J. de la Nolle, G. Picard

I00
80c,-

?
/
A
7 ~

a/m

-~ 60-

= 0
~D

40

~ 20i,

716

81o
Finol

{4

_ ~_m

9'2

916

pH

Fig. 2. Effect of final pH of flocculation efficiency: D,Dunaliella tertiolecta (at 45 mg liter -i chitosan); o, Thalassiossira nordenskoldii (at 40 mg liter -1 chitosan); z~, Skeletonema costatum (at 80 mg liter -1 chitosan); o, Chlorella sp. (at 60 mg liter -I chitosan).

reduction of flocculation efficiency. This fact is related to chitosan characteristics as well as to the physicochemical reactions between the flocculant and the algal cells. Flocculation efficiency was maximal only for pH values higher than 8.0. We did not observe any reduction in flocculation efficiency in the pH range 8.0 to 9.0 for any o f the microalgae species studied. This chitosan activity range is larger than the 0.5 pH range reported by Lavoie and de la NoiSe (1983) and Lavoie et al. (1984) for S c e n e d e s m u s cultures in Canada. Venkataraman et al. (1980) mentioned a maximal sedimentation rate of S c e n e d e s m u s culture in India for pH values between 7-5 and 8.5. C. Influence of pH preadjustment on fiocculation efficiency In the experiments described in section A, maximal flocculation efficiency was observed at pH values below 7.5, which were obtained by adding the acidic solution of chitosan. In the present experiments, a pH preadjustment was done prior to chitosan addition in order to find

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the minimal flocculant concentration leading to a sedimentation greater than 95%. These experiments were carried out with Chlorella sp. and Skeletonema costatum as representatives o f two of the main groups of marine microalgae. Algae culture features are shown in Table 3. The preadjustment o f pH was done by dropwise addition of 0-1 N HCI. Three minutes after chitosan addition, pH was readjusted to a final value of 8.0 by adding 0.1N NaOH. This preadjustment o f pH allowed a 50% reduction o f flocculant concentration for Chlorella culture, while a maximal sedimentation o f 100% was obtained on Skeletonema culture with a concentration of 2 mg liter-~; this is a flocculant concentration 40 times lower than the original concentration (Table 5). Several important observations can be reported at this point: For maximal algal flocculation, the agitation time as well as the stirring speed must be increased by 50%. - Microalgal-chitosan flocs are smaller as compared to those observed in section A experiments.
-

TABLE

Flocculation Efficiency for Chlorella sp. and Skeletonema costatum with pH Adjustment Chitosan concentration (mg liter -1) pH preadjustment {drops of HCl I N) 7-9 (9) 8-1 (8) 8.4 (8) 8-3 (7) 7.0 (10) 5-7 (15) pH after Final DO Flocculation chitosan (678 nm) efficiency addition (%) Species

10 20 30 40 10 2

7-1 6.9 6.9 6.6 6.6 5.3

0.14 0-03 0-02 0.01 0.00 0-00

82.5 96.2 97-5 98.3 100 100a

Chlorella sp. Chlorella sp. Chlorella sp. Chlorella sp. Skeletonema eostatum Skeletonema costatum

a This includes both sedimented and floated flocs (see text).

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- When pH values are brought to as low as 5.3 for S k e l e t o n e m a culture, small bubbles appear on algal flocs lifting most of the algal suspension to the surface; this could be due to chemical reactions between the acidic solution and the silica frustule of the diatoms.

DISCUSSION Since the 1950s, flocculation and coagulation phenomena have been the subject of intensive research by different authors who have developed two broad theories to explain the mechanisms of colloid precipitation. The chemical theory, which is the older one, assumed that destabilization (and final precipitation) of colloids is due to chemical interactions such as complex formation and proton transfer (Smellie and La Mer, 1958). Later on, a physical theory emphasized the importance of physical factors such as zeta potential reduction or ion pair formation in the destabilization of colloids (Stumm and Morgan, 1962). At present, the influence of both physical and chemical factors on the coagulation and flocculation processes is recognized. Clark et al. (1977) described coagulation as a reduction of net electrical repulsive forces at particle surfaces by electrolytes in solution, while flocculation should be an aggregation by chemical bridging between particles. Microalgae as well as bacteria or protozoa can be considered hydrophilic biocolloids (Tenney and Stumm, 1965). The stability of hydrophilic colloids (lack of tendency to agglomerate) depends upon a marked affinity for water rather than upon the slight mutually repulsive charges (usually negative) that they possess (Clark et al., 1977). Tenney and Stumm (1965) pointed out that the surface charge density of bacteria is strongly pH-dependent. This criterion is also valid for microalgae (Ives, 1959). Cell walls of microalgae are mainly composed of gluco- and muco-polysaccharides (Jensen, 1983); these substances have low isoelectric points (Tenney and Stumm, 1965) and they are negatively charged due to ionized carboxylic groups (Stumm and Morgan, 1962). A mechanism explaining the flocculation process of biocolloids was proposed by Tenney and Stumm (1965). Its main features are the following: natural polymers such as complex polysaccharides and polyamino acids are excreted or exposed at the surface, predominantly during the declining growth phase and endogenous respiration

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(Avnimelech et al., 1982); these polymeric molecules are of sufficient length to form bridges between microbial particles. Chitosan, as well as cationic polyelectrolytes, being positively charged, should form complex bridges between the negatively charged substances of the algae surface (Tenney and Stumm, 1965; Avnimelech et al., 1982). Tenney and Stumm (1965) reported that in the interaction of polyelectrolyres with cells, electrostatic forces are necessary to 'bind' positively charged segments of the polyelectrolyte to the microbial surface. Our experiments emphasize the importance of diminishing pH values below 7.0 prior to readjustment to 8.0 for final precipitation. This pH reduction is likely to have two main consequences: 1, increased chitosan activity by viscosity reduction; 2, induced reduction of mean surface charge of microalgae and alteration of its stability. Once the bridging is made, a final pH adjustment to 8.0 increases firstly chitosan viscosity and finally causes microalgae-chitosan floc precipitation. We have observed the necessity to extend agitation when we worked at very low chitosan concentration (i.e. 2 mg liter -1) and pH preadjustment. The first observation is in line with what Tenney and Stumm (1965) mentioned, namely ' . . . with a given microorganism-polyelectrolyre suspension, the extent of polyelectrolyte adsorption apparently increases with prolonged a g i t a t i o n . . . '. More research is needed to establish the optimal agitation times for a given chitosan concentration and the optimal minimal pH value for a maximal chitosan-microalgal bridging with a minimal flocculant concentration. Finally, it appears that the general flocculation process described for freshwater microorganisms also holds for marine algal species. ACKNOWLEDGEMENTS This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada and the Fonds FCAC of the minist6re de l'Education du Qu6bec. We thank Mr Alain Lavoie for helpful discussions, Dr N. Eidhen for improving the manuscript and the Kypro Co. (Seattle, USA) for providing us with the chitosan. REFERENCES Avnimelech, Y., Troeger, B. W. & Reed, L. W. (1982). Mutual flocculation of algae and clay: Evidence and implications. Science, 216, 63-5.

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