Microalgae biomass harvesting by bioflocculation-interpretation by classical DLVO theory

Biochemical Engineering Journal 101 (2015) 160–167

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Microalgae biomass harvesting by bioflocculation-interpretation by classical DLVO theory Theoneste Ndikubwimana a , Xianhai Zeng b,c,∗∗ , Ning He a,c , Zongyuan Xiao a , Youping Xie d , Jo-Shu Chang e,f , Lu Lin b , Yinghua Lu a,c,∗ a

Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China College of Energy, Xiamen University, Xiamen 361005, China c The Key Laboratory for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen 361005, China d College of Biological Science and Engineering, Fuzhou University, Fuzhou 350108, China e Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan f University Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan 701, Taiwan b

a r t i c l e

i n f o

Article history: Received 24 February 2015 Received in revised form 8 April 2015 Accepted 10 May 2015 Available online 14 May 2015 Keywords: Bioflocculation Microalgae Dewatering Bioflocculant DLVO theory

a b s t r a c t Poly-␥-glutamic acid (␥-PGA) broth was found to be a good bioflocculant of microalgae. However, the mechanism governing this bioflocculation process is not fully understood. In this study, Zeta potential measurement, microscopy examination and classical DLVO theory (named after Derjaguin, Landau, Verwey and Overbeek) analysis were used to explore the flocculability of microalgae induced by bacterial ␥-PGA broth bioflocculant. Microalgae flocculation could be significantly improved by modifying ionic strength of the microalgae suspension, lowering pH value and bioflocculant addition due to the stronger attraction interactions between microalgal cells. In the present study, both the pH reduction with the bioflocculant induced better the flocculation process compared to the modification of ionic strength of the microalgae suspension in the presence of the bioflocculant. The DLVO theory indicated that when the bioflocculant was introduced, the total interaction energy decreased sharply, resulting in higher flocculation efficiency (>96 %) at a separation distance of 5 nm and shorter settling time (from 2 h to 10 min) compared with that obtained only by reducing the initial pH. The microalgae interaction energy was found to be dependent on the Zeta potential. This study provided a detailed interpretation of conceivable mechanism of microalgae bioflocculation by ␥-PGA broth. © 2015 Elsevier B.V. All rights reserved.

1. Introduction In respect to compensate the increasing global demand for food, feed, biofuels, and chemicals production, microalgae has been widely regarded as one of the most promising raw materials [1–3]. However, due to the dilute nature of microalgal cultures, the microalgae dewatering process cost even more than 30 % of the entire production processes cost [4–7] and this is the bottleneck to the microalgae industry. Dewatering costs could be greatly reduced with bioflocculation compared to other conventional dewatering

∗ Corresponding author at: Department of Chemical Engineering and Biochemical Engineering, College of Chemistry and Chemical Engineering; The Key Laboratory for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen 361005, China. Tel.: +86 592 2186038; fax: +86 592 2186038. ∗∗ Corresponding author at: College of Energy, Xiamen University, Xiamen 361005, China. Tel.: +86 592 2880701; fax: +86 592 2880701. E-mail addresses: [email protected] (X. Zeng), [email protected] (Y. Lu). http://dx.doi.org/10.1016/j.bej.2015.05.010 1369-703X/© 2015 Elsevier B.V. All rights reserved.

technologies available, because much less capital and maintenance costs are incurred. Moreover, bioflocculation of microalgae is of high efficiency [8–12] and little or no energy consumption is necessary during the process compared to centrifugation method widely used in industry [13]. Bioflocculation refers to the naturally induced flocculation due to the secreted biopolymers by the microbial cells [13]. The stability of microalgal cell suspensions is ascribable to the surface charge of the cells which originates predominantly from the presence of carboxylic ( COOH), amine ( NH2 ) and phosphoryl ( POH) groups on the cell surface. The carboxylic and phosphoryl groups dissociate and are negatively charged above pH 4–5, whereas the amine groups are uncharged at this pH. This results in a net negative surface charge above pH 4–5 [2]. Generally, bioflocculation of microalgae suspensions can be attributed to three main mechanisms as depicted in Fig. 1, which can act alone or in combination. The positively charged polymers can bind partly or completely to microalgal cells. If the polymers bind partly, the unoccupied part of the polymers can bind to other

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Fig. 1. Schematic diagram of possible microalgae bioflocculation mechanisms.

microalgal cells, thereby bridging them and resulting in a network of polymers and microalgal cells [11,14]. If the polymers bind onto the microalgal cells completely, the charge of the microalgal cells surface is locally reversed, resulting in patches of opposite charge on the microalgal cells surface, and consequently the microalgal cells connect with each other through patches of opposite charge, causing them to flocculate [2,14]. Even though the microalgae dewatering by bioflocculation is considered efficient and environmental friendly, its mechanism is not yet fully understood. Very few reports are published about the mechanism of microalgae dewatering by bioflocculation. The DLVO theory [15,16], originally developed for macromolecules, particles and colloids, has been applied as both qualitative and quantitative models to explain microbial adhesion and aggregation [17]. Recently, it has been found a useful means to explore the cell flocculation/aggregation [18]. The objective of the current study is to unveil the mechanism behind the bioflocculation of Desmodesmus sp. F51 by bacterial ␥-PGA broth bioflocculant. The ␥-PGA broth bioflocculant was used over the commercial ␥-PGA bioflocculant in the view of decreasing the total process cost by removing the cost needed for further purification of the produced bioflocculant. The microalgae Zeta potential was evaluated under different flocculation conditions, and the total interaction energy between algal cells was described by the classical DLVO theory. Also the microscopic analysis was conducted. The better knowledge of interactions involved in microalgae bioflocculation would enable this dewatering technique to be more efficiently and effectively developed.

2.2. Determination of microalgae biomass concentration The microalgae biomass concentration analysis was conducted by measuring the OD685 nm using the UV Spectrophotometer (UV1800, SHIMADZU, Kyoto, Japan) and the dry cell weight (DCW) was obtained by weighing the microalgae cells after 2 times washing with deionized water and subsequent drying in an oven at 80 ◦ C overnight. The biomass concentration was then estimated by a calibration curve (Eq. (1)) relating the OD685 nm values to the dry cell weight [20]: y = 0.3x ± 0.02 where y is the DCW (g/L) and x is the OD685 nm . 2.3. Analysis of flocculating activity of the bioflocculant Kaolin clay suspension was used for assay of flocculating activity of ␥-PGA broth bioflocculant. Briefly, 1 mL of diluted ␥-PGA broth bioflocculant sample and 2.5 mL of CaCl2 solution (10 g/L) were mixed with 40 mL of kaolin clay suspension (5 g/L), gently shaken and left to stand still for 5 min at room temperature. The control was prepared as described above without the ␥-PGA broth bioflocculant. The decrease in turbidity of the upper phase was recorded by measuring the OD550 nm using the UV Spectrophotometer (UV1800, SHIMADZU, Kyoto, Japan), and the flocculating activity was expressed as flocculating rate (FR) calculated by Eq. (2) [21]: FR(U/mL) =

2. Experimental 2.1. Algae culture and production of the bioflocculant The strain Desmodesmus sp. F51 used in this study was isolated from southern Taiwan. Both the pre-culture and culture were conducted in Modified Bold 3N medium as described by Berges and Franklin (in mM) [19]: NaNO3 , 4.4; K2 HPO4 , 0.22; MgSO4 ·7H2 O, 0.3; CaCl2 ·2H2 O, 0.17; KH2 PO4 , 0.43; NaCl, 0.43. The culture of microalgae was operated according to the procedures described by Xie et al. [20]. The bioflocculant was produced by Bacillus licheniformis CGMCC 2876 isolated and identified by the Department of Chemical and Biochemical Engineering of Xiamen University, China and the cultivation process was operated by following the procedures as previously described by Xiong et al. [21]. During this study, the broth of B. licheniformis CGMCC 2876 was used as the bioflocculant without any further purification process.

(1)

C −S × 100 × F C

(2)

where C and S are the optical density values at 550 nm of the control and the sample, respectively, and F is the dilution factor of the sample. Each sample was analyzed in duplicate. 2.4. Determination of the bioflocculant concentration In order to collect the supernatant of B. licheniformis CGMCC 2876 cultures, the fermentation broth was centrifuged at 6000 rpm for 10 min, and the bioflocculant was purified by using the procedures that were modified from those in Bar-Or and Shilo [22] and Salehizadeh et al. [23]. In brief, ethanol (95 %) was added into the supernatant gradually with gentle shaking and the volume ratio of ethanol to the supernatant was 3:1. Then the whole mixture was rested overnight at 4 ◦ C. Subsequently, the resulting precipitates were separated by centrifugation at 8000 rpm for 10 min, washed twice with deionized water, and then freeze. The determined ␥-PGA bioflocculant concentration was 16.0 g/L.

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2.5. Microalgae flocculation process Flocculation experiments were run with small volumes of culture medium (50 mL) distributed in 100 mL cylindrical beakers. The pH value of each sample was gradually adjusted using either 1 M HNO3 or 1 M NaOH if necessary, thenceforth the bioflocculant was added, followed by rapid mixing at 200 rpm for 1 min, then slow mixing at 100 rpm for 20 min or 2 min and the agitation was stopped allowing the microalgal suspension to settle at room temperature. The sample of the supernatant was pipetted from half the height of the clarified layer and was used to measure the optical density at the wavelength of 685 nm (OD685 nm ). The flocculation efficiency (FE) of each test was worked out according to Eq. (3): FE(%) =

A0 − A1 × 100 A0

(3)

where A0 and A1 are the OD685 nm values of the microalgal suspension before and after flocculation, respectively. 2.6. Effect of ionic strength on flocculation behavior of Desmodesmus sp. F51 The repulsion behavior between cells can be greatly affected by changing the ionic strength of the suspension medium [18]. It have been reported that divalent cations are more effective in modifying the ionic strength compared to monovalent and trivalent cations [24]. Salts of CaCl2 , MgCl2 and ZnCl2 were used as sources of divalent cations at different concentrations.

Fig. 2. Schematic diagram of interaction between two identical spherical particles.

Fig. 2 presents the diagram of interactions between two identical spherical particles. The van der Waals attractive force is the macroscopic-scale force of attraction between condensed-phase molecules or surfaces while the electrostatic forces arise due to the double-layer interactions between two particles [27]. The total energy of two spherical interacting particles (such as microalgal cells) is therefore obtained by the summation of interaction energy resulting from van der Waal forces (UvdW ) and the overlap of electrical double layers associated with the charged surfaces (electrostatic repulsion, UER ) respectively [28–30]: UTot (D) = UvdW (D) + UER (D)

(4)

with UvdW (D) = −

AR 12D

(5)

(For the interacting sphere particles of the same radius, R)and 2.7. Zeta potential measurement and cell size determination The Zeta potential is an important parameter which characterizes the physicochemical properties of the algal cells envelope and plays an important role in aggregation and disaggregation processes [25]. The Zeta potential can easily be estimated from the mobility of the charged particles in an electric field; therefore, it is a useful indicator of the degree of repulsion between charged particles in a suspension. If the Zeta potential is relatively high (>20 mV, positive or negative), electrical repulsion between interacting particles is strong and the suspension is highly stable. Once the Zeta potential is close to zero, interacting particles can approach each other to the point where they will be attracted by van der Waals forces and consequently aggregate into heavy flocs allowing flocculation to occur [2]. Both the Zeta potential and cell size of the microalgae cells were evaluated using the Zetasizer Nano-series (Nano-ZS &MPT-2, Malvern Instruments Ltd., UK). All measurements were carried out in triplicates and the average Zeta potential value was recorded. The average diameter of microalgae cells was found to be 6.05 ␮m. 2.8. Morphological cell analysis Microscopic pictures were taken from microalgae cells employing Olympus microscope, 10/10 (OLYMPUS CX 21, Olympus, Japan) connected to the computer with the Arctam Measure 2.0 software to take pictures. 2.9. The DLVO model The DLVO theory [15,16], was firstly developed to relate the stability of colloidal suspensions to the total potential energy between two particles. The net interaction energy between particles is interpreted as a balance of attractive van der Waals force and an electrostatic force which is usually repulsive as a result of overlapping electrical double layers surrounding charged particles [26].

UER (D) = 2R

2 S In[1+exp(−D)]

(6)

(When the interaction between two sphere particles takes place at constant surface zeta potential)where D is the separation distance, A: Hamaker constant related to the properties of the interacting particles, R: cell radius, : the dielectric constant of the solution, S and  stand for the stem potential and the reciprocal of the Debye length respectively, and are related to the electric double layer interaction UER . The value of A for Desmodesmus sp. F51 is 4 × 10−21 J [18]. The average cell radius (R) determined for Desmodesmus sp. F51 was ∼3 ␮m. In order to ease the calculations of energy barrier, all cells were assumed to present a spherical structure. For aqueous solutions,  is equal to 80 × 8.854 × 10−12 C2 J−1 m−1 [18], S can be replaced by the measured Zeta potential and  (m−1 ) can be obtained by the following equation [31]: k = 3.28 × 109 × l0.5

(7)

where l is the ionic strength (or the concentration of the flocculant) in terms of molarity. Marshall et al. [32], while studying the sorption of marine bacteria to surfaces, suggested that the effect of electrolyte concentrations on the initial bacterial adhesion to surfaces could be explained using the DLVO theory. So far the DLVO theory has been applied to explain microbial aggregation and attachment in various biological systems [18,28,29,33,34]. Most microalgae cells possess net negative surface charge which allows them to form stable algal suspensions. As the stability of algal suspension seemingly depends on the interacting forces between algal cells, also algae can be considered as hydrophilic biocolloids [35], therefore the flocculation of microalgae could potentially be explained by the DLVO theory [18]. Generally, the van der Waals force is responsible for attraction; therefore the corresponding interaction energy is unremarkably negative while the interaction energy resulting from the electrostatic repulsion force is positive in classical DLVO model. The van

T. Ndikubwimana et al. / Biochemical Engineering Journal 101 (2015) 160–167 Table 1 Influence of pH on microalgae flocculation efficiency and Zeta potential (microalgae concentration: 0.5 g/L). pH

Flocculation efficiency(%)

Initial 3 4 9 10

13.50 78.50 54 27 25

± ± ± ± ±

0.71 0.71 1.41 1.41 1.41

Zeta potential (mV) −20.50 +7.12 −15.25 −17.90 −18.95

± ± ± ± ±

0.28 0.02 0.64 0.85 0.21

der Waals interaction energy decreases as the inverse power of the separation distance between cells as presented in Eq. (5), and the electrostatic repulsive interaction energy is an approximately exponential function of the separation distance between cells with a range of the order of the thickness of the double layer (−1 ) as depicted in Eq. (6). Consequently, the attraction resulting from van der Waals forces prevails at smal intercellular distance and electrostatic repulsion dominates at intermediate separation distance between cells. Conceivably, at larger energy barrier, the cell suspension is believed to be more stable, thus the flocculation if prevented [18,28]. According to the classical DLVO model, only the electrostatic double layer force can significantly be modified, and repulsion behavior between cells can be greatly affected by changing the ionic strength of the suspension medium or by modifying the surface charge of the cells by pH adjustment or addition of positively charged flocculants [18]. 3. Results and discussion 3.1. Influence of the pH on the flocculation behavior of Desmodesmus sp. F51 The pH adjustment can modify considerably the surface charge of the cells thus affecting the repulsion behavior between cells [18]. With the objective to evaluate the influence of the pH on flocculation mechanism, the effect of pH on Zeta potential has to be assessed. Microalgal cells of the freshwater Desmodesmus sp. F51 were first allowed to autoflocculate with and without the initial pH alteration. The pH values of microalgal culture (0.5 g/L) were adjusted to pH 3, 4, 9, and 10 using either 1 M HNO3 or 1 M NaOH. The flocculation efficiency and Zeta potential were measured after 2 h of the settling time, and the results are presented in Table 1. As seen in Table 1, the Zeta potential remained negative for all pH values except for pH 3, where the flocculation efficiency was the highest compared to other pH values evaluated. At physiological pH values (pH 7–9), most of algal cells are negatively charged in growth media and as a result, cells repel each other and thus suspend stably in the medium. The negative charge is due to the dissociation of functional groups at the algal cell wall. The alteration of the pH influences the degree of surface dissociation, therefore affecting the cell surface charge. As mentioned above, there are three main key functional groups on the microalgal cell surface [2,18,36], these include: carboxyl ( COOH), phosphoryl ( POH), and amino ( NH2 ) groups, all may be influenced by pH as in the following reactions: NH2 + H+ ↔ NH3 + COOH ↔ COO− + H+ POH ↔ PO− + H+ At the acidic pH, the algae cell surface charge is dominated by a positively charged amine group (NH3 + ), whilst the carboxyl and phosphoryl groups remain protonated. At the neutral pH, phospho-

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ryl and carboxyl groups are both deprotonated, as the amino group is protonated. The COO− , PO− , and NH3 + groups are introduced and result in a possibility of zero surface charge, which is called the isoelectric point. At the alkaline pH, the phosphoryl and carboxyl groups become deprotonated and add the net negative charge on the cell surface [18]. The Zeta potential of a microalgal cell is generally electronegative for pH values between 4 and 10, ranging from −10 to −35 mV [37]. The carboxylate ions would accept protons when the pH is decreased to acidic, then the surface charge of the cells is reduced resulting in the reduction of the repulsion force between cells [1], once the cells are not repelling each other, they float toward each other and join together into groups by van der Waal’s forces and when enough cells have merged together, they constitute heavy flocs which can settle out of the growth medium. As presented in Table 1, the flocculation efficiency is clearly dependent on the cell surface charge (Zeta potential) which is influenced by pH value. At initial pH (∼7.2), after 2 h of autoflocculation, the Zeta potential of microalgal cells was −20.50 ± 0.28 mV with the flocculation efficiency of 13.50 ± 0.71%. When the pH was adjusted to 3, the Zeta potential was +7.12 ± 0.02 mV resulting in flocculation efficiency of 78.50 ± 0.71 %. When the pH of the microalgal cells was adjusted to 3, the algae cell surface charge was dominated by positively charged amine groups (NH3 + ) and these will tend to neutralize with carboxyl and phosphoryl groups thus reducing the repulsion forces between cells. This charge reduction process will bring algal cells closer to one another forming flocs which will settle out, resulting in high flocculation efficiency. The same results were reported elsewhere. Liu et al. [1] reported a flocculation method induced by decreasing pH value of growth medium at pH 4 resulting in the flocculation efficiencies as high as 90 % for Chlorococcum nivale, Chlorococcum ellipsoideum and Scenedesmus sp. The authors proposed that the flocculation mechanism could be due to the fact that carboxylate ions of organic matters binding on microalgal cells accepted protons when pH decreased and the negative charges were neutralized, hence resulting in disruption of the dispersing stability of microalgal cell suspensions and subsequent flocculation of microalgal cells. Moreover, the authors found out that the Zeta potentials and flocculation efficiencies were both pH dependent where by decreasing pH from 6.5 to 4.0, Zeta potentials sharply increased to approximately 0 mV and therefore, the corresponding flocculation efficiencies greatly increased to the maximum. However, Cui et al. [18] reported the highest flocculation efficiency of Scenedesmus dimorphus achieved at moderate pH (pH 7.5) and low ionic concentration (1 ␮M Al3+ ) where cell surface charges were fully neutralized with zero Zeta potential. Aluminum salts (Al3+ ) are believed to be strong and effective chemical flocculants [38]. Therefore, Cui et al. [18] were able to achieve the highest flocculation efficiency of S. dimorphus at moderate pH with strong chemical flocculant (Al3+ ). 3.2. Influence of the bioflocculant on the flocculation behavior of Desmodesmus sp. F51 Flocculants are well known as effective candidates to induce flocculation by neutralizing the cells surface charge which consequently reduces the repulsion behavior between cells [18]. The influence of bioflocculant was conducted as described in Section 3.1 with the addition of the bioflocculant (4 × 10−6 mM), the flocculation efficiency and Zeta potential were measured after 10 min of the settling time, and the results are depicted in Table 2. The addition of the bioflocculant reduced the negative charge of Desmodesmus sp. F51 (Zeta potential of −20.15 ± 0.35 mV at initial pH) to almost zero surface charge (Zeta potential = +0.41 ± 0.15 mV at pH of 3), resulting in a flocculation efficiency as high as 96.0 ± 0.3 %. These results suggest that the addition of the bioflocculant highly decreased the cell surface charge compared to the adjustment of the pH only.

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Flocculation efficiency (%)

Initial 3 4 9

44.8 96.0 46.1 45.0

Zeta potential (mV) −20.15 +0.41 −17.80 −19.35

0.14 0.28 0.28 0.14

± ± ± ±

0.35 0.15 0.28 0.78

Another reason may be drawn from the fact that functional groups existing in the bioflocculant facilitated the chemical adsorption on the microalgae surface, thus forming bridges or patches between microalgae cells and the bioflocculant resulting in larger flocs which can easily settle out from the culture medium. The results presented in Tables 1 and 2 elucidate that the flocculation increased with the change of pH where cells had relatively low negative surface charge but the highest flocculation efficiency was achieved with low bioflocculant concentration (4 × 10−6 mM) where cell surface charges were almost completely neutralized (almost zero Zeta potential). Similar results were reported by Cui et al. [18], where the authors discovered that the flocculation was dependent on the cell surface charge (Zeta potential) which was influenced by both pH and the flocculant concentrations. 3.3. Effect of ionic strength on the flocculation behavior of Desmodesmus sp. F51 The cationic electrolytes stimulate flocculation by neutralization and stabilization of negative charges of suspended particles [24]. It can be seen from Fig. 3 that all the cationic electrolytes evaluated can enhance flocculating activity. However, flocculation efficiency above 88 % could be achieved with 2.0 mM of Zn2+ compared to 15 % and 64 % achieved with the same ionic strength of Ca2+ and Mg2+ respectively after 2 h of flocculation time. The flocculation efficiency reached 89.95 ± 0.07 % at concentration of 2.0 mM of Zn2+ in the presence of ␥-PGA bioflocculant after 1 h of flocculation time. The divalent cations compress the electric double layer of microalgae cells, thus reduce the electrostatic forces interacting 35 30

-15

25 20

-10

15 10

-5

5 0 0.0

0.5

1.0

1.5

2.0

40

80 60

-10 40 -5

20 1.5

Concentration of ZnCl 2 (mmol/L)

30 20

-5

10 0 0.0

2.0

0

Flocculation Efficiency (%)

Zeta Potential (mV)

100

1.0

50

0.5

1.0

1.5

2.0

0

Concentration of MgCl2 (mmol/L)

c

0.5

60

-10

0

-15

0 0.0

b

-15

Concentration of CaCl2 (mmol/L) -20

70

-20

Zeta Potential (mV)

a

Flocculation Efficiency (%)

Zeta Potential (mV)

-20

100

-20

Zeta Potential (mV)

± ± ± ±

-15

80

d

60

-10 40 -5 0 0.0

20 0.5

1.0

1.5

Concentration of ZnCl 2 (mmol/L)

Fig. 3. Effect of different cations on flocculation behavior of Desmodesmus sp. F51.

2.0

0

Flocculation Efficiency (%)

pH

between cells leading to destabilized state of cells associated with improved flocculation. However, looking at the Zeta potential values as presented in Fig. 3, the compressing ability of Zn2+ (Fig. 3a) on the electric double layer is more effective compared to that of Ca2+ and Mg2+ (Fig. 3a and b, respectively). In the presence of ␥-PGA bioflocculant only, the flocculation efficiency reached 44.8 ± 0.14% (Table 2); however, in the presence of both the ␥-PGA bioflocculant and Zn2+ , the flocculation efficiency reached 89.95 ± 0.07 %. Also Cui et al. [18] reported that changing the ionic strength of S. dimorphus and Nannochloropsis oculata improved their flocculability. Although the change of ionic strength can favor the flocculation process, Luo et al. [39], Aljuboori et al. [40] and Yim et al. [41] did not observe any improvement of flocculation process by the addition of any cation evaluated including Ca2+ . The microscopic pictures of freshwater Desmodesmus sp. F51 cells exhibited in Fig. 4, where (a) presents the state of cells after cultivation; (b) shows the state of cells in the presence of Zn2+ with the bioflocculant after 1 h, and (c) shows the state of cells after the bioflocculation at pH 3. Comparing (a)–(c) in Fig. 4, it is clear that more single and double cells are abundant in (a), whilst in (b) and (c) more cells connected to each other are apparent. This is due to the presence of Zn2+ in case of (b) compressing the cells double layer thus making cells close to each other hence forming heavy flocs, and in case of (c), due to charge neutralization by pH and the presence of the bioflocculant, more heavy flocs are formed. One can see that there is no visual difference between (b) and (c), meaning that the cations with the bioflocculant would have almost the same flocculating ability with the pH coupled with the bioflocculant. However, by the Zeta potential analysis, it is clear that the pH adjustment coupled with the bioflocculant reduced the microalgae cells Zeta potential to nearly zero (Table 2) which explains the highest flocculation efficiency achieved in 10 min of flocculation time, while in the presence of Zn2+ , the Zeta potential could be reduced to −8.03 ± 0.3 mV after 1 h of flocculation time.Comparing the results presented in Tables 1 and 2, Fig. 3 and Fig. 4; one can see that the pH reduction affected more the repulsion behavior between microalgae cells than the electrolytes cations. Furthermore, in the presence of bioflocculant with the pH decrease, the repulsion forces between microalgae cells is more reduced as the microalgae Zeta Flocculation Efficiency (%)

Table 2 Influence of bioflocculant on microalgae flocculation efficiency and Zeta potential (microalgae concentration: 0.5 g/L, bioflocculant concentration: 4 × 10−6 mM).

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bioflocculant produced by B. licheniformis CGMCC 2876 possibly adsorb to the cell surface, thus forming bridges or patches between microalgae cells and the bioflocculant resulting into heavy flocs which settle out of the culture medium. Salim et al. [14] suggested that patching could be the mechanism behind the flocculation of Tetraselmis suecica and Scenedesmus obliquus as cells seemed to be connected more locally. Xie et al. [29], reported that the flocculability of Rhodopseudomonas faecalis RLD-53 increased with the increase of l-cysteine concentration where the interaction energy barrier of cells dropped from 389.77 to 127.21 kT with an increase of l-cysteine concentration from 0.0 g/L to 1.0 g/L. Zeng et al. [8], conducted a study on flocculation of Chlorella vulgaris and Chlorella protothecoides using commercial ␥-PGA flocculant, and found that the flocculation efficiency increased with the increase in flocculant concentration up to the optimum concentration of 20 mg/L, resulting in the flocculation efficiency >90 % for both microalgae. The Zeta potential measurement revealed that the Zeta potentials of the microalgal suspensions before flocculation were −19.08 and −13.62 mV for C. vulgaris and C. protothecoides respectively, and those of the corresponding flocculated suspensions with optimal 20 mg/L ␥-PGA were +0.83 and +2.04 mV, respectively. Authors concluded that ␥-PGA could adsorb at the surface of the microalgae and such adsorption leaded to a reduction of surface potential (Zeta potential) by charge neutralization thus the destabilization of the microalgae. However, the authors realized that continuous adsorption beyond the point of charge neutralization by overdosing ␥-PGA could result in charge reversal and restabilization of microalgal suspensions. Considering the fact that chemicals cause environmental concerns, possible contamination of the desired end product; microalgae flocculation by the bacterial ␥-PGA bioflocculant produced by B. licheniformis CGMCC 2876 is preferable as it is safe, easy, and economical and resulted in higher flocculation efficiency. 3.4. The DLVO interaction energy

Fig. 4. Microscpic view of microalgae cells (a: cells after cultivation, b: cells in the presence of Zn2+ with the bioflocculant; c: cells at pH of 3 with the bioflocculant).

potential is reduced to nearly zero at pH 3 thus the highest flocculation efficiency of 96.0 ± 0.28 % (Table 2) was achieved compared to microalgae Zeta potential reduced to −8.03 ± 0.3 mV and the flocculation efficiency of 89.95 ± 0.07 % achieved in the presence of both the bioflocculant and Zn2+ . Our hypothesis is that protons are involved in partial charge neutralization of microalgal cell surface charge and the ␥-PGA

The classical DLVO theory has been extensively used as both qualitative and quantitative ways to explain microbial adhesion and aggregation [26,28,29]. The theory suggests that for the same ionic strength, the electrostatic repulsive energy is only related to the Zeta potential and increases with the increase of the value of Zeta potential [18]. Thus the interactions between microalgae cells of Desmodesmus sp. F51 during bioflocculation were characterized according to the DLVO approach based on the strength of two separate interactions, namely the van der Waals interaction (vdW) and electrostatic repulsion (ER). Due to the fact that interactions between microalgae cells are primarily responsible for the flocculation behavior, it was necessary to calculate the total interaction energy which help to predict whether the interaction between microalgae cells is attractive (UTot negative) or repulsive (UTot positive) as a function of separation distance (D) as depicted in Fig. 2. The total interaction energy as function of separation distance between microalgae cells at different pH values without the bioflocculant and in the presence of the bioflocculant was calculated based on the classical DLVO theory and the results are presented in Figs. 5 and 6, respectively. The energy barrier of the microalgae suspension was reduced from 891.6 kT to 64.7 kT (Fig. 5) by simply adjusting the initial pH to 3, at the separation distance of 5 nm. Therefore, the flocculation efficiency of Desmodesmus sp. F51 was expected to increase with the initial pH change according to the DLVO model. The experimental results were in agreement with the model, the flocculation efficiency of Desmodesmus sp. F51 was increased from 13.5 ± 0.7 % to 78.5 ± 0.7 % after 2 h of settling time when the initial pH (∼7.2) was adjusted to pH 3 (Table 1). Cui et al. [18] reported that the flocculation efficiency of N. oculata decreased with an increase in pH

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ing from the changes of surface charge groups on microalgae cells stimulated by pH change and the ␥-PGA bioflocculant, thus the Desmodesmus sp. F51 flocculated more effectively than flocculation at pH = 3 only. These results indicate that the microalgae cells with the lowest interaction energy (lowest energy barrier) had higher flocculation efficiency whilst those with higher energy barrier had lower flocculation efficiency. Hence the classical DLVO theory was qualitatively accurate for interpretation of flocculability of Desmodesmus sp. F51 by the bacterial ␥-PGA bioflocculant.

900 800 700 600

UTot(kT)

500 400 300 200 100 0 -100

0

5

10

15

20

25

30

Separation distance (nm)

-200

4. Conclusion initial pH pH=3 pH=4 pH=9 pH=10

-300 -400 -500 -600 -700

Fig. 5. Total interaction energy as function of separation distance at different pH values in the absence of the bioflocculant.

900 800 700 600 500

initial pH pH=3 pH=4 pH=9

UTot(kT)

400 300 200 100 0 -100

0

5

10

15

20

Nowadays, bioflocculation is considered as an efficient, environmental friendly and low cost microalgae dewatering technique. The Zeta potential results, microscopic pictures and total interaction energy according to classical DLVO theory indicate that flocculation of Desmodesmus sp. F51 is due to the cell surface charge neutralization and the adsorption of the bioflocculant to the cell surface at acidic pH. This study provide a better understanding of bioflocculation behavior of Desmodesmus sp. F51 tripped by bacterial bioflocculant as described by the classical DLVO model and enlighten further understanding and process designing of microalgae dewatering by bioflocculation means. Furthermore, the DLVO theory can be applied qualitatively to interpret the microalgae flocculation induced by bacterial bioflocculant. Acknowledgements

25

30

Separation distance (nm)

-200 -300 -400 -500 Fig. 6. Total interaction energy as function of separation distance in the presence of the bioflocculant and at different pH values.

from 5 to 9 at 100 ␮M Al3+ and similar results were observed for S. dimorphus with 0.1 ␮M ionic strengths. When the bioflocculant was employed (4 × 10−6 mM) to induce flocculation, the energy barrier of the microalgae suspension was reduced from 846.7 kT to −48.2 kT when bioflocculation was induced at pH of 3 (Fig. 6) at the separation distance of 5 nm, according to the DLVO model, the higher flocculation efficiency is anticipated. The experimental results were in full agreement with the predictions of the model as the flocculation efficiency of Desmodesmus sp. F51 was increased from 44.8 ± 0.1 % to 96.0 ± 0.3% after only 10 min of settling time when 4 × 10−6 mM of the ␥-PGA bioflocculant was used to induce the flocculation at pH 3 (Table 2). The highest flocculation efficiency was observed at pH 3 when 4 × 10−6 mM of the ␥-PGA bioflocculant was added, all cells possessed almost zero surface charge (Zeta potential = +0.41 ± 0.15 mV) and Utot was found negative at all separation distances. Even though the pH adjustment resulted in considerable flocculation efficiency (78.5%), long time (2 h) exposure of cells at acidic environmental may affect the desired end product. However flocculation by ␥-PGA bioflocculant achieved relatively higher flocculation efficiency (96%) for only 10 min of settling time. The electrostatic repulsive forces between cells decreased considerably and this is indicated by the decreasing of the Zeta potential result-

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