Flocculation of microalgal cells by pH decrease
Flocculation efficiencies for the three species of freshwater microalgal cells were studied in terms of pH variations (Figure 1). The microalgal cells began to coagulate when the pH decreased from pH 6.7 to about 5.0 Coagulating of algal cells could still be observed when pH further decreased to 4.5, but the coagulated cells remained suspended in the growth medium. The flocculation efficiencies were relatively low. When the pH value was further decreased to pH 4.0, the cells further coagulated and rapidly subsided within a few minutes, resulting in a higher flocculation efficiencies of 90%. Thus, a flocculation zone was attained when pH was lower than 5.0. The efficiencies reached a maximum at about pH 4.0 and then reached a plateau, with a slight decrease when pH was lower than 2.0. These results showed that flocculation induced by pH decrease is a useful method to harvest the three species of freshwater microalgae. Moreover, the biomass concentrations (>1g/L) in this method were much higher those [23, 28] in other harvesting methods (Additional file 1: Table S1). For example, it is 20 times higher than those flocculated using cationic starch [28] and FeCl3[29], and about 10 times higher than that flocculated using poly(γ-glutamic acid) [23], indicating that it could be applied to practical uses.
Mechanisms of flocculation
Role of metal ions in flocculation process
Earlier studies [25, 26, 30] reported that multivalent metal ions such as Mg2+ and Ca2+ played an important role in flocculating microalgae by pH increase. It was found that such metal ions in the growth medium were hydrolyzed to form positive precipitates, which coagulated negative microalgal cells by sweeping flocculation and charge neutralization. To evaluate the role of multivalent metal ions in flocculation induced by pH decrease, their concentrations before and after flocculation for the three microalgae species were measured (Additional file 2: Table S2). Fe3+, Mg2+ and Ca2+ concentrations kept constant before and after flocculation. It suggested that in contrast to the important role of multivalent metal ions in the flocculation by pH increase, they played little part in this flocculation method by pH decrease.
Role of RPS in flocculation process
It has been reported that many microalgae release large amount of RPS during growth [31], and most RPS can interfere with flocculation due to their complexation with multivalent metal ions [26, 32]. However, some RPS are helpful to flocculation owing to the bridging mechanism [33, 34]. All of the studied microalgae here released large amount of RPS which was measured and listed in Additional file 3: Table S3. In order to investigate whether RPS was a promoter or hindrance to this flocculation process, the flocculation efficiencies for microalgae were studied in terms of RPS dosages. The presence of RPS caused negligible changes in the flocculation efficiencies for the three microalgae species, suggesting that RPS has little influence (neither a promoter nor a hindrance) on this flocculation process (Figure 2). As the detrimental effect of RPS on microalgal flocculation result from the complexation of RPS with multivalent metal ions, the little influence of RPS on flocculation efficiencies further testified that multivalent metal ions played little part in this flocculation process.
Mechanism of flocculation
As metal ions and RPS played little part in the flocculation process, mechanism of flocculation might be with the physical-chemical properties of microalgal cells. The most important characteristic of microalgal cells is their surface charge [27]. Thus, zeta potentials of microalgae during flocculation were determined to explore the mechanism.
Zeta potentials and flocculation efficiencies were both pH dependant (Figure 3). From pH 6.5 to 4.0, zeta potentials showed a sharp increase to approximately 0 mV and the corresponding flocculation efficiencies greatly increased to the maximum with pH decrease.
It has been reported that organic matters adhering on microalgal cell surfaces contain carboxyl groups and amino groups. The amounts of the groups and their pKa values were calculated and listed in Additional file 4: Table S4 The cells usually receive their charge and exhibit dispersing stability from ionization of carboxyl groups into carboxylate ions [27]. The concentrations of the functional groups as a function of pH value were calculated (Additional file 5: Figure S1). For pH>6.0, the microalgae surface charge is dominated by negatively charged carboxylate ions and neutral amine groups. As pH decreased, carboxylate ions would accept protons [Eq. (1)] [29]. Then, the surface charge of the cells reduced and the cells became instable in growth medium and coagulated to form big flocs. When the surface charge was totally neutralized at pH 4.0, flocculation efficiencies reached the maximum.
However, from pH 4.0 to 1.5, zeta potentials continuously increased while the corresponding flocculation efficiencies slightly decreased. It might be because that the concentrations of the neutral carboxyl groups increased sharply while the concentrations of the positively charged amine groups (-NH3+) remained constant [Eq. (2)]. This caused the zeta potential increasing, and the positive surface charge made the microalgal cells resuspend so flocculation efficiencies slightly decreased.
The above proposed mechanism involved only protons and functional groups. But, growth medium also contains large amounts of metal salts and extracellular organic matter (EOM). The contributions of metal salts and EOM to the mechanism were evaluated. The average zeta potentials and flocculation efficiencies in deionized water as a function of pH value were also shown in Figure 3. Compared with those in growth medium, the variation trends of zeta potentials and flocculation efficiencies were extremely similar. From pH 6.5 to 1.5, zeta potentials continuously increased but flocculation efficiencies firstly increased then decreased. pH 4.0 was the transiting point, at which flocculation efficiencies reached peak and the cells were electrically neutral. The results further confirmed that flocculation was induced only by neutralizing cells surface charges with protons while metal salts and EOM had no contribution to the mechanism.
(1)
(2)
Flocculation dependence on microalgae biomass concentration
In our previous report [26], microalgae biomass concentrations have effects on the flocculation efficiencies induced by pH increase and the efficiencies decreased considerably with the increase of biomass concentrations. However, in this study, the flocculation efficiencies at the same pH value for the microalgae were all increased with the increase of biomass concentrations (Figure 4). Low biomass concentrations correspond to lag phase, while high biomass concentrations correspond to exponential growth phase and stationary phase. During lag phase, microalgae are unicells. But during exponential growth phase and stationary phase, microalgal cells usually coagulate by threes and fours to form cell clusters. As the surface charge of cells are generally neutralized, it is easier for the heavier cell clusters than the unicells to settle (Figure 5). So, the flocculation efficiencies increased with the increase of biomass concentrations.
Relationship between biomass concentration and amount of HNO3
As mentioned above, flocculation efficiencies for the three microalgae species were increased with the increase of biomass concentrations. However, the minimum dosages of HNO3 resulting in the same flocculation efficiencies also rose accordingly. Hence, the biomass concentration is a very important parameter affecting the optimal dosage of HNO3. Experiments were run at different biomass concentrations to determine the correlation between biomass concentration and HNO3 dosage (Figure 6). A linear correlation was thus observed, which was expressed by the following equations [Eqs. (3), (4) and (5) for Chlorococcum nivale and Chlorococcum ellipsoideum and Scenedesmus sp.] Hence, in practical uses, acid dosage needed for flocculating microalgae can be calculated according to biomass concentration and their relationship.
(3)
(4)
(5)
Cells viability during pH decrease process
Viability of microalgal cells was determined by the Evans blue assay and the cells seemed to be very resistant to relatively low pH values (6.0-3.0). A positive control is also provided. As shown in Figures 7a, 7b and 7c, the controlled cells are light green and the yellow liquid in the cells can be seen clearly. However, for the cells heated at 121 in Figures 7d, 7e and 7f, the dead cells (solid arrows) are dark green which were dyed by Evans blue and the yellow liquid in the cells are not visible. While the alive cells (dash arrows) are light green and similar to the cells shown in Figures 7a, 7b and 7c. As for the cells flocculated by adjusting pH value of growth medium to 0.5 with nitric acid (Figure 7g, 7h and 7i), the dead cell nuclei turned black, the cytoplasm turned green-yellow and the materials surrounding the cells were dyed blue . The cells flocculated by adjusting pH value of growth medium to 3.5 in Figures 7j, 7k and 7l, are similar to the controlled cells in Figure 7a, 7b and 7c, except that few cells were dyed blue. The above results indicated there were no cell lysis and the cell walls were intact. Thus, the cells were not damaged during the process of pH decreasing to 3.5.
Recycling of flocculated culture medium for cultivation
Ideally, medium recovered from flocculation could be recycled for next cultivation. The problem with medium recycling is that residual flocculant such as ferric salts and aluminum salts can cause contamination, which eventually cause environmental problems and a great loss of water [35]. However, in this flocculation method induced by pH decrease, since no flocculants were used and the medium was not contaminated, the growth medium after flocculation might be recycled by neutralizing pH and then adding nutrients. The product of neutralizing pH of flocculated BG-11 medium with NaOH was NaNO3, which was a necessary nutrient. So, the recycling of flocculated medium could minimize the cost of nutrients and demand for water. In this respect, the possibility of recycling the flocculated medium was examined. Some microalgal cells flocculated were cultivated in the recycled culture solution and the biomass as a function of growth phase was shown in Figure 8. It was observed that the biomass of each microalgal species cultivated in the recycled growth medium was close to that cultivated in the fresh medium, indicating the flocculated medium could be successfully recycled for cultivation. The fact that the flocculated microalgal cells could be recultivated further suggested that there was no cell lysis during the flocculation process and the molecular function and structure of the photosynthetic apparatus were not affected.
Comparison with other harvesting methods
The flocculation method presented here is simple and effective. The traditional harvesting methods, such as ultrasound, flotation, centrifugation and filtration, were also successfully applied to a range of microalgal species. But they are energy-intensive and cost-intensive [36]. Moreover, microalgae can also be harvested by using flocculants, including inorganic and organic flocculants. Inorganic flocculants such as aluminum salts, ferric salts and zinc salts were often used, but caused an environmental problem and a great loss of water due to the contamination of residual inorganic flocculants. Organic flocculants, such as cationic polyacrylamide, cationic starch and chitosan, are biodegradable and low toxic [37], but are of high cost owing to their high prices ($US10 per kilogram for chitosan and $US 1~3 per kilogram for cationic starch [28]). In this study, microalgae were flocculated induced by HNO3. In contrast, HNO3, is inexpensive and about $US 0.28 per kilogram. And what’s more, HNO3 does not contaminate growth medium, which can be recycled to reduce not only the cost and the demand for water, but also the extra operational costs for reusing growth medium. Additionally, comparison of the cost of cultivation and flocculation for per kilogram microalgae has been listed in Additional file 6: Table S5. Thereby this method is helpful to lower the production cost from algae to biodiesel.