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Effect of different organic matters on flocculation of Chlorella sorokiniana and optimization of flocculation conditions in swine manure wastewater
Bioresource Technology 192 (2015) 774–780
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Effect of different organic matters on flocculation of Chlorella sorokiniana and optimization of flocculation conditions in swine manure wastewater Bo Zhang, Sanfeng Chen ⇑ State Key Laboratory for Agrobiotechnology and College of Biological Sciences, China Agricultural University, Beijing 100193, PR China
h i g h l i g h t s Concentrations of AOM were increased in the medium with glucose. Flocculation in swine manure wastewater was depressed significantly. Tryptone could cause similar inhibitory effect on flocculation as in wastewater. Urea as nitrogen source would scarcely affect flocculation of Chlorella. The best harvest condition in sewage: pH 8.5, dilution (7-folds) and Al
a r t i c l e
i n f o
Article history: Received 29 April 2015 Received in revised form 5 June 2015 Accepted 7 June 2015 Available online 18 June 2015 Keywords: Chlorella Flocculation Organic matters Response surface methodology (RSM) Swine manure wastewater
3+
a b s t r a c t In this study, flocculation of Chlorella sorokiniana cultivated in swine manure wastewater, BG-11 medium and BG-11 medium supplemented with different organic matters (glucose, urea and tryptone) was investigated. The results demonstrated that the minimum amount of Al3+ required for complete flocculation in wastewater would increase substantially, and flocculation efficiency became highly sensitive to pH. Tryptone could cause similar extent of inhibition on flocculation as in wastewater. Meanwhile, glucose could increase concentrations of Algogenic Organic Matter (AOM), inhibiting flocculation strongly at higher pH, including flocculation induced by Al3+ and autoflocculation. However, urea had little effect on flocculation of C. sorokiniana. Moreover, the major factors: dilution times, pH and flocculants dosage, which had significant impact on flocculation efficiency of C. sorokiniana in piggery wastewater, were optimized using response surface methodology (RSM). The optimal flocculation efficiency (100%) was achieved at pH 8.5, 7-folds of dilution and 52.14 mg L1 of Al3+. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Microalgae are today considered to be the most promising new source of biomass for production of biofuels due to their high oil contents and high growth rates (Benemann et al., 1977; Chisti, 2008; Oswald and Golueke, 1960; Wijffels and Barbosa, 2010). However, highly expensive culture medium and energy-intensive harvesting processes result in the huge cost for microalgae biomass application (Sander and Murthy, 2010). For microalgal biomass to become a widely used commodity like most agricultural crops, the cost of production has to be reduced greatly (Vandamme et al., 2013). The integration of wastewater treatment and algal biomass culture could significantly reduce cost of nutrient input (Hu et al., 2012). For example, high concentrations of nitrogen and phosphorus and some organic pollutants in swine manure ⇑ Corresponding author. E-mail address: [email protected] (S. Chen). http://dx.doi.org/10.1016/j.biortech.2015.06.068 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
(52 mg/L).
wastewater could be assimilated by algae (Burrell et al., 1984; Lau et al., 1995). Harvesting is another critical step in the process of microalgae biomass production, as it accounts for 20–30% of the total process costs (Molina Grima et al., 2003). At the same time, efficient harvesting process is one of the bottlenecks to cost reduction, due to the small size (5–20 lm), low biomass concentrations (0.5–5 g L1) and negative surface charge (about 7.5 to 40 mV) of microalgal cells (Chen et al., 2011; Georgianna and Mayfield, 2012; Larkum et al., 2012). Flocculation technology is undoubtedly the best choice due to low costs and energy demand. During flocculation, the cells could form larger aggregates that can be separated from system by simple gravity action (Vandamme et al., 2013). After flocculation, a dilute suspension of 0.5 g L1 biomass could be concentrated 20–100 times to a slurry of 10–50 g L1. The energy requirements for further mechanical dewatering step are acceptable because the volumes of water to process are relatively small (Schlesinger et al., 2012).
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Different methods have been reported for algae aggregation during decades of research. These approaches range from traditional flocculation methods used in water treatment to the use of emerging technologies, including autoflocculation (e.g., pH changes), physical flocculation methods (e.g., electrocoagulation flocculation and use of magnetic nanoparticles) and bioflocculation. Among the various flocculation approaches, metal flocculants turn out to be a good model system to study the interaction between microalgal cells and flocculants because their properties have been extensively studied (Wyatt et al., 2012; Zhang et al., 2012). Previous studies focused mainly on flocculation efficiency of various methods in specific system, such as in synthetic medium. But the influence of organic matters, such as glucose, urea and tryptone, on flocculation of algae is not well investigated. Moreover, characteristics of flocculation for the culture of Chlorella in swine manure wastewater were poorly studied, although microalgae biomass accumulation based on wastewater treatment was widely researched and applied. The present research was undertaken to determine the optimal flocculation condition in swine manure wastewater and the impacts of organic matters (glucose, urea, and tryptone) on flocculation of Chlorella. 2. Methods 2.1. Microalgal strain and culture condition
Table 2 Variables and experimental levels for Box–Behnken design. Factor
A/dilution times B/pH C/concentration of Al3+ (mg L1)
Level 1
0
+1
2 5 22
6 7 44
10 9 66
20 g L1), and then left undisturbed for 20 min at room temperature. Recovery efficiency was calculated based on optical density at 540 nm according to Eq. (1):
Flocculation efficiency ð%Þ ¼ ð1 ODfinal =ODinitial Þ 100
ð1Þ
where ODfinal is the optical density of sample taken after the flocculation experiment, ODinitial is the optical density of untreated microalgae culture. 2.4. Flocculation of C. sorokiniana at different flocculants dosage After cultivation, at natural pH, different dosages of flocculants were used to carry out the flocculation experiment in the five kinds of media. The flocculation efficiency was detected and the minimum amount of flocculants for achieving effective flocculation was determined. 2.5. Flocculation of C. sorokiniana at different pH
Algae strain indentified as Chlorella sorokiniana (18S r DNA, Genbank No. KJ734869) was isolated from Xiaoqing River in Beijing of China in this study. The algae seeds were cultivated in 250 mL Erlenmeyer flasks each containing 100 mL BG-11 medium (Rippka et al., 1979). The seeds were transferred into diluted swine manure wastewater and other four kinds of media (BG-11, BG-11 containing 5 g L1 glucose, BG-11 with 1 g L1 urea instead of sodium nitrate, BG-11 with 2 g L1 tryptone instead of sodium nitrate). Microalgal inoculation amount was approximately 150 mg L1 (dry weight concentration). The flasks were incubated at 27 ± 2 °C, with 3500 lux cool-white fluorescent light illumination (light:dark = 14:10) and shaken at 100 rpm. 2.2. Wastewater Swine manure wastewater was collected from Hongyun pig farm at Changping District in Beijing. All the wastewater was filtered using multi-layer gauze to remove large particles. The wastewater was diluted 5 times with tap water, and then the diluted wastewater was used to cultivate the microalgae biomass. The levels of the characteristics of undiluted wastewater are shown in Table 1. 2.3. Determination of flocculation efficiency After a seven-day culture of microalgae, flocculation process was carried out in 250 mL Erlenmeyer flasks with 200 mL culture. The cultures were immediately shaken using a vortex for 30 s after addition of mother liquid of aluminum chloride (AlCl36H2O, Table 1 Characteristics of swine manure wastewater.
At the end of cultivation, pH in each kind of media was adjusted to 4, 5, 6, 7, 8, 9, 10, 11 and 12, respectively, using solution of 1 N HCl or 1 N NaOH. Then observe and record whether autoflocculation phenomenon occurred after change of pH. Flocculation efficiency at various pH was recorded with 22 mg L1 of Al3+. 2.6. The effect of glucose in algae cultivation process on flocculation Centrifugation and subsequent resuspension was used to remove AOM (Algogenic Organic Matter) generated during metabolic processes of microalgae (Beuckels et al., 2013; Vandamme et al., 2012). To determine the effect of glucose in algae cultivation process on flocculation, after centrifugation of algae culture in BG-11 with glucose, Chlorella cells were resuspended in fresh BG-11 medium without AOM, in centrifugal supernatant of algae culture in BG-11 and in their original supernatant, respectively. Flocculation efficiency in the three groups was compared with addition of 22 mg L1 of Al3+ at pH 11 and 12. 2.7. Experiment design and analysis of the response surfaces Three factors: dilution times of wastewater, pH and concentration of Al3+, which had significant impact on algae flocculation, were selected in RSM (response surface methodology) experiment for optimization for flocculation efficiency. Box–Behnken, a method of response surface design, was used to perform the experimental design for the optimization. The levels of the 3 factors used here are shown in Table 2. The results were analyzed and the optimal values were predicted by Design-Expert 8.0.4. 3. Results and discussion
Parameter
Swine manure wastewater
pH Total nitrogen (mg L1) Ammonia nitrogen (mg L1) Total phosphorous (mg L1) Chemical oxygen demand (mg L1)
7.8 605 520 82 1660
3.1. Flocculation at different flocculants dosage The natural pH after cultivation of Chlorella was 8.9 in BG-11, 5.6 in medium with glucose, 7.8 in medium with tryptone, 9.5 in medium with urea and 8.5 in diluted swine manure wastewater,
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respectively. To determine the influence of flocculants dosage on flocculation, 0–70 mg L1 of Al3+ was added to the five culture systems under natural pH, respectively. As shown in Fig. 1, for the flocculation of microalgae in BG-11 system, only 11 mg L1 of Al3+ could achieve nearly 100% flocculation efficiency. Similarly, 11 mg L1 of Al3+ could achieve nearly 100% flocculation efficiency in both BG-11 with glucose and BG-11 with urea, indicating that glucose or urea did not produce influence on flocculation by AlCl3 under natural pH. However, the minimum concentration of Al3+ needed for complete flocculation in BG-11 with tryptone and in diluted swine manure wastewater had increased to 33 mg L1 and 54 mg L1, respectively. Meanwhile, a linear correlation between flocculation efficiency and addition amount of flocculants was found (R2 = 0.99, p < 0.05) in diluted swine manure wastewater, indicating that amount of flocculants was an important factor affecting the flocculation efficiency of microalgae cells. Compared with flocculation in BG-11 medium, the substantial increase in flocculants dose required in wastewater would raise the cost of microalgae harvest. Thus, it is necessary to understand the effect of components of wastewater on flocculation and to optimize the related conditions. 3.2. Flocculation at different pH 3.2.1. Autoflocculation induced by pH change As displayed in Fig. 2a, for algae culture in BG-11, only at pH 12, autoflocculation emerged with harvesting efficiency of 71%. Meanwhile, for algae culture in diluted swine manure wastewater, when pH was adjusted to 11 or higher (Fig. 2b), aggregation and sedimentation of algae cells occurred rapidly, and the solution became clear and transparent in 2 min. Flocculation efficiency of 98% was obtained only by increasing pH in a very short time. However, for algae cells in BG-11 with organic additives (glucose, urea and tryptone), phenomenon of autoflocculation did not occur, suggesting that the addition of organic matters had significant inhibition on autoflocculation induced by high pH. Most studies have demonstrated that for autoflocculation induced by higher pH, magnesium hydroxide is the key precipitates causing algae flocculation through charge neutralization, bridging, or sweeping flocculation (Besson and Guiraud, 2013; Castrillo et al., 2013; Chen et al., 2013; Elmaleh et al., 1991; Sirin et al., 2012; Smith and Davis, 2012; Wu et al., 2012; Yahi et al.,
1994). Generally, there are a large number of various metal ions in sewage, as to the diluted wastewater used in this research, the concentration of Mg2+ could reach 35 mg L1, which was 5 times of BG-11 (Table 3). Therefore, algae autoflocculation is much easier to occur in the culture system with wastewater. 3.2.2. Aluminum chloride-induced flocculation at different pH It was reported that the minimum pH required for effective flocculation and negative surface charge of Chlorella is 4.0 (Wyatt et al., 2012). Here, flocculation efficiency for algae culture in various systems was determined with addition of 22 mg L1 of Al3+ at different pH (4–12). As shown in Fig. 2, at pH 4, the algae flocculation efficiency for kinds of culture media was only between 10% and 40%. In the range of pH from 5 to 11, the flocculation efficiency for algae culture in BG-11 medium was around 90% (Fig. 2a). For algae culture in diluted swine manure wastewater, flocculation efficiency responded to the change of pH sensitively (Fig. 2b). The maximum flocculation efficiency (98%) was reached at pH 6, and then began to decline rapidly with the increase of pH. The value had fallen to 30% at pH 10. As shown in Fig. 2c, the pattern of flocculation in BG-11 with urea was similar to that in BG-11, indicating that the addition of urea had little influence on flocculation efficiency at different pH. Interestingly, algae flocculation in BG-11 with tryptone had a highly similar variation trend with change of pH as in wastewater (Fig. 2d). Therefore, inhibitory effect on flocculation process in wastewater might result from the complex organic components as tryptone, making flocculation efficiency highly sensitive to pH. For algae culture in BG-11 with glucose (Fig. 2e), when pH was between 5 and 10, flocculation efficiency was maintained at a higher level (>95%), and the flocculation efficiency decreased to 10%, with pH increased to 11 or 12. The reason for disappearance of autoflocculation and decrease of flocculation efficiency might be that the secretion of AOM by algae cells became stronger due to addition of glucose during cultivation process and higher concentrations of AOM made flocculation more difficult. Researches of recent years demonstrate that AOM generated during metabolic processes of microalgae has significant inhibition effect on various flocculation methods including autoflocculation induced by pH increase and flocculation based on metal ions, resulting in an increase of flocculants dosage demand (Beuckels et al., 2013; Vandamme et al., 2012; Wu et al., 2012).
Fig. 1. Flocculation efficiency for algae culture in various systems at different flocculants dosage.
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Fig. 2. Flocculation efficiency for algae culture in various systems at different pH; (a) flocculation efficiency in BG-11; (b) flocculation efficiency in diluted swine manure wastewater; (c) flocculation efficiency in BG-11 with urea; (d) flocculation efficiency in BG-11 with tryptone; (e) flocculation efficiency in BG-11 with glucose.
Table 3 Metal elemental characteristics of BG-11 medium and 5 swine manure wastewater.
1
Mg (mg L ) Ca (mg L1) Al (mg L1) Fe (mg L1) Cu (mg L1) Mn (mg L1) Zn (mg L1) K (mg L1) Na (mg L1)
BG-11
5 swine manure wastewater
7.3 9.8 – 1.2 0.02 0.5 0.05 13.66 414.84
35.3 19.7 0.17 0.14 0.06 0.018 0.09 81.46 35.68
3.3. The effect of glucose in algae cultivation process on flocculation In order to investigate the influence of AOM, algae cells were resuspended in fresh BG-11 medium without AOM (without algal cultivation), in supernatant of algae culture in BG-11 and in supernatant of algae culture in BG-11 with glucose, respectively, and
then flocculation efficiency at pH 11 and 12 in the three groups was determined. As displayed in Fig. 3, at pH 12, only for cells resuspended in fresh BG-11 medium without AOM, autoflocculation emerged with flocculation efficiency of 90%, suggesting that AOM in the supernatant of algae culture in BG-11 and BG-11 with glucose had strong inhibition on autoflocculation of microalgae. When 22 mg L1 of Al3+ was added, at pH 11, flocculation efficiency could reach 99% in the group with fresh BG-11, a lower efficiency of 20% was obtained in the group with supernatant of algae culture in BG-11, and the lowest recovery efficiency of 10% showed the greatest difficulty of flocculation in algae culture with glucose. The data also demonstrate that AOM in the algae culture in BG-11 and BG-11 with glucose had strong inhibition on flocculation of microalgae based on Al3+. Meanwhile, at pH 12, the minimum Al3+ concentration needed for algae culture in BG-11 with glucose had increased to 220 mg L1, which was 2 times of that in the group with supernatant of algae culture in BG-11. Compared with the fresh BG-11 medium without AOM, flocculation was suppressed in the
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Fig. 3. Flocculation for algae cells resuspended in fresh BG-11 medium, in supernatant of algae culture in BG-11 and in supernatant of algae culture in BG-11 with glucose.
supernatant of algae culture in BG-11 suggesting that algae cells had secreted some substances during the autotrophic growth process in BG-11 to inhibit harvest of algae. And this inhibition appeared to be stronger for the culture in BG-11 containing glucose due to the increase of content of AOM. Therefore, the assimilation of organic matters may have a huge impact on subsequent microalgae recovery by affecting metabolic products of cells. Further studies should investigate how flocculation is influenced by organic matters in the culture medium to design flocculation systems for algae culture in different nutrition environment. 3.4. Statistical analysis using RSM In order to achieve high flocculation efficiency, the major factors (dilution times of wastewater, pH value and concentration of Al3+) involved in flocculation of Chlorella in swine manure wastewater were optimized. Table 4 shows the flocculation efficiency corresponding to the combined effect of three components in the specified ranges through Box–Behnken Design of Design-Expert
Table 4 Box–Behken design matrix with experimental and predicted values for flocculation efficiency. Standard order
Run order
Coded level
Flocculation efficiency
A
B
C
Experimental value
Predicted value
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
12 15 3 11 8 2 17 6 1 13 4 5 9 7 16 10 14
1 +1 1 +1 1 +1 1 +1 0 0 0 0 0 0 0 0 0
1 1 +1 +1 0 0 0 0 1 +1 1 +1 0 0 0 0 0
0 0 0 0 1 1 +1 +1 1 1 +1 +1 0 0 0 0 0
0.73 0.96 0.23 0.99 0.23 0.99 0.73 0.99 0.98 0.49 0.98 1.00 0.99 0.99 0.99 0.98 1.00
0.73 0.96 0.22 0.99 0.23 0.98 0.74 0.99 0.98 0.49 0.98 1.00 0.99 0.99 0.99 0.99 0.99
8.0.4. The mathematical regression model for flocculation efficiency fitted in terms of coded factors was as Eq. (2):
Flocculation efficiency ¼ 0:99 þ 0:25 A 0:12 B þ 0:13 C þ 0:13 AB 0:12 AC þ 0:13 BC 0:20 A2 0:067 B2 0:060 C 2
ð2Þ
The results of the second-order response surface model fitting in the form of ANOVA (analysis of variance) are given in Table 5. The R2 coefficient was 0.9998 suggesting that it was a reliable model and that 99.98% of the sample variation of flocculation efficiency was attributed to a high correlation between the independent variables. The relatively high adjusted determination coefficient (R2Adj = 0.9997) accounted for significance of the model. This revealed that the equation was a suitable model to describe the response of experiment. ANOVA showed that all the three factors and the interaction between them had significant effects on the response value. The response surfaces of flocculation efficiency of Chlorella in swine manure wastewater were established according to the model. Fig. 4a and b show the mutual influences of dilution times and pH on flocculation efficiency. In brief, greater degree of dilution of sewage could cause higher flocculation efficiency. Dilution could reduce the concentration of organic compounds effectively relieving the inhibition on flocculation. When the dilution ratio was less than 4-folds, the effect of pH on flocculation efficiency was significant, but when wastewater was diluted more than 8-folds, the influence of pH became greatly weakened. Fig. 4c and d show the mutual influences of dilution times and flocculants dosage on flocculation efficiency. When wastewater was diluted 10-folds, a lower concentration (22 mg L1) of flocculants was enough to achieve complete flocculation (Run 2), but when wastewater was diluted 2-folds, even a higher concentration (66 mg L1) of Al3+ only realized a flocculation efficiency of 73% (Run 17). Fig. 4e and f show the mutual influences of flocculants dosage and pH on flocculation efficiency. Flocculation induced by Al3+ was highly dependent on pH, and flocculants dosage required to achieve complete flocculation would change greatly at different pH (Run 1 and 5). Meanwhile, microalgae biomass productivity was affected dramatically by concentration of organic matters in the wastewater. Previous study (Travieso et al., 2006) reported that in a range of
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B. Zhang, S. Chen / Bioresource Technology 192 (2015) 774–780 Table 5 Analysis of variance for response. Source
Sum of squares
DF
Mean square
F value
p-Value
Significance
Model A B C AB AC BC A2 B2 C2 Residual Lack of fit Pure error Corrected total
1.15 0.5 0.11 0.13 0.071 0.062 0.064 0.16 0.019 0.015 1.743E004 6.609E005 1.082E004 1.15
9 1 1 1 1 1 1 1 1 1 7 3 4 16
0.13 0.50 0.11 0.13 0.071 0.062 0.064 0.16 0.019 0.015 2.490E005 2.203E005 2.705E005 –
5120.56 20020.72 4590.96 5060.68 2871.02 2496.18 2570.55 6514.05 767.84 599.89 – 0.81 – –
<0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 – 0.5490 – –
Significant Significant Significant Significant Significant Significant Significant Significant Significant Significant – Not significant – –
R2 = 0.9998 AdjR2 = 0.9997.
Fig. 4. 3D response surface and contour line of dilution times of wastewater, pH and concentration of Al3+; (a and b) demonstrated the effects of dilution times and pH on flocculation efficiency; (c and d) showed the effects of dilution times and concentration of Al3+ on flocculation efficiency; (e and f) displayed the effects of pH and concentration of Al3+ on flocculation efficiency.
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initial substrate concentration from 250 to 800 mg L1 COD, microalgae concentration increased, nevertheless, when the initial COD concentration increased to 1100 mg L1, the biomass concentration decreased considerably, showing an inhibition phenomenon. Therefore, for cultivation of microalgae with wastewater, dilution times would affect biomass accumulation and flocculation of cells at the same time. It is necessary to determine a suitable scope of dilution for higher algae growth rates with lower flocculation costs. Thus, the scope of factors was defined before the optimal condition was proposed by the model as follows: dilution times, in range (5–7); pH, in range (8.5–9); flocculants dosage (to minimize). Thus, on the basis of these response surfaces, the optimal condition for flocculation efficiency was at pH 8.5, 7-folds of dilution and 52.14 mg L1 of Al3+, and the maximum recovery efficiency was 100%. The accuracy of the model was validated with at least three replicates giving the flocculation efficiency of 0.99 ± 0.01, which concurred with the model prediction. The study demonstrates that microalgae cultivated in swine manure wastewater can be harvested efficiently through flocculation by increasing pH or by adding Al3+. It should be noted that Al3+ would remain as residual metals in the algal biomass residue after extraction of lipids, which may interfere with the use of this residue as animal feed or fertilizer. Thus, for the use of Al3+ in flocculation, further research on related technological process is necessary to ensure that the amount of Al in the biomass residue is lower than the maximum tolerable levels of Al in the feed for poultry and swine (1000 mg/kg) (Mandik et al., 2015; Mineral Tolerance of Animals, 2005). Meanwhile, safer polymers such as chitosan and cationic starch should be extensively studied to establish effective algae flocculation system in swine manure wastewater (Buelna et al., 1990; Masse and Massé, 2010; Vandamme et al., 2010). 4. Conclusions Microalgae cultivated in swine manure wastewater can be harvested efficiently through flocculation by increasing pH or by adding Al3+. The optimal condition for flocculation of C. sorokiniana in swine manure wastewater was at pH 8.5, 7-folds of dilution and 52.14 mg L1 of Al3+, with a recovery efficiency of 100%. Addition of organic matters, such as glucose and tryptone, in microalgae cultivation system could cause significant inhibition on flocculation efficiency by affecting metabolic processes of cells. Acknowledgement This research was supported by the 863 High Technology Program (Grant No. 2013AA065802). References Benemann, J.R., Weissman, J.C., Koopman, B.L., Oswald, W.J., 1977. Energy production by microbial photosynthesis. Nature 268 (5615), 19–23. Besson, A., Guiraud, P., 2013. High-pH-induced flocculation-flotation of the hypersaline microalga Dunaliella salina. Bioresour. Technol. 147, 464–470. Beuckels, A., Depraetere, O., Vandamme, D., Foubert, I., Smolders, E., Muylaert, K., 2013. Influence of organic matter on flocculation of Chlorella vulgaris by calcium phosphate precipitation. Biomass Bioenergy 54, 107–114. Buelna, G., Bhattarai, K.K., de la Noue, J., Taiganides, E.P., 1990. Evaluation of various flocculants for the recovery of algal biomass grown on pig-waste. Biol. Wastes 31, 211–222. Burrell, R.E., Inniss, W.E., Mayfield, C.I., 1984. Development of an optimal heterotrophic growth medium for Chlorella vulgaris. Appl. Microbiol. Biotechnol. 20 (4), 281–283.
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Influence of organic matter generated by Chlorella vulgaris on five different modes of flocculation. Bioresour. Technol. 124, 508–511. Vandamme, D., Foubert, I., Meesschaert, B., Muylaert, K., 2010. Flocculation of microalgae using cationic starch. J. Appl. Phycol. 22, 525–530. Vandamme, D., Foubert, I., Muylaert, K., 2013. Flocculation as a low-cost method for harvesting microalgae for bulk biomass production. Trends Biotechnol. 31 (4), 233–239. Wijffels, R.H., Barbosa, M.J., 2010. An outlook on microalgal biofuels. Science 329 (5993), 796–799. Wu, Z., Zhu, Y., Huang, W., Zhang, C., Li, T., Zhang, Y., Li, A., 2012. Evaluation of flocculation induced by pH increase for harvesting microalgae and reuse of flocculated medium. Bioresour. Technol. 110, 496–502. Wyatt, N.B., Gloe, L.M., Brady, P.V., Hewson, J.C., Grillet, A.M., Hankins, M.G., Pohl, P.I., 2012. Critical conditions for ferric chloride-induced flocculation of freshwater algae. Biotechnol. Bioeng. 109 (2), 493–501. 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Effect of different organic matters on flocculation of Chlorella sorokiniana and optimization of flocculation conditions in swine manure wastewater Bo Zhang, Sanfeng Chen ⇑ State Key Laboratory for Agrobiotechnology and College of Biological Sciences, China Agricultural University, Beijing 100193, PR China
h i g h l i g h t s Concentrations of AOM were increased in the medium with glucose. Flocculation in swine manure wastewater was depressed significantly. Tryptone could cause similar inhibitory effect on flocculation as in wastewater. Urea as nitrogen source would scarcely affect flocculation of Chlorella. The best harvest condition in sewage: pH 8.5, dilution (7-folds) and Al
a r t i c l e
i n f o
Article history: Received 29 April 2015 Received in revised form 5 June 2015 Accepted 7 June 2015 Available online 18 June 2015 Keywords: Chlorella Flocculation Organic matters Response surface methodology (RSM) Swine manure wastewater
3+
a b s t r a c t In this study, flocculation of Chlorella sorokiniana cultivated in swine manure wastewater, BG-11 medium and BG-11 medium supplemented with different organic matters (glucose, urea and tryptone) was investigated. The results demonstrated that the minimum amount of Al3+ required for complete flocculation in wastewater would increase substantially, and flocculation efficiency became highly sensitive to pH. Tryptone could cause similar extent of inhibition on flocculation as in wastewater. Meanwhile, glucose could increase concentrations of Algogenic Organic Matter (AOM), inhibiting flocculation strongly at higher pH, including flocculation induced by Al3+ and autoflocculation. However, urea had little effect on flocculation of C. sorokiniana. Moreover, the major factors: dilution times, pH and flocculants dosage, which had significant impact on flocculation efficiency of C. sorokiniana in piggery wastewater, were optimized using response surface methodology (RSM). The optimal flocculation efficiency (100%) was achieved at pH 8.5, 7-folds of dilution and 52.14 mg L1 of Al3+. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Microalgae are today considered to be the most promising new source of biomass for production of biofuels due to their high oil contents and high growth rates (Benemann et al., 1977; Chisti, 2008; Oswald and Golueke, 1960; Wijffels and Barbosa, 2010). However, highly expensive culture medium and energy-intensive harvesting processes result in the huge cost for microalgae biomass application (Sander and Murthy, 2010). For microalgal biomass to become a widely used commodity like most agricultural crops, the cost of production has to be reduced greatly (Vandamme et al., 2013). The integration of wastewater treatment and algal biomass culture could significantly reduce cost of nutrient input (Hu et al., 2012). For example, high concentrations of nitrogen and phosphorus and some organic pollutants in swine manure ⇑ Corresponding author. E-mail address: [email protected] (S. Chen). http://dx.doi.org/10.1016/j.biortech.2015.06.068 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
(52 mg/L).
wastewater could be assimilated by algae (Burrell et al., 1984; Lau et al., 1995). Harvesting is another critical step in the process of microalgae biomass production, as it accounts for 20–30% of the total process costs (Molina Grima et al., 2003). At the same time, efficient harvesting process is one of the bottlenecks to cost reduction, due to the small size (5–20 lm), low biomass concentrations (0.5–5 g L1) and negative surface charge (about 7.5 to 40 mV) of microalgal cells (Chen et al., 2011; Georgianna and Mayfield, 2012; Larkum et al., 2012). Flocculation technology is undoubtedly the best choice due to low costs and energy demand. During flocculation, the cells could form larger aggregates that can be separated from system by simple gravity action (Vandamme et al., 2013). After flocculation, a dilute suspension of 0.5 g L1 biomass could be concentrated 20–100 times to a slurry of 10–50 g L1. The energy requirements for further mechanical dewatering step are acceptable because the volumes of water to process are relatively small (Schlesinger et al., 2012).
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Different methods have been reported for algae aggregation during decades of research. These approaches range from traditional flocculation methods used in water treatment to the use of emerging technologies, including autoflocculation (e.g., pH changes), physical flocculation methods (e.g., electrocoagulation flocculation and use of magnetic nanoparticles) and bioflocculation. Among the various flocculation approaches, metal flocculants turn out to be a good model system to study the interaction between microalgal cells and flocculants because their properties have been extensively studied (Wyatt et al., 2012; Zhang et al., 2012). Previous studies focused mainly on flocculation efficiency of various methods in specific system, such as in synthetic medium. But the influence of organic matters, such as glucose, urea and tryptone, on flocculation of algae is not well investigated. Moreover, characteristics of flocculation for the culture of Chlorella in swine manure wastewater were poorly studied, although microalgae biomass accumulation based on wastewater treatment was widely researched and applied. The present research was undertaken to determine the optimal flocculation condition in swine manure wastewater and the impacts of organic matters (glucose, urea, and tryptone) on flocculation of Chlorella. 2. Methods 2.1. Microalgal strain and culture condition
Table 2 Variables and experimental levels for Box–Behnken design. Factor
A/dilution times B/pH C/concentration of Al3+ (mg L1)
Level 1
0
+1
2 5 22
6 7 44
10 9 66
20 g L1), and then left undisturbed for 20 min at room temperature. Recovery efficiency was calculated based on optical density at 540 nm according to Eq. (1):
Flocculation efficiency ð%Þ ¼ ð1 ODfinal =ODinitial Þ 100
ð1Þ
where ODfinal is the optical density of sample taken after the flocculation experiment, ODinitial is the optical density of untreated microalgae culture. 2.4. Flocculation of C. sorokiniana at different flocculants dosage After cultivation, at natural pH, different dosages of flocculants were used to carry out the flocculation experiment in the five kinds of media. The flocculation efficiency was detected and the minimum amount of flocculants for achieving effective flocculation was determined. 2.5. Flocculation of C. sorokiniana at different pH
Algae strain indentified as Chlorella sorokiniana (18S r DNA, Genbank No. KJ734869) was isolated from Xiaoqing River in Beijing of China in this study. The algae seeds were cultivated in 250 mL Erlenmeyer flasks each containing 100 mL BG-11 medium (Rippka et al., 1979). The seeds were transferred into diluted swine manure wastewater and other four kinds of media (BG-11, BG-11 containing 5 g L1 glucose, BG-11 with 1 g L1 urea instead of sodium nitrate, BG-11 with 2 g L1 tryptone instead of sodium nitrate). Microalgal inoculation amount was approximately 150 mg L1 (dry weight concentration). The flasks were incubated at 27 ± 2 °C, with 3500 lux cool-white fluorescent light illumination (light:dark = 14:10) and shaken at 100 rpm. 2.2. Wastewater Swine manure wastewater was collected from Hongyun pig farm at Changping District in Beijing. All the wastewater was filtered using multi-layer gauze to remove large particles. The wastewater was diluted 5 times with tap water, and then the diluted wastewater was used to cultivate the microalgae biomass. The levels of the characteristics of undiluted wastewater are shown in Table 1. 2.3. Determination of flocculation efficiency After a seven-day culture of microalgae, flocculation process was carried out in 250 mL Erlenmeyer flasks with 200 mL culture. The cultures were immediately shaken using a vortex for 30 s after addition of mother liquid of aluminum chloride (AlCl36H2O, Table 1 Characteristics of swine manure wastewater.
At the end of cultivation, pH in each kind of media was adjusted to 4, 5, 6, 7, 8, 9, 10, 11 and 12, respectively, using solution of 1 N HCl or 1 N NaOH. Then observe and record whether autoflocculation phenomenon occurred after change of pH. Flocculation efficiency at various pH was recorded with 22 mg L1 of Al3+. 2.6. The effect of glucose in algae cultivation process on flocculation Centrifugation and subsequent resuspension was used to remove AOM (Algogenic Organic Matter) generated during metabolic processes of microalgae (Beuckels et al., 2013; Vandamme et al., 2012). To determine the effect of glucose in algae cultivation process on flocculation, after centrifugation of algae culture in BG-11 with glucose, Chlorella cells were resuspended in fresh BG-11 medium without AOM, in centrifugal supernatant of algae culture in BG-11 and in their original supernatant, respectively. Flocculation efficiency in the three groups was compared with addition of 22 mg L1 of Al3+ at pH 11 and 12. 2.7. Experiment design and analysis of the response surfaces Three factors: dilution times of wastewater, pH and concentration of Al3+, which had significant impact on algae flocculation, were selected in RSM (response surface methodology) experiment for optimization for flocculation efficiency. Box–Behnken, a method of response surface design, was used to perform the experimental design for the optimization. The levels of the 3 factors used here are shown in Table 2. The results were analyzed and the optimal values were predicted by Design-Expert 8.0.4. 3. Results and discussion
Parameter
Swine manure wastewater
pH Total nitrogen (mg L1) Ammonia nitrogen (mg L1) Total phosphorous (mg L1) Chemical oxygen demand (mg L1)
7.8 605 520 82 1660
3.1. Flocculation at different flocculants dosage The natural pH after cultivation of Chlorella was 8.9 in BG-11, 5.6 in medium with glucose, 7.8 in medium with tryptone, 9.5 in medium with urea and 8.5 in diluted swine manure wastewater,
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respectively. To determine the influence of flocculants dosage on flocculation, 0–70 mg L1 of Al3+ was added to the five culture systems under natural pH, respectively. As shown in Fig. 1, for the flocculation of microalgae in BG-11 system, only 11 mg L1 of Al3+ could achieve nearly 100% flocculation efficiency. Similarly, 11 mg L1 of Al3+ could achieve nearly 100% flocculation efficiency in both BG-11 with glucose and BG-11 with urea, indicating that glucose or urea did not produce influence on flocculation by AlCl3 under natural pH. However, the minimum concentration of Al3+ needed for complete flocculation in BG-11 with tryptone and in diluted swine manure wastewater had increased to 33 mg L1 and 54 mg L1, respectively. Meanwhile, a linear correlation between flocculation efficiency and addition amount of flocculants was found (R2 = 0.99, p < 0.05) in diluted swine manure wastewater, indicating that amount of flocculants was an important factor affecting the flocculation efficiency of microalgae cells. Compared with flocculation in BG-11 medium, the substantial increase in flocculants dose required in wastewater would raise the cost of microalgae harvest. Thus, it is necessary to understand the effect of components of wastewater on flocculation and to optimize the related conditions. 3.2. Flocculation at different pH 3.2.1. Autoflocculation induced by pH change As displayed in Fig. 2a, for algae culture in BG-11, only at pH 12, autoflocculation emerged with harvesting efficiency of 71%. Meanwhile, for algae culture in diluted swine manure wastewater, when pH was adjusted to 11 or higher (Fig. 2b), aggregation and sedimentation of algae cells occurred rapidly, and the solution became clear and transparent in 2 min. Flocculation efficiency of 98% was obtained only by increasing pH in a very short time. However, for algae cells in BG-11 with organic additives (glucose, urea and tryptone), phenomenon of autoflocculation did not occur, suggesting that the addition of organic matters had significant inhibition on autoflocculation induced by high pH. Most studies have demonstrated that for autoflocculation induced by higher pH, magnesium hydroxide is the key precipitates causing algae flocculation through charge neutralization, bridging, or sweeping flocculation (Besson and Guiraud, 2013; Castrillo et al., 2013; Chen et al., 2013; Elmaleh et al., 1991; Sirin et al., 2012; Smith and Davis, 2012; Wu et al., 2012; Yahi et al.,
1994). Generally, there are a large number of various metal ions in sewage, as to the diluted wastewater used in this research, the concentration of Mg2+ could reach 35 mg L1, which was 5 times of BG-11 (Table 3). Therefore, algae autoflocculation is much easier to occur in the culture system with wastewater. 3.2.2. Aluminum chloride-induced flocculation at different pH It was reported that the minimum pH required for effective flocculation and negative surface charge of Chlorella is 4.0 (Wyatt et al., 2012). Here, flocculation efficiency for algae culture in various systems was determined with addition of 22 mg L1 of Al3+ at different pH (4–12). As shown in Fig. 2, at pH 4, the algae flocculation efficiency for kinds of culture media was only between 10% and 40%. In the range of pH from 5 to 11, the flocculation efficiency for algae culture in BG-11 medium was around 90% (Fig. 2a). For algae culture in diluted swine manure wastewater, flocculation efficiency responded to the change of pH sensitively (Fig. 2b). The maximum flocculation efficiency (98%) was reached at pH 6, and then began to decline rapidly with the increase of pH. The value had fallen to 30% at pH 10. As shown in Fig. 2c, the pattern of flocculation in BG-11 with urea was similar to that in BG-11, indicating that the addition of urea had little influence on flocculation efficiency at different pH. Interestingly, algae flocculation in BG-11 with tryptone had a highly similar variation trend with change of pH as in wastewater (Fig. 2d). Therefore, inhibitory effect on flocculation process in wastewater might result from the complex organic components as tryptone, making flocculation efficiency highly sensitive to pH. For algae culture in BG-11 with glucose (Fig. 2e), when pH was between 5 and 10, flocculation efficiency was maintained at a higher level (>95%), and the flocculation efficiency decreased to 10%, with pH increased to 11 or 12. The reason for disappearance of autoflocculation and decrease of flocculation efficiency might be that the secretion of AOM by algae cells became stronger due to addition of glucose during cultivation process and higher concentrations of AOM made flocculation more difficult. Researches of recent years demonstrate that AOM generated during metabolic processes of microalgae has significant inhibition effect on various flocculation methods including autoflocculation induced by pH increase and flocculation based on metal ions, resulting in an increase of flocculants dosage demand (Beuckels et al., 2013; Vandamme et al., 2012; Wu et al., 2012).
Fig. 1. Flocculation efficiency for algae culture in various systems at different flocculants dosage.
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Fig. 2. Flocculation efficiency for algae culture in various systems at different pH; (a) flocculation efficiency in BG-11; (b) flocculation efficiency in diluted swine manure wastewater; (c) flocculation efficiency in BG-11 with urea; (d) flocculation efficiency in BG-11 with tryptone; (e) flocculation efficiency in BG-11 with glucose.
Table 3 Metal elemental characteristics of BG-11 medium and 5 swine manure wastewater.
1
Mg (mg L ) Ca (mg L1) Al (mg L1) Fe (mg L1) Cu (mg L1) Mn (mg L1) Zn (mg L1) K (mg L1) Na (mg L1)
BG-11
5 swine manure wastewater
7.3 9.8 – 1.2 0.02 0.5 0.05 13.66 414.84
35.3 19.7 0.17 0.14 0.06 0.018 0.09 81.46 35.68
3.3. The effect of glucose in algae cultivation process on flocculation In order to investigate the influence of AOM, algae cells were resuspended in fresh BG-11 medium without AOM (without algal cultivation), in supernatant of algae culture in BG-11 and in supernatant of algae culture in BG-11 with glucose, respectively, and
then flocculation efficiency at pH 11 and 12 in the three groups was determined. As displayed in Fig. 3, at pH 12, only for cells resuspended in fresh BG-11 medium without AOM, autoflocculation emerged with flocculation efficiency of 90%, suggesting that AOM in the supernatant of algae culture in BG-11 and BG-11 with glucose had strong inhibition on autoflocculation of microalgae. When 22 mg L1 of Al3+ was added, at pH 11, flocculation efficiency could reach 99% in the group with fresh BG-11, a lower efficiency of 20% was obtained in the group with supernatant of algae culture in BG-11, and the lowest recovery efficiency of 10% showed the greatest difficulty of flocculation in algae culture with glucose. The data also demonstrate that AOM in the algae culture in BG-11 and BG-11 with glucose had strong inhibition on flocculation of microalgae based on Al3+. Meanwhile, at pH 12, the minimum Al3+ concentration needed for algae culture in BG-11 with glucose had increased to 220 mg L1, which was 2 times of that in the group with supernatant of algae culture in BG-11. Compared with the fresh BG-11 medium without AOM, flocculation was suppressed in the
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Fig. 3. Flocculation for algae cells resuspended in fresh BG-11 medium, in supernatant of algae culture in BG-11 and in supernatant of algae culture in BG-11 with glucose.
supernatant of algae culture in BG-11 suggesting that algae cells had secreted some substances during the autotrophic growth process in BG-11 to inhibit harvest of algae. And this inhibition appeared to be stronger for the culture in BG-11 containing glucose due to the increase of content of AOM. Therefore, the assimilation of organic matters may have a huge impact on subsequent microalgae recovery by affecting metabolic products of cells. Further studies should investigate how flocculation is influenced by organic matters in the culture medium to design flocculation systems for algae culture in different nutrition environment. 3.4. Statistical analysis using RSM In order to achieve high flocculation efficiency, the major factors (dilution times of wastewater, pH value and concentration of Al3+) involved in flocculation of Chlorella in swine manure wastewater were optimized. Table 4 shows the flocculation efficiency corresponding to the combined effect of three components in the specified ranges through Box–Behnken Design of Design-Expert
Table 4 Box–Behken design matrix with experimental and predicted values for flocculation efficiency. Standard order
Run order
Coded level
Flocculation efficiency
A
B
C
Experimental value
Predicted value
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
12 15 3 11 8 2 17 6 1 13 4 5 9 7 16 10 14
1 +1 1 +1 1 +1 1 +1 0 0 0 0 0 0 0 0 0
1 1 +1 +1 0 0 0 0 1 +1 1 +1 0 0 0 0 0
0 0 0 0 1 1 +1 +1 1 1 +1 +1 0 0 0 0 0
0.73 0.96 0.23 0.99 0.23 0.99 0.73 0.99 0.98 0.49 0.98 1.00 0.99 0.99 0.99 0.98 1.00
0.73 0.96 0.22 0.99 0.23 0.98 0.74 0.99 0.98 0.49 0.98 1.00 0.99 0.99 0.99 0.99 0.99
8.0.4. The mathematical regression model for flocculation efficiency fitted in terms of coded factors was as Eq. (2):
Flocculation efficiency ¼ 0:99 þ 0:25 A 0:12 B þ 0:13 C þ 0:13 AB 0:12 AC þ 0:13 BC 0:20 A2 0:067 B2 0:060 C 2
ð2Þ
The results of the second-order response surface model fitting in the form of ANOVA (analysis of variance) are given in Table 5. The R2 coefficient was 0.9998 suggesting that it was a reliable model and that 99.98% of the sample variation of flocculation efficiency was attributed to a high correlation between the independent variables. The relatively high adjusted determination coefficient (R2Adj = 0.9997) accounted for significance of the model. This revealed that the equation was a suitable model to describe the response of experiment. ANOVA showed that all the three factors and the interaction between them had significant effects on the response value. The response surfaces of flocculation efficiency of Chlorella in swine manure wastewater were established according to the model. Fig. 4a and b show the mutual influences of dilution times and pH on flocculation efficiency. In brief, greater degree of dilution of sewage could cause higher flocculation efficiency. Dilution could reduce the concentration of organic compounds effectively relieving the inhibition on flocculation. When the dilution ratio was less than 4-folds, the effect of pH on flocculation efficiency was significant, but when wastewater was diluted more than 8-folds, the influence of pH became greatly weakened. Fig. 4c and d show the mutual influences of dilution times and flocculants dosage on flocculation efficiency. When wastewater was diluted 10-folds, a lower concentration (22 mg L1) of flocculants was enough to achieve complete flocculation (Run 2), but when wastewater was diluted 2-folds, even a higher concentration (66 mg L1) of Al3+ only realized a flocculation efficiency of 73% (Run 17). Fig. 4e and f show the mutual influences of flocculants dosage and pH on flocculation efficiency. Flocculation induced by Al3+ was highly dependent on pH, and flocculants dosage required to achieve complete flocculation would change greatly at different pH (Run 1 and 5). Meanwhile, microalgae biomass productivity was affected dramatically by concentration of organic matters in the wastewater. Previous study (Travieso et al., 2006) reported that in a range of
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B. Zhang, S. Chen / Bioresource Technology 192 (2015) 774–780 Table 5 Analysis of variance for response. Source
Sum of squares
DF
Mean square
F value
p-Value
Significance
Model A B C AB AC BC A2 B2 C2 Residual Lack of fit Pure error Corrected total
1.15 0.5 0.11 0.13 0.071 0.062 0.064 0.16 0.019 0.015 1.743E004 6.609E005 1.082E004 1.15
9 1 1 1 1 1 1 1 1 1 7 3 4 16
0.13 0.50 0.11 0.13 0.071 0.062 0.064 0.16 0.019 0.015 2.490E005 2.203E005 2.705E005 –
5120.56 20020.72 4590.96 5060.68 2871.02 2496.18 2570.55 6514.05 767.84 599.89 – 0.81 – –
<0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 – 0.5490 – –
Significant Significant Significant Significant Significant Significant Significant Significant Significant Significant – Not significant – –
R2 = 0.9998 AdjR2 = 0.9997.
Fig. 4. 3D response surface and contour line of dilution times of wastewater, pH and concentration of Al3+; (a and b) demonstrated the effects of dilution times and pH on flocculation efficiency; (c and d) showed the effects of dilution times and concentration of Al3+ on flocculation efficiency; (e and f) displayed the effects of pH and concentration of Al3+ on flocculation efficiency.
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initial substrate concentration from 250 to 800 mg L1 COD, microalgae concentration increased, nevertheless, when the initial COD concentration increased to 1100 mg L1, the biomass concentration decreased considerably, showing an inhibition phenomenon. Therefore, for cultivation of microalgae with wastewater, dilution times would affect biomass accumulation and flocculation of cells at the same time. It is necessary to determine a suitable scope of dilution for higher algae growth rates with lower flocculation costs. Thus, the scope of factors was defined before the optimal condition was proposed by the model as follows: dilution times, in range (5–7); pH, in range (8.5–9); flocculants dosage (to minimize). Thus, on the basis of these response surfaces, the optimal condition for flocculation efficiency was at pH 8.5, 7-folds of dilution and 52.14 mg L1 of Al3+, and the maximum recovery efficiency was 100%. The accuracy of the model was validated with at least three replicates giving the flocculation efficiency of 0.99 ± 0.01, which concurred with the model prediction. The study demonstrates that microalgae cultivated in swine manure wastewater can be harvested efficiently through flocculation by increasing pH or by adding Al3+. It should be noted that Al3+ would remain as residual metals in the algal biomass residue after extraction of lipids, which may interfere with the use of this residue as animal feed or fertilizer. Thus, for the use of Al3+ in flocculation, further research on related technological process is necessary to ensure that the amount of Al in the biomass residue is lower than the maximum tolerable levels of Al in the feed for poultry and swine (1000 mg/kg) (Mandik et al., 2015; Mineral Tolerance of Animals, 2005). Meanwhile, safer polymers such as chitosan and cationic starch should be extensively studied to establish effective algae flocculation system in swine manure wastewater (Buelna et al., 1990; Masse and Massé, 2010; Vandamme et al., 2010). 4. Conclusions Microalgae cultivated in swine manure wastewater can be harvested efficiently through flocculation by increasing pH or by adding Al3+. The optimal condition for flocculation of C. sorokiniana in swine manure wastewater was at pH 8.5, 7-folds of dilution and 52.14 mg L1 of Al3+, with a recovery efficiency of 100%. Addition of organic matters, such as glucose and tryptone, in microalgae cultivation system could cause significant inhibition on flocculation efficiency by affecting metabolic processes of cells. Acknowledgement This research was supported by the 863 High Technology Program (Grant No. 2013AA065802). References Benemann, J.R., Weissman, J.C., Koopman, B.L., Oswald, W.J., 1977. Energy production by microbial photosynthesis. Nature 268 (5615), 19–23. Besson, A., Guiraud, P., 2013. High-pH-induced flocculation-flotation of the hypersaline microalga Dunaliella salina. Bioresour. Technol. 147, 464–470. Beuckels, A., Depraetere, O., Vandamme, D., Foubert, I., Smolders, E., Muylaert, K., 2013. Influence of organic matter on flocculation of Chlorella vulgaris by calcium phosphate precipitation. Biomass Bioenergy 54, 107–114. Buelna, G., Bhattarai, K.K., de la Noue, J., Taiganides, E.P., 1990. Evaluation of various flocculants for the recovery of algal biomass grown on pig-waste. Biol. Wastes 31, 211–222. Burrell, R.E., Inniss, W.E., Mayfield, C.I., 1984. Development of an optimal heterotrophic growth medium for Chlorella vulgaris. Appl. Microbiol. Biotechnol. 20 (4), 281–283.
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