Introduction

Microalgae have potential as feedstocks for biodiesel (Ratha and Prasanna 2012; Singh and Dhar 2011), besides their value as a source of bioactive molecules, pigments, and nutraceuticals (Gupta et al. 2013); however, most of the technologies currently available are not economically feasible. Intensive cultivation for production of large quantities of microalgal biomass requires an efficient harvesting technique, especially for minimizing the energy consumption of harvesting microalgae (Benemann 1997). One of the major hurdles in large-scale cost-effective production of microalgae is the development of effective downstream process to enable efficient separation of cells from culture broth. Additionally, maintaining their viability and bioactivity prior to use as biofuels or as sources of value added products becomes integral to their utility. Low cell densities, small size of cells, and the electronegative surface of cells makes recovery of microalgal biomass difficult (Brennan and Owende 2010), and efficient biomass harvesting is one of the bottlenecks to develop a cost-effective process (Chen et al. 2011; Georgianna and Mayfield 2012; Larkum et al. 2012). The major techniques presently applied in the harvesting of microalgae include centrifugation, flocculation, filtration, gravity sedimentation, flotation, and electrolytic methods (Chen et al. 2011; Christenson and Sims 2011). The process options and economics of the different methods for the recovery of microalgal biomass have been reviewed by Molina Grima et al. (2003).

Flocculation is a widely used process for microalgae harvesting, which can be induced by inorganic or polymeric flocculants. Despite versatile applications of such flocculants, there are some limitations with their use. Inorganic flocculants (e.g., alum and ferric chloride) are toxic and produce large amount of sludge, while polymeric flocculants are expensive to use. Biopolymers, on the other hand, are cheap, efficient, and environmentally friendly. An efficient flocculation process that can maintain high cell viability could be a method of choice, as it is rapid, inexpensive, and a more simple method for harvesting large quantity of microalgae cells from culture broth, prior to utilization of microalgal biomass for extraction of biochemicals or biofuels. The flocculation efficiency of microalgae for separation of cells from the culture broth was found to be greatly influenced by pH, NaOH, and varying concentrations of inorganic (ferric chloride, aluminium sulphate, and calcium chloride) and organic amendments.

Evaluation of several harvesting methods has shown that flocculation combined with flotation or sedimentation and subsequent further dewatering by centrifugation or filtration is the most promising cost- and energy-efficient alternative (Schenk et al. 2008). Flocculation induced in different ways and chemical flocculation using Zn2+, Al3+, Fe3+, or other flocculants has been studied extensively (e.g., McGarry 1970; Lee et al. 1998; Papazi et al. 2010; Wyatt et al. 2012). Inorganic flocculants such as alum and iron chloride are efficient, but these are required in high doses and result in contamination of the biomass with aluminium or iron (Becker 1994; Wyatt et al. 2012).

Biodegradable organic flocculants may not contaminate the algal biomass and are often required in lower doses (Singh et al. 2000; Rashid et al. 2013). They are based on biopolymers like chitosan, guar gum, alginic acid, or starch. Among these, chitosan has been shown to be an effective flocculant for microalgae (Divakaran and Pillai 2002), which has no apparent toxic effects on the harvested algae (Knuckey et al. 2006). During flocculation, the dispersed microalgal cells aggregate and form larger particles with higher sedimentation rate. Flocculation occurs when solid particles aggregate into large but loose particles resulting from the interaction of the flocculants with the surface charge of the suspended solid and subsequent coalescing of these aggregates into large flocs that settle out of suspension (Knuckey et al. 2006). This process has been extensively used in the industry to remove suspended solids such as in wastewater treatment (Mahvi and Razavi 2005), clarification of drinking water, color removal in paper making industry, and mineral processing (Yoon and Deng 2004).

Several factors such as the flocculant type and its dose, settling time, and culture pH affect the harvesting efficiency of microalgae (Xu et al. 2011). Scattered information is available in published literature regarding the use of different organic/inorganic chemicals or pH or comparative analyses of different unicellular microalgal strains. In the present investigation, we have compared the potential of promising organic and inorganic chemicals (based on available literature) and pH as a flocculant for harvesting of biomass of seven unicellular microalgae strains.

Materials and methods

Seven microalgae, namely Chlamydomonas sp. MCC28, Chlorella sp. MCC29, Chlorococcum sp. MCC30, Botryococcus sp. MCC31, Botryococcus sp. MCC32, Chlorella sp. MCC6, and Chlorella sorokiniana MIC-G5 were obtained from the culture collection of Division of Microbiology and CCUBGA, IARI, New Delhi. These cultures were grown in trays under greenhouse conditions of 25 °C and natural sunlight (61–68 μmol photons m−2 s−1) in a commercial medium containing NPK fertilizers (i.e., urea, single super phosphate, and muriate of potash in appropriate ratio). Batch cultures were maintained by inoculating a known volume of fresh medium every 2 weeks for use as seed culture.

Determination of flocculation efficiency

Inorganic (pH, aluminium sulphate, calcium chloride, and ferric chloride) as well as organic (chitosan, carboxymethyl cellulose, maize starch, cationic starch, potato starch, tapioca starch, yellow dextrin, rice starch, oxidized starch, and pregelatinized starch) amendments were added to the 2-week-old microalgae cultures.

Microalgal suspension (50 mL) in a 250 mL conical flask either alone as reference or with inorganic/organic amendments were kept on shaker at 50 rpm for 10 min at room temperature and allowed to settle for 30 min in an Imhoff cone. After the flocculation of microalgal cells, an aliquot of culture was withdrawn at a level two-thirds from the bottom of the Imhoff cone. The absorbance of remaining suspension of the clear region was measured at 680 nm. The flocculation efficiency was evaluated by comparing this with the absorbance before treatment. The flocculation or harvesting efficiency (%) was calculated using Eq (1) (Harith et al. 2009).

$$ \mathrm{Flocculation}/\mathrm{harvest}\;\mathrm{efficiency}\left(\%\right)=\frac{{\mathrm{C}}_{\mathrm{i}}-{\mathrm{C}}_{\mathrm{f}}}{{\mathrm{C}}_{\mathrm{i}}}\times 100 $$
(1)

Where C i is the absorbance of cells in suspension before treatment and C f is the final absorbance of cells in suspension after flocculation.

Cost economics of organic and inorganic amendments used for harvesting microalgae

The economics of use of all the inorganic as well as organic amendments for harvesting of microalgae was calculated based upon the costs incurred for procurement of the chemicals and related requirements.

Results

Effect of pH on flocculation efficiency

Alkaline pH values of 10 and 11 were found to be most effective in enhancing the flocculation efficiency of the microalgal cultures. pH 10 was optimum for all cultures except for Chlorella sp. MCC29 and Botryococcus sp. MCC32. Modulation of pH did not bring about any significant change in the efficiency of flocculation of Chlorella sp. MCC6 and C. sorokiniana MIC-G5 (Fig. 1a).

Fig. 1
figure 1

Flocculation efficiency of seven microalgal strains (Chlamydomonas sp. MCC28, Chlorella sp. MCC29, Chlorococcum sp. MCC30, Botryococcus sp. MCC31, Botryococcus sp. MCC32, Chlorella sp. MCC6, and Chlorella sorokiniana MIC-G5) as influenced by a different pH levels, b aluminium sulphate concentrations, c calcium chloride concentrations, and d ferric chloride concentrations. Level of significance was determined at P < 0.05. Each value is the mean of three replicates

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Effect of inorganics on flocculation efficiency

Inorganic chemicals viz, aluminium sulphate, calcium chloride, and ferric chloride were more effective than pH adjustment for the recovery of algae. Botryococcus sp. MCC32 formed dense flocs and quickly precipitated during the settling phase of the test at 200 mg L−1. The supernatant was almost clear and the removal rate reached up to 81.9 % with aluminium sulphate. Low concentration (50 mg L−1) of aluminium sulphate was more effective in enhancing flocculation of most of the microalgal cultures except for Botryococcus sp. MCC31 and MCC32, which showed enhanced flocculation at 150 and 200 mg L−1 concentration (Fig. 1b).

Calcium chloride concentrations had a variable effect on flocculation efficiency of microalgae, but flocculation of Chlorella sp. MCC6 and C. sorokiniana MIC-G5 was not influenced significantly (Fig. 1c). The change in ferric chloride concentrations showed a differential effect on the flocculation efficiency of microalgae. Flocculation efficiency of above 80 % was achieved for most of the microalgae except for Botryococcus sp. MCC32 and Chlorella sp. MCC6 (Fig. 1d).

Effect of organics on flocculation efficiency

Higher concentrations of 120 and 150 mg L−1 were more effective in enhancing flocculation of microalgal cultures, except for Chlorella sp. MCC6 and C. sorokiniana MIC-G5, which showed enhanced flocculation at 60 and 10 mg L−1 concentration. Flocculation efficiency was at par using 10 and 120 mg L−1 concentration of chitosan for Chlamydomonas sp. MCC28.

The effect of carboxymethyl cellulose (CMC) concentrations on flocculation efficiency of microalgae was variable. CMC was not effective for most of the microalgal cultures, except for Botryococcus sp. MCC32 and Chlorella sp. MCC6, which showed enhanced flocculation of 82 and 62 % at 10 mg L−1 concentration. Low concentrations of maize starch were not very effective in enhancing flocculation, whereas concentration of 120 mg L−1 showed higher flocculation efficiency for most of the microalgal cultures. Cationic starch concentrations had a variable influence on the flocculation efficiency of microalgae (Fig. 2a).

Fig. 2
figure 2

Dose-response curves for different flocculation methods in seven microalgal strains (Chlamydomonas sp. MCC28, Chlorella sp. MCC29, Chlorococcum sp. MCC30, Botryococcus sp. MCC31, Botryococcus sp. MCC32, Chlorella sp. MCC6, and Chlorella sorokiniana MIC-G5) on flocculation efficiency using a chitosan, CMC, maize starch and cationic starch, b potato starch, tapioca starch and yellow dextrin, and c rice starch, oxidized starch, and pregelitinized starch. Level of significance was determined at P < 0.05. Each value is the mean of three replicates

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A higher concentration (150 mg L−1) of potato starch was more effective in enhancing the flocculation of the microalgal cultures except for Chlamydomonas sp. MCC28, Botryococcus sp. MCC31, and C. sorokiniana MIC-G5, which showed enhanced flocculation at 120 mg L−1 potato starch. Tapioca starch concentrations showed a variable effect on flocculation efficiency.

Yellow dextrin at a concentration of 120 mg L−1 was more effective in enhancing flocculation of Chlamydomonas sp. MCC28, Chlorella sp. MCC29, Botryococcus sp. MCC32, Chlorella sp. MCC6, and C. sorokiniana MIC-G5. However, Chlorococcum sp. MCC30 and Botryococcus sp. MCC31 showed enhanced flocculation at 60 and 30 mg L−1 concentration (Fig. 2b).

Addition of rice starch into microalgal suspension at various concentrations showed a differential effect on the flocculation efficiency. A concentration of 90 mg L−1 was more effective in enhancing flocculation of Chlamydomonas sp. MCC28, Botryococcus sp. MCC31, and Botryococcus sp. MCC32. However, Chlorella sp. MCC29, Chlorococcum sp. MCC30, and Chlorella sp. MCC6 showed enhanced flocculation at 120 mg L−1 concentration. Oxidized starch at a concentration of 150 mg L−1 was most effective in flocculation of the algae, except for Chlamydomonas sp. MCC28 and C. sorokiniana MIC-G5, which showed enhanced flocculation at 120 and 90 mg L−1, respectively. Lower concentration of 10 mg L−1 of pregelatinized starch was more effective in enhancing flocculation of Chlorococcum sp. MCC30 and Chlorella sp. MCC6. In Botryococcus sp. MCC31, maximum flocculation was observed at 90 mg L−1 concentration; while for the remaining microalgal suspensions, a concentration of 120 mg L−1 exhibited enhanced flocculation (Fig. 2c).

Cost economics

The cost of all the inorganic and organic amendments was obtained from local suppliers and calculated. Comparison of cost and the efficiency indicated that rice starch was more economically feasible for its utilization as a flocculant for efficient harvesting of microalgae (Table 1). The cost of inorganic chemicals, CMC, and chitosan varied on a per kilogram basis. The price of other organic chemicals like different starches and yellow dextrin was only US$0.7 on a per kilogram basis, while the cost for rice starch was only US$0.1 per 1,000 L culture harvested. The price of ferric chloride was US$0.7 per 1,000 L. In general, the price of inorganic amendments was 20 times more than organic amendments per liter of suspension.

Table 1 Cost economics of inorganic and organic amendments used for harvesting microalgae
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Discussion

Mass production technologies for microalgae require efficient methods for harvesting, which determine the economics of the available process options (Molina Grima et al. 2003). The nature of flocculants and their concentrations play a critical role in flocculation process, as these can influence the extent as well as the rate of flocculation. Therefore, preliminary experiments were undertaken to determine the effects of pH, salinity, and different concentrations of various organic and inorganic amendments on the flocculation of 2-week-old cultures of selected microalgae.

Buelna et al. (1990) reported that higher pH levels were effective in algae sedimentation. Millamena et al. (1990) also observed precipitation, when culture pH was raised to 10. Wu et al. (2012) hypothesized that the mechanism underlying flocculation mediated by high pH may be related to the presence of magnesium ions in the growth medium, which is hydrolyzed to form magnesium hydroxide precipitate, which coagulates cells by sweeping flocculation and charge neutralization. The present investigation is in agreement with the previous studies of Blanchemain et al. (1994), Yahi et al. (1994), and Vandamme et al. (2012) on different algae. Flocculation induced by high pH is considered as a potentially useful method to preconcentrate freshwater microalgal biomass during harvesting (Vandamme et al. 2012). However, as microalgae generally carry a negative surface charge, an increase in pH will cause an increase in surface charge rather than a decrease, which might be the possible cause for flocculation induced by high pH. The use of flocculation induced by high pH for harvesting microalgae may have an additional advantage in that the high pH effectively sterilizes the microalgal biomass as well as the process water. This may be advantageous when microalgae are used in wastewater treatment, as the high pH may kill pathogenic microorganisms (Semerjian and Ayoub 2003). It has been observed that, irrespective of the method, an increase in pH within the range of 8.5 to 10.5 allows the recovery of microalgae such as Phaeodactylum tricornutum (Sirin et al. 2011), Anabaena marina (González-López et al. 2009), or Dunaliella tertiolecta (Horiuchi et al. 2003) with biomass recovery values higher than 90 %.

Lee et al. (1998) also reported that a high pH of 11 was more effective for the flocculation of the green alga Botryococcus braunii than using aluminium sulfate as a flocculant.

Although in the present study, the growth medium was discarded after the harvesting of biomass, since no flocculants were used and medium was not contaminated, the growth medium after flocculation can be reused by neutralizing pH and supplementing with nutrients. The recycling of flocculated medium can minimize the cost of nutrients and the demand for water, besides being useful for growth and value addition purposes (Castrillo et al. 2013). The flocculated microalgal cells exhibit growth which demonstrates that no cell lysis occurs during the flocculation by pH increase, and the function and structure of the photosynthetic apparatus are not affected (Wu et al. 2012).

The flocculation mechanism depends on the nature of the cell and the charge of the flocculant. Numerous chemical coagulants or flocculants have been tested in literature (e.g., McGarry 1970; Papazi et al. 2010). Metal salts (aluminium sulphate, ferric chloride, etc.) are generally preferred because they lead to improved harvesting efficiency.

Alum, which has proven to be an efficient flocculant for other species of algae (Clasen et al. 2000), showed an increase in turbidity of algal cultures at high concentration of 200 mg L−1. Avnimelech et al. (1982) reported that the variable flocculation potential of different algae depends on the composition and properties of the cell wall, the extent and type of excretions, physiological conditions, age, and other factors. The biochemical composition of microalgae also changed when culture parameters were modified (Brown et al. 1996).

Granados et al. (2012) observed that polyelectrolytes at low doses of 2–2.5 mg g−1 biomass were most efficient for recovery of biomass, but this may not be economically viable, as these products are costly and availability is restricted. Multivalent aluminium and iron metal salts have been widely used to flocculate algal biomass (Bernhardt and Clasen 1994; Papazi et al. 2010), and di- or trivalent cationic flocculants such as CaCl2 and FeCl3 were found to be more effective than the monovalent cationic flocculants.

The results of ferric chloride as flocculant compared well with the reported efficiencies of over 90 % using ferric chloride for the flocculation of marine algae (Sukenik et al. 1988). For any given algae species, effective flocculation with FeCl3 may be obtained, if the conditions of negative surface charge and sufficient ferric chloride concentration are met. Since different algae species vary in their concentrations of functional groups on the algal surface, the minimum amount of FeCl3 required for effective flocculation may differ (Wyatt et al. 2012). Kwon et al. (1996) reported that the flocculating activity of a bioflocculant from Pestalotiopsis sp. was highest on addition of cationic solutions, especially 8 mM CaCl2 or FeCl3. It was observed that such concentrations of Fe3+ ions contribute to microalgal floc formation and results in a separation efficiency of 62 % after 5 min of sedimentation (Sun et al. 1998).

In general, a minimum amount of ferric chloride is required to promote coagulation of algae cells. In solution, ferric chloride forms a positively charged hydroxide precipitate (at pH <8) which associates with the negative algae cell surface. The ferric hydroxide precipitates form bridges between algae cells which bind them together into flocs. At low algal concentrations, the amount of FeCl3 required to achieve coagulation increases linearly with algal concentration. However, at higher concentrations, the minimum amount of FeCl3 required for flocculation becomes independent of algal concentration, as the dominant mechanism changes from electrostatic bridging to sweep flocculation by large coagulated algal flocs (Wyatt et al. 2012). Inorganic flocculants, including alum and iron chloride, also lead to contamination of growth medium with aluminium or iron (Oh et al. 2001); however, they may be useful in treatment of wastewaters, wherein the spent water after mass multiplication of microalgae can be passed through columns to remove the Fe ions and then reused for growth purposes.

In the last few decades, chitosan has emerged as a favorable flocculating agent as a cationic polysaccharide in the harvesting of microalgae. Compared with other commercial flocculants, it presents various advantages, including producing larger flocs (Zeng et al. 2008), resulting in faster sedimentation rates, providing a clearer residual solution after harvesting for higher flocculation efficiency, and being nontoxic (Knuckey et al. 2006), which makes it possible to reuse the residual solution again to grow microalgae.

Earlier studies using chitosan revealed similar differential response on flocculation efficiency of microalgae with change in concentrations with an efficiency of 75 % having been reported for Lubian (1989). Rashid and co-workers (2013) found that 120 mg L−1 of chitosan showed the highest flocculation efficiency (within 3 min), and pH of 6 was optimal for Chlorella vulgaris. Suspensions of unicellular microalgae are known to be stabilized by the negative surface charge of the algal cells and organic flocculants, viz starches which can induce flocculation of negatively charged particles through bridging and/or patch charge neutralization (Sharma et al. 2006; Bratby 2006). Cationic starch can be used as a cheaper and more widely available alternative to chitosan (Vandamme et al. 2010). Organic polymer/polyelectrolyte flocculants, such as cationic starch and chitosan, have no toxic effects and do not contaminate growth medium. They are, however, high-value products with bulk costs of about US$2 kg−1 for chitosan (Rashid et al. 2013) and US$1–3 kg−1 for cationic starch (Vandamme et al. 2010), and therefore seem to hold promise for their large-scale use.

In conclusion, among the inorganic flocculants, ferric chloride was the most promising, with a flocculation efficiency of 87 % at 600 μM concentration; while among the organics, rice starch was most effective in terms of flocculation, with an efficiency of about 86 % at 90 mg L−1 concentration. Enhanced sedimentation rate was the obvious advantage of flocculation, which significantly reduced the sedimentation time. The comparative costs involved revealed that although flocculation efficiency with inorganic flocculant, viz FeCl3, was at par with organic flocculant, viz rice starch (87.4 compared to 86.8 %), organic flocculants are economically more feasible, besides being environmentally safe options. Other organic flocculants such as maize starch (81.7 %) and potato starch (82.2 %) also showed promise in terms of their flocculation efficiency. In our experiments, microalgae which were harvested by high pH induced increased flocculation efficiency. Moreover, pH does not contaminate growth medium, which can be recycled to reduce the cost and the demand for water at larger scale. Thereby, the pH-induced flocculation method can be a suitable option for economic production from algae to biodiesel.