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Flocculation: An effective way to harvest microalgae for biodiesel production
Journal of Environmental Chemical Engineering 7 (2019) 103221
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Flocculation: An effective way to harvest microalgae for biodiesel production
T
M. Mubaraka, , A. Shaijab, T.V Suchithrac ⁎
a
Department of Mechanical Engineering, Karpagam College of Engineering, Coimbatore, 641032, India Department of Mechanical Engineering, National Institute of Technology Calicut, Kerala, 673601, India c School of Biotechnology, National Institute of Technology Calicut, Kerala, 673601, India b
ARTICLE INFO
ABSTRACT
Keywords: Biodiesel Autoflocculation Lipid content Microalgae Electrolytic flocculation
Microalgae are considered the most suitable feedstock for biodiesel production due to their higher areal productivity and lipid content. The economical production of biofuels from microalgae is important. This paper is a review of different techniques used for flocculating microalgae such as use of inorganic salts, organic salts, bioflocculants, autoflocculation and electrolytic flocculation for harvesting microalgae for biodiesel production. It was found that the usage of natural flocculants for harvesting microalgae eliminates the toxicity of biomass harvested but the cost of flocculant makes it uneconomical for large scale applications. The economic method of harvesting large quantity of microalgae still requires further research and development.
1. Introduction The production of biodiesel from microalgae are promising due to the advantages such as very short harvesting life capable of continuous harvesting year around, higher photosynthetic yield with superior lipid productivity with major portion of lipid as neutral which is more suitable for biodiesel production [1]. Microalgae can double their weight with respect to biomass within 24 h [2]. The key processes involved in biofuels production from microalgae biomass are cultivation, harvest, oil extraction, and conversion of algal lipids into advanced biodiesel [3,4]. Among these processes the harvesting and oil extraction contributes 60% of the total biodiesel production cost. The harvesting and dewatering of microalgae is a cost determining steps in converting algal biomass into biodiesel [5]. The cost involved in microalgae harvest is as high as 20% of the total production cost of biodiesel and varies according to the type of harvest methods used [6]. The major challenge in microalgae harvesting is due to the small size of cell and their low biomass concentration in the media which ranges from 0.5 to 1.5 g/l [7]. The common techniques currently employed in microalgae harvesting and recovery are centrifugation, coagulation, electrophoresis technique, flocculation, filtration, flotation, gravity sedimentation and magnetic separation [6,8–10]. As shown in Table 1 the flocculation method of harvesting microalgae were more suitable due to their reduced cost and easiness for execution and it allows large quantities of culture with reduced energy
⁎
requirement. Besides, this method has more option for selection of flocculants i.e, the agent which flocculates the cells, depending upon the type of flocculation methods. This method can be induced through different forms of flocculants such as inorganic, organic, polymeric [17] or through processes such as auto-flocculation [18], electro flocculation [19] or microbial flocculation [14]. The flocculation of microalgae can be performed in different ways such as chemical flocculation, bioflocculation, and magnetic nanoparticles. This review paper discusses the different flocculation techniques used for harvesting microalgae such as inorganic flocculants, organic flocculants which can be either by using natural organic flocculants or synthetic organic flocculants, bioflocculation, autoflocculation and electrolytic flocculation for biodiesel production and their efficiencies and impact on lipid extracted. 2. Flocculation Flocculation can be defined as the process in which where a solute particle in a solution forms aggregates called floc [8]. The flocculation of microalgae can occur in four mechanisms acting alone or in combination; (i) Phenomenon of charge neutralization in which charged ions, polymers or colloids strongly absorb on the opposite charged surface of the microalgae cells, (ii) The electrostatic patch mechanism in which a charged polymer binds to microalgae cells with opposite charge resulting in patches of opposite charge on the microalgae surface. The
Corresponding author. E-mail address: [email protected] (M. Mubarak).
https://doi.org/10.1016/j.jece.2019.103221 Received 27 March 2019; Received in revised form 16 June 2019; Accepted 17 June 2019 Available online 18 June 2019 2213-3437/ © 2019 Elsevier Ltd. All rights reserved.
Journal of Environmental Chemical Engineering 7 (2019) 103221
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Table 1 Advantages and disadvantages of different harvesting methods for harvesting microalgae. Method used
Advantages
Disadvantages
References
Centrifugation
Less time required,
[11,12]
Coagulation Electrophoresis
Allows treatment of large quantities of microalgae cultures, Selectivity, energy efficiency, and cost effectiveness
Flotation
More effective and beneficial than sedimentation
Magnetic separation Gravity sedimentation
Low running cost, energy saving and simple operation Low energy requirement
Filtration
Selective filtration is possible
Flocculation
Cost and energy required are less, large quantity of culture can be used, Can use for wide variety of microalgae
High energy consuming, causes damage of cells due to gravitational and shear forces, less quantity of culture only used, requires costly equipment Expensive, at high dosages causes contamination problem. Fouling of the cathodes and systems getting damaged by high temperature, scale up problem Requires high particulate size of the algal biomass, oversized bubbles break up the floc, expensive process Fabrication was complex and expensive, practical limitability Requires large amount of land, very slow process, cell composition may change Requires periodical membrane washing or replacement, high cost, species specific Some types of flocculants like chitosan can be expensive, at high dosages causes contamination
microalgae cells connect with each other through patches of opposite charge causing flocculation, (iii) the phenomenon of bridging in which polymers or charged colloids simultaneously bind to the surface of two different microalgae cells to form a bridge between them brings the microalgae cells together, (iv) the massive precipitation of mineral present in the cultivation media causes the microalgae cells to get entrapped know as sweeping flocculation [20]. An ideal flocculant should be inexpensive, nontoxic and effective in low concentration [21]. The microalgae carry negative charge that prevents the aggregation of cells in suspension. The surface charge of microalgae can be minimized or neutralized by adding suitable flocculants such as multivalent cations and cationic polymers in the broth.
[12] [13,14] [13,14] [9] [8] [8] [12,15,16]
performed using Aluminum sulfate (Al2(SO4)3) [29]. The flocculation of Chlroella vulgaris and Phaeodactylum tricornutum was performed using magnesium hydroxide in which precipitates were formed at high pH of 10–10.5 [30]. The flocculation of Chlamydomonas reinhardtii was performed using inorganic flocculants such as aluminum sulfate, ferric chloride, polyalluminium chloride, plyaluminium chlorosulfate and cationic polyacrylamide and reported 95% efficiency with cationic polyacrylamide [31]. A new flocculant, combination of polymer and FeCl3 was introduced for flocculating Chlorella vulgaris GKV1 increased the sedimentation time of 80% in the first five minutes [32]. The metal salts such as NaOH, Ca(OH)2 and FeCl3 were used for the flocculation of Dunaliella salina and reported that FeCl3 as flocculant is most promising one [33]. A continuous flocculation of Nannochloropsis salina was performed with Al2(SO4)3) as flocculant and obtained efficiency above 85% [34]. The slurries of microalgae such as Chlorella vulgaris, Choricystis minor, Cylindrotheca fusiformis, Neochloris sp., Nannochloropsis salina was effectively flocculated using aluminium sulfate and ferric chloride as flocculants and gained 95% efficiency [35]. The flocculation of Scenedesmus sp. grown in fresh media and waste water was performed using ferric chloride as flocculant under reduced pH below 6.5 improved the harvesting efficiency [36]. The flocculation of microalgae, Chlorella sp. KR-1 was achieved with 98% efficiency using metal coagulant (Fe2(SO4)3) and sulfuric acid (H2SO4) [37]. Cost-efficient harvesting of microalgae is a major challenge for large-scale biomass production. The optimization of flocculation of Scenedesmus obliquus grown in wastewater was performed using ferric sulphate as flocculant [38]. As per the opinion of authors flocculation using iron and alum is most effective with better efficiency for harvesting mass culture of microalgae for biofuel production.
3. Methods used for flocculation 3.1. Inorganic flocculation The mechanism involved by the usage of inorganic flocculants is that the charge neutralization due to the absorption of equal amount of opposite charge by microalgae cell. The pH of the medium should be low to form cationic hydrolysis products for inducing flocculation. Examples of such inorganic flocculants are alum, ammonia, FeCl3, Fe2 (SO4)3, polyaluminium chloride etc. Ammonia is the first flocculant used for harvesting of microalgae like Chlorella sorokiniana (freshwater), Nannochloropsis oculata (marine), Dunaliella with an efficiency of 99% [22]. FeCl3 and Fe2(SO4)3 are also suitable for 98% efficient flocculation of Chlorella sorokiniana, Scenedesmus obliquus, Chlorococcum sp and a wild type Chlorella in symbiosis with bacteria [23] 99% and flocculated media were reused for cultivation. A minimum concentration of ferric chloride is required to overcome the electrostatic stabilization of negative surface charge of algae. As a flocculant for Chlorella zofingiensis, FeCl3 possess a linear proportionality with a dosage of flocculant with biomass concentration and pH [24]. The significant effect of 143 mg/L of FeCl3 and pH 8.1 of Chlorella has proved by response surface methodology (RSM) [25]. Similar to FeCl3, alum also has a linear correlation between dosage of flocculant, biomass concentration and pH suitable for harvesting Scenedesmus sp cultivated in open pond achieved flocculation efficiency of 90% at pH 6 [26]. The usage of alum at different dosages for flocculating Nannochloropsis salina with a biomass concentration 15–20 g/L achieved an efficiency of 79–99% and measurement of lipids and fatty acids were done using differential pulse absorptive voltametry [27]. Inorganic flocculant polyaluminium chloride (PACl) were used by considering effect of dosage, biomass concentration, lipid content, pH, mixing speed and time for harvesting Chlorella vulgaris ESP-31and gave more harvesting efficiency with reduced cost than chitosan [28]. The flocculation of Scenedesmus spp., Chlamydomonas reinhardtii,and Schizochytriumlimacinum cells at three different concentrations were
3.2. Organic flocculation The organic flocculation is based on the method of neutralization of surface charge on the algal cells by forming physical linkage between algal cells and organic flocculants. The organic flocculants are either natural organic flocculants or synthetic organic flocculants. The natural organic flocculants consist of Moringa oleifera. The synthetic organic flocculants consist of polyelectrolytes. 3.2.1. Natural organic flocculants 3.2.1.1. Moringa oleifera as flocculant. Moringa oleifera seed contain active peptides of molecular weight ranging from 6 to 20 kDa, with pH value between 9 and 10 or higher and can in saline environment it can act as a polyelectrolyte to induce flocculation [39]. The flocculation potential of M. oleifera has been confirmed with the jar test [40–42]. According to Teixeria et al. [43], the flocculation efficiency can be increased by increasing the pH for harvesting Chlorella vulgaris by using M. oleifera seeds. 2
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were 10 ppm, 20 min, 150 rpm, 20 min respectively and arrived an harvesting efficiency of 99% for Chlorella sp using jar test [54]. Usage of multiple dosage 1:300 w/w of chitosan: biomass ratio and 6.3–6.8 of pH range is used for Chlorella sorokiniana cultivated in waste water gave harvesting efficiency of above 99% [55]. A bioflocculant from Cobetia marina L03 was used for flocculation of Chlorella vulgaris with an efficiency of 92.7% and observed that bioflocculant was stable at wide range of pH and temperature [56]. The fungal assisted flocculation using co-cultivation of filamentous fungi with freshwater microalgae, Chlorella protothecoides and marine microalgae, Tetraselmis suecica were performed for the efficient and cost effective harvesting [57]. The harvesting of microalgae such as Chlorophyta species, Chlorella vulgaris, Nannochloropsis granulate and Dunaliella salina was performed using dinoflagellate Heterocapsa circularisquama as flocculant [58]. Chitosan flocculation worked generally very well for freshwater microalgae, but not for marine species [59]. The co-culture of microalgae with bacteria or flocculating microalgae with the use of bacteria was already reported for the purpose of flocculation [60,61]. Microbial bioflocculant has been widely used for wastewater treatment [62–64]. The harvesting of microalgae using microbes such as bacteria were also reported [65,66]. The bioflocculant produced by Paenibacillus polymyxa AM49 were used with inorganic flocculants for harvesting high density cultures of Scenedesmus sp. and the reuse of flocculated medium showed 8% decrease in growth and improved by the addition of 20%, 50% fresh BG11 medium [66]. Wan et al. [67] reported 90% of bioflocculation efficiency of microalgae Nannochloropsis oceanic by using the bacterial strain Solibacillus silvestris from activated sludge and the necessity of culture media without any effect on the growth. The presence of bacterial extracellular substances enhanced the flocculation efficiency of Chlorella vulgaris cultured in the presence of bacteria such as Flavobacterium sp., Terrimonas sp., Sphingobacterium sp., Rhizobium sp. and Hyphomonas sp. [68]. The feasibility of using microbial flocculant poly c-glutamic acid (c-PGA) for harvesting marine Chlorella vulgaris and fresh water Chlorella protothecoides were optimized using response surface methodology [69].
3.2.2. Synthetic organic flocculants 3.2.2.1. Polyelectrolyte flocculation. Polyelectrolyte flocculants are polymeric flocculants that includes cationic, non-ionic and anionic polymers, in addition to bentonites and active carbon generally used in waste water treatment [44]. The cationic polyelectrolytes generally used are poly DADMAC (PDADMAC), polymer from epichlorohydrin and dimethylamine (ECH/DMA), cationic Polyacrylamide (CPAM) and anionic polyelectrolyte are anionic polyacrylamide (APAM), polystyrene sulfonic acid (PSSA), and 2 acrylamido 2 methyl propane sulfonic acid (AAMPSA) [45]. Their mechanism of flocculation is due to the combination of charge neutralization and particle bridging and it depends on charge density and chain length of the polymer. The polymer molecule attached on the microalgae cell surface due to electrostatic or chemical forces causes the aggregation of cells [8]. This makes the polyelectrolytes more advantageous. The extent of aggregation of cells by the polymer depends on the key polymer characteristics such as charge, molecular weight and concentration [46]. 2–2.5 mg/g of polyelectrolyte Actipol range of flocculants such as EM1, EM16, EM22 and FB1 is sufficient for comparatively efficient recovery of freshwater microalgae (Muriellopsis sp, C. vulgaris, C. fusca, S. subspicatus and Scenedesmus sp) than metal salts like iron chloride, aluminium sulphate, iron sulphate and chitosan [44]. 3.3. Bioflocculation The spontaneous flocculation due to the presence of extracellular polymeric substances in the medium of microalgae and using chitosan is named as bioflocculation [47]. Some naturally bioflocculating microalgae can be mixed with other species of microalgae to induce flocculation [48]. As a bioflocculant, chitosan biopolymer have superior technical and environmental performance in the flocculation of Neochloris oleoabundans compared to ferric sulfate and alum [49]. Similar to chemical flocculants, the efficiency of bioflocculant is influenced by dosage and pH [50]. For example, chitosan is effective at pH range of 4–9 for flocculating Spirulina, Oscillatoria, Chlorella, and one brackish alga, Synechocystis [50]. Xu et al. [51] achieved more than 99% of harvesting efficiency of Chlorella sorokiniana using chitosan (approximately 10 mg/g algal dry weight) with cultivation media pH below 7. Table 2 shows the comparison of inorganic flocculants with recovery efficiency. Bioflocculation followed by centrifugation were used with reduced energy demand for harvesting Chlorella vulgaris (SAG211-11b), S. obliquus (SAG276-3a), A. falcatus (SAG202-9) and Ettlia texensis (SAG79.80) by considering the effect of ratio between sedimentation rate and energy demand for harvesting [52]. Table 3 shows the comparison of harvesting efficiency of microalgae with different bioflocculants. The usage of nano-chitosan (chitosan cross linked with sodium triphosphate) for flocculating Nannochloropsis sp. is reduced the consumption of flocculant by 40% with increased flocculation efficiency [53]. Chitosan dissolved in hydrochloric acid showed highest flocculation efficiency for flocculating Chlorella vulgaris at exponential and stationary growth phase compared to the usage of chitosan dissolved in citric, phosphoric, nitric acids [15]. The optimized conditions such as chitosan dosage, mixing time, mixing speed, sedimentation time
3.4. Organoclay flocculation The organoclays are basically formed from 3-aminopropyl functionalized magnesium phyllosilicate and consists of cationic metals Mg2+,Ca2+, Al3+, and Fe3+ in its center which are sandwiched organo functional entities that contain, by sol–gel reaction under ambient conditions, a high density of amino sites. In aqueous solution, organoclay particles are delaminated by protonated amine groups, as the result of which forms positively charged nano-clusters [70]. The advantages of using aminoclays for flocculation were due to its fast harvesting yield for a wide variety of microalgae species without affecting the pH of the microalgae feedstock [71]. The harvesting efficiency of about 100% were achieved with the use of cationic charged aluminum and magnesium backboned organoclays in aqueous form for harvesting 0.6 g/L concentrated Chlorella sp. KR -1 [72]. The usage of acids with aminoclay can induce flocculation with higher efficiency. Lee et al. [73] reported harvesting efficiency of approximately 100% using aminoclay induced humic acid for flocculating Chlorella sp. with a dosage of 5 g/L.
Table 2 Comparison of inorganic flocculants with recovery efficiency. Inorganic flocculant
Microalgae
Dosage
Recovery efficiency, %
References
Alum Ammonia
Nannochloropsis salina Chlorella sorokiniana Nannochloropsis oculata Dunaliella` Mixture of Chlorella sorokiniana, Scenedesmus obliquus, Chlorococcum sp. and a wild type Chlorella Chlorella sp
10.8 mg /L 57.31 mmol/L
79-99 99
[27] [22]
150–250 mg/L
66-98
[23]
143 mg/L
56.54
[25]
FeCl3 Fe2(SO4)3 Aluminium sulfate, ferric chloride
3
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Table 3 Comparison of different bioflocculants used and its recovery efficiency. Bioflocculant used
Algae species
Quantity used
Chitosan with Citric acid Nitric acid Phosphoric Hydrochloric Combination of CaCl2, FeCl3 and Paenibacillus polymyxa AM49.
Chlorella vulgaris
30 mg/L
Scenedesmus sp
8.5 mM CaCl2 0.2 mM FeCl3 1% P. polymyxa AM49
Recovery efficiency
Reference [15]
5.1 54.5 69.3 84.5 95
[66]
3.5. Auto flocculation
3.6. Electrolytic flocculation
The mechanism behind auto flocculation, increased pH due to the presence of photosynthetic CO2 consumption and precipitation of inorganic precipitates. This precipitation causes the formation of aggregates by excreted organic macromolecules that inhibit the release of microalgae daughter cells. When pH of the microalgae increases above 9, the flocculation occurs spontaneously [74]. The principle behind flocculation by increased pH of growth media due to the presence of Mg2+ in the growth medium hydrolyzed to form magnesium hydroxide precipitate which causes coagulation of micro algal cells by sweeping flocculation and charge neutralization. The effect of increased pH (8.610.6) for harvesting freshwater microalgae (Chlorella vulgaris, Scenedesmus sp., Chlorococcum sp) with low/medium biomass concentration. and marine microalgae (Nannochloropsis oculata, Phaeodactylum tricornutum) yielded better harvesting efficiency with the possibility of reuse of flocculated medium by supplementing nutrients for cultivation [75]. The usage of calcium hydroxide was most cost efficient for increasing pH to 11 in flocculating Chlorella vulgaris compared to other bases such as sodium hydroxide, potassium hydroxide, magnesium hydroxide and sodium carbonate [7]. The naturally available ions such as Mg2+,Ca2+, and CO32− in brackish water were used for harvesting Chlorella vulgaris gave settling rate of 100 cm/h more than the unflocculated cells [76]. The presence of algal organic matter and the increased biomass concentration of Chlorella vulgaris reduced flocculation efficiency with pH above 8.5 using precipitation of calcium phosphate. The mild acidification method was used to remove calcium phosphate culture from the media [77]. The cell wall associated with polysaccharides act as biopolymer were responsible for self-flocculation of S. obliquus AS-6-1 [78]. The bases such as NaOH and Ca(OH)2 can be used for pH induced flocculation of Scenedesmus obliquus and Chlorella vulgaris. The growth rate of reused flocculated media gave 1.79 times higher than fresh media which saves water and nutrients for cultivation [79]. A recovery efficiency of 90% and concentration factor of 20 were achieved by adding NaOH continuously using syringe for harvesting of marine microalgae Dunaliella salina. The flow rate of NaOH has no effect on recovery efficiency due to the auto flocculation mechanism with precipitation of magnesium ions present in the cultivation media [80]. The polysaccharides present in the unique cell wall of C.vulgaris JSC-7 induced flocculation and it does not interfere with the quality of harvested algae [81]. The autoflocculation of Chlorella vulgaris JSC-7 was used for removing Zn2+ and Cd2+ using surface adsorption from contaminated waste water [82]. Another study used self-flocculating microalgae, S. obliquus FSP-3 as biosorbent for removing heavy metal ions from waste water [83].
The electrolytic flocculation does not require any flocculants. The principle behind electrolytic flocculation is based on the movement of negatively charged algae towards anode loses their charge and forms aggregates [8]. The harvesting efficiency of 95% with a current consumption of 0.3 kW h/m3 were obtained with algal suspensions containing blue green algae such as Coelosphaerium sp. and Aphanizomenon sp., green algae (Closterium sp., Pediastrum sp., Cryptomonas sp. and Staurastrum sp.) and most of all diatoms (Asterionella sp., Cyclotella sp. and Melosira sp.) using electrolytic flocculation [84]. The electrodes subjected to polarity exchange in the middle of the harvesting process improved harvesting efficiency in continuous electrolytic microalgae (CEM) harvesting system under optimized conditions such as applied current, initial pH and mechanical mixing [85]. The harvesting efficiency of Nannochloris oculata (KMMCC-16) was better with aluminum and dimensionally stable anode (Al–DSA) compared to Al-platinum (AlPt) electrode in used continuous electrolytic microalgae (CEM) harvesting system. The harvesting efficiency were improved by optimizing the timing of polarity exchange [86]. The incorporation of mixing and settling of marine microalgae Tetraselmis sp has no effect on electroflocculation method while electrode separation has effect on harvesting efficiency [87]. The cell density and coagulant dosage has significant effect on harvesting efficiency of Nannochloris oculata in inorganic electrolytic flocculation with AlCl3 compared to other factors such as ionic strength, media pH and cell surface charge [88]. The usage of pulsed or continuous current in electro-coagulation- flocculation (ECF) reactor reduced the energy input and treatment time drastically with harvesting efficiency 90% for harvesting Nannochloropsis sp [89]. The microalgae, Chlorella vulgaris was used electro-coagulation flocculation using aluminum and iron electrodes [90]. The electrolytic flocculation of D. salina was performed using aluminium air battery and during the process the along with separation energy also produced which is usable for charging the battery [91]. Table 4 compares the flocculation efficiency of microalgae with electrolytic flocculation method. The influence of algal organic matter in the flocculation efficiency was reported. The presence of algal organic matter (AOM) inhibits flocculation efficiency and caused more dosage of flocculants for harvesting Chlorella vulgaris using methods such as aluminum sulfate, chitosan, cationic starch, pH induced flocculation and electro-coagulation flocculation [92]. The advantages and disadvantages of different flocculation methods were shown in Table 5.
Table 4 Comparison of electrolytic flocculation of microalgae. Method
Type of algae used
Recovery efficiency
References
Electrolytic flocculation Continuous electrolytic Electrolytic Coagulation flocculation
Coelosphaerium sp. Aphanizomenon sp. Closterium sp., Pediastrum sp. Cryptomonas sp. and Staurastrum sp. Nannochloropsis oculata Nannochloropsis sp.
95% 99.9% 95%
[84] [86] [89]
4
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Table 5 Comparison of advantages and disadvantages of different flocculation methods. Method
Advantages
Disadvantages
References
Electrolytic flocculation
No risk of contamination of algae with chemical flocculant Less expensive Rapid method of flocculation Environmental friendly, no contamination problem
Requires reactor, electrodes which may cost higher initial investment, scale up problem, electrodes need to replace periodically
[84]
Contamination of harvested biomass Higher production cost limit scale up, at higher pH ineffective
[27] [50]
Inorganic flocculation Organic flocculation
3.7. Determination of flocculation effectiveness
depreciation time A (year), Ce the energy costs, Cc the costs for consumables, Cl the costs for labour and Y total is the total produced biomass in a year. The above equations can be used for flocculation method of harvesting microalgae by considering the cost of flocculants and other capital investments.
The effectiveness of flocculation can be determined by using recovery efficiency and the concentration factor during microalgal separation [60]. The recovery efficiency or algal removal efficiency is the ratio of mass of cells recovered to the total mass of the cells [60]. The flocculation experiments generally uses narrow necked wide bodied geometrical forms for faster settling of culture than cylindrical reservoirs [93]. The flocculation activity of particular algae was measured by calculating algae removal efficiency or harvesting efficiency and concentration factor. The concentration factor provides quantification measurement of how many folds algae cells were concentrated from an initial volume. Some of the work were reported with algae removal efficiency by measuring the optical density of the culture at 680 nm before and after flocculation [7].
4. Conclusions The development of an efficient flocculation method for harvesting microalgae can reduce the cost and energy in biodiesel production from microalgae. The numerous studies have already investigated various methods for flocculating microalgae. The flocculation of microalgae using chemicals (organic) results in the contamination of biomass and it can be eliminated by using naturally available polymers as flocculants. The usage of chitosan as flocculant is most promising but the for large scale applications the cost of chitosan makes it unfeasible. The electrolytic flocculation is characterized as the flocculant free method of harvesting microalgae but it requires the reactor and electrodes. The usage of bacteria as flocculant for microalgae harvesting were getting more attention due to its environmental friendly nature. The algal organic matter influences the flocculation efficiency of the microalgae. Even though number of flocculation methods available there is no more flocculation methods are used in large scale production of biodiesel from microalgae explores the requirement of further research to make the economic harvesting of microalgae for biodiesel production.
3.8. Effect of flocculants on quality of lipid extracted A few works were reported the effect of flocculants on the quality of lipid extracted. The reduced production of unsaturated fatty acids were obtained with polyacrylamide Magnafloc LT-25 compared to cationic polyacrylamide flocculant Flopam as flocculants for harvesting of two marine microalgae Nannochloropsis oculata and Thalassiosira weissflogii. The anionic flocculants for harvesting N. oculata gave an increase of C14:0 and a decrease of C20:5 fatty acids and for T. weissflogii a decrease of C18:0 [94]. The usage of cationic starch as flocculant for flocculating S. obliquus yielded more efficiency compared to alum as flocculant for biodiesel production [95]. Thus, the type of flocculant used can differs the quality of lipid extracted from microalgae. For producing more stable biodiesel with high saturation level of fatty acids, the anionic flocculants are to be used.
Conflict of interest Nothing declared. Acknowledgment The authors are thankful to Kerala State Council for Science, Technology, and Environment (KSCSTE), Kerala, India for financially supporting (Order No. 1237/2015/KSCSTE) this investigation.
3.9. Economic analysis of flocculation method of harvesting microalgae The cost analysis of flocculation method of harvesting is vital for the economic way of harvesting microalgae. The cost of chitosan was approximately 50 to 100 times higher than aluminium sulphate [55]. The higher initial investment were required for using Tangential Flow Filtration (TFF) method of harvesting microalgae Tetraselmis suecica compared to polymeric flocculation obtained from the economic analysis [96]. The harvesting and dewatering of dilute microalgae culture with flocculation requires lower energy compared to other harvesting methods. But due to the requirement of flocculants as chemicals and loss of flocculants, these systems end at the same cost level as mechanical harvesting systems [97]. According to Lee et al. [98] the yearly cost of harvesting microalgae and production cost per kg of microalgae can be estimated using Eqs.(1) and (2):
Pc = (0.5I+M) × C1 +
Pc,algae =
Pc Ytotal
C1 + Ce + Cc + C1 + (Closs) A
References [1] Y. Chisti, Biodiesel from microalgae, Biotechnol. Adv. 25 (May-June) (2007) 294–306. [2] I. Rawat, R. Ranjith Kumar, T. Mutanda, F. Bux, Biodiesel from microalgae: a critical evaluation from laboratory to large scale production, Appl. Energy 103 (2013) 444–467. [3] T.M. Mata, A.A. Martins, N.S. Caetano, Microalgae for biodiesel production and other applications: a review, Renew. Sustain. Energy Rev. 14 (2010) 217–232. [4] M. Mubarak, A. Shaija, T.V. Suchithra, Optimization of lipid extraction from Salvinia molesta for biodiesel production using RSM and its FAME analysis, Environ. Sci. Pollut. Res. - Int. 23 (2016) 14047–14055. [5] P.M. Schenk, S.R. Thomas-Hall, E. Stephens, U.C. Marx, J.H. Mussgnug, C. Posten, et al., Second generation biofuels: high-efficiency microalgae for biodiesel production, Bioenergy Res. 1 (2008) 20–43. [6] J. Kim, G. Yoo, H. Lee, J. Lim, K. Kim, C.W. Kim, et al., Methods of downstream processing for the production of biodiesel from microalgae, Biotechnol. Adv. 31 (November) (2013) 862–876. [7] D. Vandamme, I. Foubert, I. Fraeye, B. Meesschaert, K. Muylaert, Flocculation of Chlorella vulgaris induced by high pH: role of magnesium and calcium and practical implications, Bioresour. Technol. 105 (February) (2012) 114–119. [8] N. Uduman, Y. Qi, M.K. Danquah, G.M. Forde, A. Hoadley, Dewatering of microalgal cultures: a major bottleneck to algae-based fuels, J. Renew. Sustain. Energy 2 (2010) 012701.
(1) (2)
where CI is the investment cost, I the rate of interest (%) and M is the maintenance costs (%), CI/A is the yearly depreciation over the 5
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Flocculation: An effective way to harvest microalgae for biodiesel production
T
M. Mubaraka, , A. Shaijab, T.V Suchithrac ⁎
a
Department of Mechanical Engineering, Karpagam College of Engineering, Coimbatore, 641032, India Department of Mechanical Engineering, National Institute of Technology Calicut, Kerala, 673601, India c School of Biotechnology, National Institute of Technology Calicut, Kerala, 673601, India b
ARTICLE INFO
ABSTRACT
Keywords: Biodiesel Autoflocculation Lipid content Microalgae Electrolytic flocculation
Microalgae are considered the most suitable feedstock for biodiesel production due to their higher areal productivity and lipid content. The economical production of biofuels from microalgae is important. This paper is a review of different techniques used for flocculating microalgae such as use of inorganic salts, organic salts, bioflocculants, autoflocculation and electrolytic flocculation for harvesting microalgae for biodiesel production. It was found that the usage of natural flocculants for harvesting microalgae eliminates the toxicity of biomass harvested but the cost of flocculant makes it uneconomical for large scale applications. The economic method of harvesting large quantity of microalgae still requires further research and development.
1. Introduction The production of biodiesel from microalgae are promising due to the advantages such as very short harvesting life capable of continuous harvesting year around, higher photosynthetic yield with superior lipid productivity with major portion of lipid as neutral which is more suitable for biodiesel production [1]. Microalgae can double their weight with respect to biomass within 24 h [2]. The key processes involved in biofuels production from microalgae biomass are cultivation, harvest, oil extraction, and conversion of algal lipids into advanced biodiesel [3,4]. Among these processes the harvesting and oil extraction contributes 60% of the total biodiesel production cost. The harvesting and dewatering of microalgae is a cost determining steps in converting algal biomass into biodiesel [5]. The cost involved in microalgae harvest is as high as 20% of the total production cost of biodiesel and varies according to the type of harvest methods used [6]. The major challenge in microalgae harvesting is due to the small size of cell and their low biomass concentration in the media which ranges from 0.5 to 1.5 g/l [7]. The common techniques currently employed in microalgae harvesting and recovery are centrifugation, coagulation, electrophoresis technique, flocculation, filtration, flotation, gravity sedimentation and magnetic separation [6,8–10]. As shown in Table 1 the flocculation method of harvesting microalgae were more suitable due to their reduced cost and easiness for execution and it allows large quantities of culture with reduced energy
⁎
requirement. Besides, this method has more option for selection of flocculants i.e, the agent which flocculates the cells, depending upon the type of flocculation methods. This method can be induced through different forms of flocculants such as inorganic, organic, polymeric [17] or through processes such as auto-flocculation [18], electro flocculation [19] or microbial flocculation [14]. The flocculation of microalgae can be performed in different ways such as chemical flocculation, bioflocculation, and magnetic nanoparticles. This review paper discusses the different flocculation techniques used for harvesting microalgae such as inorganic flocculants, organic flocculants which can be either by using natural organic flocculants or synthetic organic flocculants, bioflocculation, autoflocculation and electrolytic flocculation for biodiesel production and their efficiencies and impact on lipid extracted. 2. Flocculation Flocculation can be defined as the process in which where a solute particle in a solution forms aggregates called floc [8]. The flocculation of microalgae can occur in four mechanisms acting alone or in combination; (i) Phenomenon of charge neutralization in which charged ions, polymers or colloids strongly absorb on the opposite charged surface of the microalgae cells, (ii) The electrostatic patch mechanism in which a charged polymer binds to microalgae cells with opposite charge resulting in patches of opposite charge on the microalgae surface. The
Corresponding author. E-mail address: [email protected] (M. Mubarak).
https://doi.org/10.1016/j.jece.2019.103221 Received 27 March 2019; Received in revised form 16 June 2019; Accepted 17 June 2019 Available online 18 June 2019 2213-3437/ © 2019 Elsevier Ltd. All rights reserved.
Journal of Environmental Chemical Engineering 7 (2019) 103221
M. Mubarak, et al.
Table 1 Advantages and disadvantages of different harvesting methods for harvesting microalgae. Method used
Advantages
Disadvantages
References
Centrifugation
Less time required,
[11,12]
Coagulation Electrophoresis
Allows treatment of large quantities of microalgae cultures, Selectivity, energy efficiency, and cost effectiveness
Flotation
More effective and beneficial than sedimentation
Magnetic separation Gravity sedimentation
Low running cost, energy saving and simple operation Low energy requirement
Filtration
Selective filtration is possible
Flocculation
Cost and energy required are less, large quantity of culture can be used, Can use for wide variety of microalgae
High energy consuming, causes damage of cells due to gravitational and shear forces, less quantity of culture only used, requires costly equipment Expensive, at high dosages causes contamination problem. Fouling of the cathodes and systems getting damaged by high temperature, scale up problem Requires high particulate size of the algal biomass, oversized bubbles break up the floc, expensive process Fabrication was complex and expensive, practical limitability Requires large amount of land, very slow process, cell composition may change Requires periodical membrane washing or replacement, high cost, species specific Some types of flocculants like chitosan can be expensive, at high dosages causes contamination
microalgae cells connect with each other through patches of opposite charge causing flocculation, (iii) the phenomenon of bridging in which polymers or charged colloids simultaneously bind to the surface of two different microalgae cells to form a bridge between them brings the microalgae cells together, (iv) the massive precipitation of mineral present in the cultivation media causes the microalgae cells to get entrapped know as sweeping flocculation [20]. An ideal flocculant should be inexpensive, nontoxic and effective in low concentration [21]. The microalgae carry negative charge that prevents the aggregation of cells in suspension. The surface charge of microalgae can be minimized or neutralized by adding suitable flocculants such as multivalent cations and cationic polymers in the broth.
[12] [13,14] [13,14] [9] [8] [8] [12,15,16]
performed using Aluminum sulfate (Al2(SO4)3) [29]. The flocculation of Chlroella vulgaris and Phaeodactylum tricornutum was performed using magnesium hydroxide in which precipitates were formed at high pH of 10–10.5 [30]. The flocculation of Chlamydomonas reinhardtii was performed using inorganic flocculants such as aluminum sulfate, ferric chloride, polyalluminium chloride, plyaluminium chlorosulfate and cationic polyacrylamide and reported 95% efficiency with cationic polyacrylamide [31]. A new flocculant, combination of polymer and FeCl3 was introduced for flocculating Chlorella vulgaris GKV1 increased the sedimentation time of 80% in the first five minutes [32]. The metal salts such as NaOH, Ca(OH)2 and FeCl3 were used for the flocculation of Dunaliella salina and reported that FeCl3 as flocculant is most promising one [33]. A continuous flocculation of Nannochloropsis salina was performed with Al2(SO4)3) as flocculant and obtained efficiency above 85% [34]. The slurries of microalgae such as Chlorella vulgaris, Choricystis minor, Cylindrotheca fusiformis, Neochloris sp., Nannochloropsis salina was effectively flocculated using aluminium sulfate and ferric chloride as flocculants and gained 95% efficiency [35]. The flocculation of Scenedesmus sp. grown in fresh media and waste water was performed using ferric chloride as flocculant under reduced pH below 6.5 improved the harvesting efficiency [36]. The flocculation of microalgae, Chlorella sp. KR-1 was achieved with 98% efficiency using metal coagulant (Fe2(SO4)3) and sulfuric acid (H2SO4) [37]. Cost-efficient harvesting of microalgae is a major challenge for large-scale biomass production. The optimization of flocculation of Scenedesmus obliquus grown in wastewater was performed using ferric sulphate as flocculant [38]. As per the opinion of authors flocculation using iron and alum is most effective with better efficiency for harvesting mass culture of microalgae for biofuel production.
3. Methods used for flocculation 3.1. Inorganic flocculation The mechanism involved by the usage of inorganic flocculants is that the charge neutralization due to the absorption of equal amount of opposite charge by microalgae cell. The pH of the medium should be low to form cationic hydrolysis products for inducing flocculation. Examples of such inorganic flocculants are alum, ammonia, FeCl3, Fe2 (SO4)3, polyaluminium chloride etc. Ammonia is the first flocculant used for harvesting of microalgae like Chlorella sorokiniana (freshwater), Nannochloropsis oculata (marine), Dunaliella with an efficiency of 99% [22]. FeCl3 and Fe2(SO4)3 are also suitable for 98% efficient flocculation of Chlorella sorokiniana, Scenedesmus obliquus, Chlorococcum sp and a wild type Chlorella in symbiosis with bacteria [23] 99% and flocculated media were reused for cultivation. A minimum concentration of ferric chloride is required to overcome the electrostatic stabilization of negative surface charge of algae. As a flocculant for Chlorella zofingiensis, FeCl3 possess a linear proportionality with a dosage of flocculant with biomass concentration and pH [24]. The significant effect of 143 mg/L of FeCl3 and pH 8.1 of Chlorella has proved by response surface methodology (RSM) [25]. Similar to FeCl3, alum also has a linear correlation between dosage of flocculant, biomass concentration and pH suitable for harvesting Scenedesmus sp cultivated in open pond achieved flocculation efficiency of 90% at pH 6 [26]. The usage of alum at different dosages for flocculating Nannochloropsis salina with a biomass concentration 15–20 g/L achieved an efficiency of 79–99% and measurement of lipids and fatty acids were done using differential pulse absorptive voltametry [27]. Inorganic flocculant polyaluminium chloride (PACl) were used by considering effect of dosage, biomass concentration, lipid content, pH, mixing speed and time for harvesting Chlorella vulgaris ESP-31and gave more harvesting efficiency with reduced cost than chitosan [28]. The flocculation of Scenedesmus spp., Chlamydomonas reinhardtii,and Schizochytriumlimacinum cells at three different concentrations were
3.2. Organic flocculation The organic flocculation is based on the method of neutralization of surface charge on the algal cells by forming physical linkage between algal cells and organic flocculants. The organic flocculants are either natural organic flocculants or synthetic organic flocculants. The natural organic flocculants consist of Moringa oleifera. The synthetic organic flocculants consist of polyelectrolytes. 3.2.1. Natural organic flocculants 3.2.1.1. Moringa oleifera as flocculant. Moringa oleifera seed contain active peptides of molecular weight ranging from 6 to 20 kDa, with pH value between 9 and 10 or higher and can in saline environment it can act as a polyelectrolyte to induce flocculation [39]. The flocculation potential of M. oleifera has been confirmed with the jar test [40–42]. According to Teixeria et al. [43], the flocculation efficiency can be increased by increasing the pH for harvesting Chlorella vulgaris by using M. oleifera seeds. 2
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were 10 ppm, 20 min, 150 rpm, 20 min respectively and arrived an harvesting efficiency of 99% for Chlorella sp using jar test [54]. Usage of multiple dosage 1:300 w/w of chitosan: biomass ratio and 6.3–6.8 of pH range is used for Chlorella sorokiniana cultivated in waste water gave harvesting efficiency of above 99% [55]. A bioflocculant from Cobetia marina L03 was used for flocculation of Chlorella vulgaris with an efficiency of 92.7% and observed that bioflocculant was stable at wide range of pH and temperature [56]. The fungal assisted flocculation using co-cultivation of filamentous fungi with freshwater microalgae, Chlorella protothecoides and marine microalgae, Tetraselmis suecica were performed for the efficient and cost effective harvesting [57]. The harvesting of microalgae such as Chlorophyta species, Chlorella vulgaris, Nannochloropsis granulate and Dunaliella salina was performed using dinoflagellate Heterocapsa circularisquama as flocculant [58]. Chitosan flocculation worked generally very well for freshwater microalgae, but not for marine species [59]. The co-culture of microalgae with bacteria or flocculating microalgae with the use of bacteria was already reported for the purpose of flocculation [60,61]. Microbial bioflocculant has been widely used for wastewater treatment [62–64]. The harvesting of microalgae using microbes such as bacteria were also reported [65,66]. The bioflocculant produced by Paenibacillus polymyxa AM49 were used with inorganic flocculants for harvesting high density cultures of Scenedesmus sp. and the reuse of flocculated medium showed 8% decrease in growth and improved by the addition of 20%, 50% fresh BG11 medium [66]. Wan et al. [67] reported 90% of bioflocculation efficiency of microalgae Nannochloropsis oceanic by using the bacterial strain Solibacillus silvestris from activated sludge and the necessity of culture media without any effect on the growth. The presence of bacterial extracellular substances enhanced the flocculation efficiency of Chlorella vulgaris cultured in the presence of bacteria such as Flavobacterium sp., Terrimonas sp., Sphingobacterium sp., Rhizobium sp. and Hyphomonas sp. [68]. The feasibility of using microbial flocculant poly c-glutamic acid (c-PGA) for harvesting marine Chlorella vulgaris and fresh water Chlorella protothecoides were optimized using response surface methodology [69].
3.2.2. Synthetic organic flocculants 3.2.2.1. Polyelectrolyte flocculation. Polyelectrolyte flocculants are polymeric flocculants that includes cationic, non-ionic and anionic polymers, in addition to bentonites and active carbon generally used in waste water treatment [44]. The cationic polyelectrolytes generally used are poly DADMAC (PDADMAC), polymer from epichlorohydrin and dimethylamine (ECH/DMA), cationic Polyacrylamide (CPAM) and anionic polyelectrolyte are anionic polyacrylamide (APAM), polystyrene sulfonic acid (PSSA), and 2 acrylamido 2 methyl propane sulfonic acid (AAMPSA) [45]. Their mechanism of flocculation is due to the combination of charge neutralization and particle bridging and it depends on charge density and chain length of the polymer. The polymer molecule attached on the microalgae cell surface due to electrostatic or chemical forces causes the aggregation of cells [8]. This makes the polyelectrolytes more advantageous. The extent of aggregation of cells by the polymer depends on the key polymer characteristics such as charge, molecular weight and concentration [46]. 2–2.5 mg/g of polyelectrolyte Actipol range of flocculants such as EM1, EM16, EM22 and FB1 is sufficient for comparatively efficient recovery of freshwater microalgae (Muriellopsis sp, C. vulgaris, C. fusca, S. subspicatus and Scenedesmus sp) than metal salts like iron chloride, aluminium sulphate, iron sulphate and chitosan [44]. 3.3. Bioflocculation The spontaneous flocculation due to the presence of extracellular polymeric substances in the medium of microalgae and using chitosan is named as bioflocculation [47]. Some naturally bioflocculating microalgae can be mixed with other species of microalgae to induce flocculation [48]. As a bioflocculant, chitosan biopolymer have superior technical and environmental performance in the flocculation of Neochloris oleoabundans compared to ferric sulfate and alum [49]. Similar to chemical flocculants, the efficiency of bioflocculant is influenced by dosage and pH [50]. For example, chitosan is effective at pH range of 4–9 for flocculating Spirulina, Oscillatoria, Chlorella, and one brackish alga, Synechocystis [50]. Xu et al. [51] achieved more than 99% of harvesting efficiency of Chlorella sorokiniana using chitosan (approximately 10 mg/g algal dry weight) with cultivation media pH below 7. Table 2 shows the comparison of inorganic flocculants with recovery efficiency. Bioflocculation followed by centrifugation were used with reduced energy demand for harvesting Chlorella vulgaris (SAG211-11b), S. obliquus (SAG276-3a), A. falcatus (SAG202-9) and Ettlia texensis (SAG79.80) by considering the effect of ratio between sedimentation rate and energy demand for harvesting [52]. Table 3 shows the comparison of harvesting efficiency of microalgae with different bioflocculants. The usage of nano-chitosan (chitosan cross linked with sodium triphosphate) for flocculating Nannochloropsis sp. is reduced the consumption of flocculant by 40% with increased flocculation efficiency [53]. Chitosan dissolved in hydrochloric acid showed highest flocculation efficiency for flocculating Chlorella vulgaris at exponential and stationary growth phase compared to the usage of chitosan dissolved in citric, phosphoric, nitric acids [15]. The optimized conditions such as chitosan dosage, mixing time, mixing speed, sedimentation time
3.4. Organoclay flocculation The organoclays are basically formed from 3-aminopropyl functionalized magnesium phyllosilicate and consists of cationic metals Mg2+,Ca2+, Al3+, and Fe3+ in its center which are sandwiched organo functional entities that contain, by sol–gel reaction under ambient conditions, a high density of amino sites. In aqueous solution, organoclay particles are delaminated by protonated amine groups, as the result of which forms positively charged nano-clusters [70]. The advantages of using aminoclays for flocculation were due to its fast harvesting yield for a wide variety of microalgae species without affecting the pH of the microalgae feedstock [71]. The harvesting efficiency of about 100% were achieved with the use of cationic charged aluminum and magnesium backboned organoclays in aqueous form for harvesting 0.6 g/L concentrated Chlorella sp. KR -1 [72]. The usage of acids with aminoclay can induce flocculation with higher efficiency. Lee et al. [73] reported harvesting efficiency of approximately 100% using aminoclay induced humic acid for flocculating Chlorella sp. with a dosage of 5 g/L.
Table 2 Comparison of inorganic flocculants with recovery efficiency. Inorganic flocculant
Microalgae
Dosage
Recovery efficiency, %
References
Alum Ammonia
Nannochloropsis salina Chlorella sorokiniana Nannochloropsis oculata Dunaliella` Mixture of Chlorella sorokiniana, Scenedesmus obliquus, Chlorococcum sp. and a wild type Chlorella Chlorella sp
10.8 mg /L 57.31 mmol/L
79-99 99
[27] [22]
150–250 mg/L
66-98
[23]
143 mg/L
56.54
[25]
FeCl3 Fe2(SO4)3 Aluminium sulfate, ferric chloride
3
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Table 3 Comparison of different bioflocculants used and its recovery efficiency. Bioflocculant used
Algae species
Quantity used
Chitosan with Citric acid Nitric acid Phosphoric Hydrochloric Combination of CaCl2, FeCl3 and Paenibacillus polymyxa AM49.
Chlorella vulgaris
30 mg/L
Scenedesmus sp
8.5 mM CaCl2 0.2 mM FeCl3 1% P. polymyxa AM49
Recovery efficiency
Reference [15]
5.1 54.5 69.3 84.5 95
[66]
3.5. Auto flocculation
3.6. Electrolytic flocculation
The mechanism behind auto flocculation, increased pH due to the presence of photosynthetic CO2 consumption and precipitation of inorganic precipitates. This precipitation causes the formation of aggregates by excreted organic macromolecules that inhibit the release of microalgae daughter cells. When pH of the microalgae increases above 9, the flocculation occurs spontaneously [74]. The principle behind flocculation by increased pH of growth media due to the presence of Mg2+ in the growth medium hydrolyzed to form magnesium hydroxide precipitate which causes coagulation of micro algal cells by sweeping flocculation and charge neutralization. The effect of increased pH (8.610.6) for harvesting freshwater microalgae (Chlorella vulgaris, Scenedesmus sp., Chlorococcum sp) with low/medium biomass concentration. and marine microalgae (Nannochloropsis oculata, Phaeodactylum tricornutum) yielded better harvesting efficiency with the possibility of reuse of flocculated medium by supplementing nutrients for cultivation [75]. The usage of calcium hydroxide was most cost efficient for increasing pH to 11 in flocculating Chlorella vulgaris compared to other bases such as sodium hydroxide, potassium hydroxide, magnesium hydroxide and sodium carbonate [7]. The naturally available ions such as Mg2+,Ca2+, and CO32− in brackish water were used for harvesting Chlorella vulgaris gave settling rate of 100 cm/h more than the unflocculated cells [76]. The presence of algal organic matter and the increased biomass concentration of Chlorella vulgaris reduced flocculation efficiency with pH above 8.5 using precipitation of calcium phosphate. The mild acidification method was used to remove calcium phosphate culture from the media [77]. The cell wall associated with polysaccharides act as biopolymer were responsible for self-flocculation of S. obliquus AS-6-1 [78]. The bases such as NaOH and Ca(OH)2 can be used for pH induced flocculation of Scenedesmus obliquus and Chlorella vulgaris. The growth rate of reused flocculated media gave 1.79 times higher than fresh media which saves water and nutrients for cultivation [79]. A recovery efficiency of 90% and concentration factor of 20 were achieved by adding NaOH continuously using syringe for harvesting of marine microalgae Dunaliella salina. The flow rate of NaOH has no effect on recovery efficiency due to the auto flocculation mechanism with precipitation of magnesium ions present in the cultivation media [80]. The polysaccharides present in the unique cell wall of C.vulgaris JSC-7 induced flocculation and it does not interfere with the quality of harvested algae [81]. The autoflocculation of Chlorella vulgaris JSC-7 was used for removing Zn2+ and Cd2+ using surface adsorption from contaminated waste water [82]. Another study used self-flocculating microalgae, S. obliquus FSP-3 as biosorbent for removing heavy metal ions from waste water [83].
The electrolytic flocculation does not require any flocculants. The principle behind electrolytic flocculation is based on the movement of negatively charged algae towards anode loses their charge and forms aggregates [8]. The harvesting efficiency of 95% with a current consumption of 0.3 kW h/m3 were obtained with algal suspensions containing blue green algae such as Coelosphaerium sp. and Aphanizomenon sp., green algae (Closterium sp., Pediastrum sp., Cryptomonas sp. and Staurastrum sp.) and most of all diatoms (Asterionella sp., Cyclotella sp. and Melosira sp.) using electrolytic flocculation [84]. The electrodes subjected to polarity exchange in the middle of the harvesting process improved harvesting efficiency in continuous electrolytic microalgae (CEM) harvesting system under optimized conditions such as applied current, initial pH and mechanical mixing [85]. The harvesting efficiency of Nannochloris oculata (KMMCC-16) was better with aluminum and dimensionally stable anode (Al–DSA) compared to Al-platinum (AlPt) electrode in used continuous electrolytic microalgae (CEM) harvesting system. The harvesting efficiency were improved by optimizing the timing of polarity exchange [86]. The incorporation of mixing and settling of marine microalgae Tetraselmis sp has no effect on electroflocculation method while electrode separation has effect on harvesting efficiency [87]. The cell density and coagulant dosage has significant effect on harvesting efficiency of Nannochloris oculata in inorganic electrolytic flocculation with AlCl3 compared to other factors such as ionic strength, media pH and cell surface charge [88]. The usage of pulsed or continuous current in electro-coagulation- flocculation (ECF) reactor reduced the energy input and treatment time drastically with harvesting efficiency 90% for harvesting Nannochloropsis sp [89]. The microalgae, Chlorella vulgaris was used electro-coagulation flocculation using aluminum and iron electrodes [90]. The electrolytic flocculation of D. salina was performed using aluminium air battery and during the process the along with separation energy also produced which is usable for charging the battery [91]. Table 4 compares the flocculation efficiency of microalgae with electrolytic flocculation method. The influence of algal organic matter in the flocculation efficiency was reported. The presence of algal organic matter (AOM) inhibits flocculation efficiency and caused more dosage of flocculants for harvesting Chlorella vulgaris using methods such as aluminum sulfate, chitosan, cationic starch, pH induced flocculation and electro-coagulation flocculation [92]. The advantages and disadvantages of different flocculation methods were shown in Table 5.
Table 4 Comparison of electrolytic flocculation of microalgae. Method
Type of algae used
Recovery efficiency
References
Electrolytic flocculation Continuous electrolytic Electrolytic Coagulation flocculation
Coelosphaerium sp. Aphanizomenon sp. Closterium sp., Pediastrum sp. Cryptomonas sp. and Staurastrum sp. Nannochloropsis oculata Nannochloropsis sp.
95% 99.9% 95%
[84] [86] [89]
4
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Table 5 Comparison of advantages and disadvantages of different flocculation methods. Method
Advantages
Disadvantages
References
Electrolytic flocculation
No risk of contamination of algae with chemical flocculant Less expensive Rapid method of flocculation Environmental friendly, no contamination problem
Requires reactor, electrodes which may cost higher initial investment, scale up problem, electrodes need to replace periodically
[84]
Contamination of harvested biomass Higher production cost limit scale up, at higher pH ineffective
[27] [50]
Inorganic flocculation Organic flocculation
3.7. Determination of flocculation effectiveness
depreciation time A (year), Ce the energy costs, Cc the costs for consumables, Cl the costs for labour and Y total is the total produced biomass in a year. The above equations can be used for flocculation method of harvesting microalgae by considering the cost of flocculants and other capital investments.
The effectiveness of flocculation can be determined by using recovery efficiency and the concentration factor during microalgal separation [60]. The recovery efficiency or algal removal efficiency is the ratio of mass of cells recovered to the total mass of the cells [60]. The flocculation experiments generally uses narrow necked wide bodied geometrical forms for faster settling of culture than cylindrical reservoirs [93]. The flocculation activity of particular algae was measured by calculating algae removal efficiency or harvesting efficiency and concentration factor. The concentration factor provides quantification measurement of how many folds algae cells were concentrated from an initial volume. Some of the work were reported with algae removal efficiency by measuring the optical density of the culture at 680 nm before and after flocculation [7].
4. Conclusions The development of an efficient flocculation method for harvesting microalgae can reduce the cost and energy in biodiesel production from microalgae. The numerous studies have already investigated various methods for flocculating microalgae. The flocculation of microalgae using chemicals (organic) results in the contamination of biomass and it can be eliminated by using naturally available polymers as flocculants. The usage of chitosan as flocculant is most promising but the for large scale applications the cost of chitosan makes it unfeasible. The electrolytic flocculation is characterized as the flocculant free method of harvesting microalgae but it requires the reactor and electrodes. The usage of bacteria as flocculant for microalgae harvesting were getting more attention due to its environmental friendly nature. The algal organic matter influences the flocculation efficiency of the microalgae. Even though number of flocculation methods available there is no more flocculation methods are used in large scale production of biodiesel from microalgae explores the requirement of further research to make the economic harvesting of microalgae for biodiesel production.
3.8. Effect of flocculants on quality of lipid extracted A few works were reported the effect of flocculants on the quality of lipid extracted. The reduced production of unsaturated fatty acids were obtained with polyacrylamide Magnafloc LT-25 compared to cationic polyacrylamide flocculant Flopam as flocculants for harvesting of two marine microalgae Nannochloropsis oculata and Thalassiosira weissflogii. The anionic flocculants for harvesting N. oculata gave an increase of C14:0 and a decrease of C20:5 fatty acids and for T. weissflogii a decrease of C18:0 [94]. The usage of cationic starch as flocculant for flocculating S. obliquus yielded more efficiency compared to alum as flocculant for biodiesel production [95]. Thus, the type of flocculant used can differs the quality of lipid extracted from microalgae. For producing more stable biodiesel with high saturation level of fatty acids, the anionic flocculants are to be used.
Conflict of interest Nothing declared. Acknowledgment The authors are thankful to Kerala State Council for Science, Technology, and Environment (KSCSTE), Kerala, India for financially supporting (Order No. 1237/2015/KSCSTE) this investigation.
3.9. Economic analysis of flocculation method of harvesting microalgae The cost analysis of flocculation method of harvesting is vital for the economic way of harvesting microalgae. The cost of chitosan was approximately 50 to 100 times higher than aluminium sulphate [55]. The higher initial investment were required for using Tangential Flow Filtration (TFF) method of harvesting microalgae Tetraselmis suecica compared to polymeric flocculation obtained from the economic analysis [96]. The harvesting and dewatering of dilute microalgae culture with flocculation requires lower energy compared to other harvesting methods. But due to the requirement of flocculants as chemicals and loss of flocculants, these systems end at the same cost level as mechanical harvesting systems [97]. According to Lee et al. [98] the yearly cost of harvesting microalgae and production cost per kg of microalgae can be estimated using Eqs.(1) and (2):
Pc = (0.5I+M) × C1 +
Pc,algae =
Pc Ytotal
C1 + Ce + Cc + C1 + (Closs) A
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(1) (2)
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