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Efficient harvesting of Chaetoceros calcitrans for biodiesel production a
b
Sema Şirin , Ester Clavero & Joan Salvadó
ab
a
Departament d'Enginyeria Química, Universitat Rovira i Virgili, 43007 Tarragona, Catalonia, Spain b
Bioenergy and Biofuels Division, Institut de Recerca de l'Energia de Catalunya (IREC), C/ Marcel lí Domingo, 2, 43007 Tarragona, Catalonia, Spain Accepted author version posted online: 06 Feb 2015.Published online: 09 Mar 2015.
Click for updates To cite this article: Sema Şirin, Ester Clavero & Joan Salvadó (2015): Efficient harvesting of Chaetoceros calcitrans for biodiesel production, Environmental Technology, DOI: 10.1080/09593330.2015.1015456 To link to this article: http://dx.doi.org/10.1080/09593330.2015.1015456
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Environmental Technology, 2015 http://dx.doi.org/10.1080/09593330.2015.1015456
Efficient harvesting of Chaetoceros calcitrans for biodiesel production Sema Sirin ¸ a∗ , Ester Claverob and Joan Salvadóa,b a Departament
d’Enginyeria Química, Universitat Rovira i Virgili, 43007 Tarragona, Catalonia, Spain; b Bioenergy and Biofuels Division, Institut de Recerca de l’Energia de Catalunya (IREC), C/Marcel lí Domingo, 2, 43007 Tarragona, Catalonia, Spain
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(Received 12 November 2014; accepted 1 February 2015 ) Harvesting is one of the key challenges to determine the feasibility of producing biodiesel from algae. This paper presents experimental results for a cost-effective system to harvest Chaetoceros calcitrans, using natural sedimentation, flocculation, and inducing pH. No efficient sedimentation of microalgal cells was observed only by gravity. By alkalinity-induced flocculation, at a pH value of 9.51, 86% recovery of the cells was achieved with a sedimentation rate of 125 cm/h and a concentration factor (CF) of 4 (volume/volume (v/v)) in 10 min. The maximum photochemical quantum yield of photosystem II (F v /F m ) of concentrated cells was almost the same as fresh culture (0.621). Commercial flocculants, aluminium sulphate and poly-aluminium chloride (PAC), were also successful in harvesting the studied algal cells. Optimum concentration of aluminium sulphate (AS) could be concluded as 10 ppm with 87.6% recovery and 7.10 CF (v/v) in 30 min for cost-efficient harvesting, whereas for PAC it was 20 ppm with 74% recovery and 6.6 CF (v/v). F v /F m yields of concentrated cells with AS and PAC showed a 1% reduction compared to fresh culture. Mg+2 was the triggering ion for alkalinity-induced flocculation in the conditions studied. The rheology behaviour of the concentrated cells was Newtonian with values between 2.2 × 10−3 and 2.3 × 10−3 Pa s at 30°C. Keywords: pre-concentration; Chaetoceros calcitrans; flocculation; harvesting; microalgae
1. Introduction Microalgae have recently received considerable attention with potential uses as a sustainable energy source for biodiesel production. Although producing microalgae is a well-known process,[1] past studies have concluded that efficient harvesting is still the major challenge of commercializing biodiesel from microalgae,[2] due to 20– 30% (min.) of the total cost of biomass production being attributed to the recovery process. Generally, the biochemical composition of microalgal species depends on their growth rate and on the phase of their life cycle in which they are harvested.[3] In this study, the diatom Chaetoceros calcitrans (Paulsen) Takano was selected, which is known as a potential species for producing biodiesel,[4] with high growth rates even at low light intensities.[5] And therewithal, C. calcitrans is known to be favourable as live feed for larvae culture in aquaculture with stable cultivation and high nutritional value.[6] Several studies [7,8] have focused on the growth and cultivation conditions of C. calcitrans in order to have higher production rates and/or high lipid contents. For harvesting, from the various methods of dewatering microalgae [9] for the production of biofuel, a combination of potential methods should be considered to make the production process most economically feasible. It is essential
*Corresponding author. Email: [email protected] © 2015 Taylor & Francis
to reduce the costs and base the harvesting strategy on the lowest energy method.[10] Among the several methods (centrifugation, filtration, flocculation, bio-flocculation, etc.) that have been developed for harvesting microalgae,[9] flocculation is one of the common methods considered to be an effective and convenient process, which allows for fast treatment of large volumes.[11] Numerous chemical coagulants or flocculants have been tested in the literature.[12,13] Metal salts (aluminium sulphate (AS), ferric chloride, ferric sulphate, and others) are generally preferred in flocculation processes, because of their high harvesting efficiency. Besides, for microalgae, harvesting by changing the properties of growth medium is not a novel technique. Several studies have reported that increasing the pH value of the growth medium induced the formation of metallic hydroxide precipitates, which microalgal cells precipitated by sweeping flocculation and charge neutralization.[14] pH-induced flocculation is also a mimic of auto-flocculation, which is an alternative to high-energy consuming harvesting methods. In auto-flocculation, raised pH of the medium is promoted by prolonging the cultivation in sunlight with a limited CO2 supply, whereas in pH-induced flocculation caustic soda, lime, etc., are preferred to raise the pH levels.[15]
Ca: 11.20 ± 1.8
Mg: 55.78 ± 1.28 8.5–9.5 ∼ 38 95.33 ± 16.97
Cylindrical with two long pairs of setae
Shape of cell
4–10 (diameter)
2.20 ± 0.52 × 106
46.44 ± 5.98
Maximum photochemical quantum yield of PSII-F v /F m Mg2+ and Ca2+ ion concentration (mM) pH range Salinity (‰) Ash-free DW (mg L−1 ) Dry weight (DW) (mg L−1 ) Cell concentration (cells m L−1 )
2. Materials and methods 2.1. Cultures C. calcitrans is a roughly cylindrical alga, elliptical in valve view and rectangular in girdle view. The cells bear long cell wall prolongations (seta) at their poles which join cells together to form chains. C. calcitrans was obtained from the Institut de Recerca i Tecnologia Agroalimentàries (IRTA) in Sant Carles de la Rapita (Tarragona, Spain). Experiments were performed with aliquots of 300 L cultures obtained using a scale-up process from 200 mL to 4 L cultures. Cultures of 200 mL (conical flask) to 4 L (6 L volumetric flasks) were grown in an autoclaved Walne’s medium under the continuous illumination of daylight fluorescent lights (TDL18 W-840) at 100140 μmol photons m−2 s−1 . A stream of air was injected to provide CO2 and culture agitation. Cultures of 300 L were grown with seawater (filtered through 25, 10, 5, and 1 μm pore size filters (polyKLEAN, MICRO-KLEAN, 3M/Cuno); sterilized with bleach; and then neutralized with Na2 S2 O3 ), commercial fertilizer (0.3 mL L−1 Codafol 14.6.5, Sustainable Agro Solutions S.A., Lleida, Spain), B group vitamins (0.02 mL L−1 AquavitaB, Syva, Leon, Spain), and sodium silicate (107 μM, SigmaAldrich) in polyethylene bags at 20 ± 2°C and exposed to a continuous illumination at 100 μmol photon m−2 s−1 with cool white fluorescents. Aeration was also used to provide CO2 and agitation. The cultures were harvested at the end of exponential growth, which was after 7 days of inoculation of the diatom. Cell density (cells m L−1 ), dry weight, and ash-free dry weight (g L−1 ) protocols were described in detail by earlier studies.[10] The correlation used to estimate cell density from absorbance at 750 nm was y = 33.074*106 x + 1282020 (R2 = 0.99), where y represents the cell density (cells m L−1 ) and x is the absorbance value. Samples for absorbance measurements were fixed with formaldehyde at 0.4% final concentration. Light micrographs were taken using a Zeiss
Table 1. C. calcitrans cultures.
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The main objective of this study was to evaluate the use of different harvesting methods to concentrate the biomass of the diatom C. calcitrans. More specifically, we wanted to determine the flocculation efficiency (FE), concentration factor (CF), and settleable solids volume fraction (SSVF) of natural sedimentation; flocculation by commercial flocculants; and pH-induced flocculation methods. Sedimentation rates of the author’s chosen method are also presented. The morphological condition of the cells was examined after the application of the harvesting methods. The photosynthetic activity of the settled algal cells with chlorophyll fluorescence was studied to support the microscopic observations. Viscosity and Ca2+ and Mg2+ ion determination analyses were performed after applying different harvesting methods to elucidate the flocculation mechanism of C. calcitrans.
0.600–0.660
S. S¸ irin et al.
Mean size (μm)
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Environmental Technology Axio Scope A1 microscope equipped with Nomarski interference contrast optics. The culture properties of C. calcitrans are detailed in Table 1. 2.2.
Experiments
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2.2.1. Sedimentation by gravity Sedimentation by gravity (natural sedimentation) in C. calcitrans cultures was observed under different light (daylight and darkness) and temperature (ambient temperatures, T = 4°C and 12°C) conditions. After harvesting, the cultures were poured into 2 L graduated cylinders (height/diameter ratio of ≈ 8) and allowed to settle. All experiments were conducted at the original pH of the culture. Experimental conditions, the calculations (FE, CF, and SSVF), and sampling heights are explained elsewhere.[10] 2.2.2. Flocculation by pH adjustment Experiments of flocculation by pH adjustment (pHinduced flocculation) were run with 250 mL volumes of culture. The target pH (3 ≤ pH ≤ 11) was achieved simply by adjusting the pH with HCl or NaOH solutions (0.1 and 1 N). In order to obtain homogeneous pH, NaOH or HCl was added to cultures under agitation (300–350 rpm), followed by slower mixing and settling. Then, the culture was poured into graduated cylinders and allowed to stand at room temperature. Sampling and calculations were performed as in natural sedimentation experiments. 2.2.3. Flocculation by commercial flocculants The effect of flocculants on harvesting was studied by using two commercial flocculants: AS and poly-aluminium chloride (PAC). The flocculants were purchased from Kemira Iberica S.A., Spain. The required concentrations of AS and PAC solutions were prepared by diluting stock solutions of flocculants to a reasonable and effective dilution ratio (e.g. considering algal cell damage and the corrosivity factor). Properties of flocculants can be found in the supplementary material (S1). Flocculation experiments were all run with smaller volumes of culture. Fifty millilitre of algal culture suspension was placed in a 100 mL beaker. Flocculant concentrations between 5 and 200 mg L−1 were added to 50 mL aliquots. The test beaker was stirred at 150 rpm for 2 min at room temperature, the contents poured into polypropylene tubes with (height/diameter) ratio of ≈ 4, and left to settle. Sampling, measurement, and calculations were performed according to Sirin et al.[10] 2.2.4. Analyses for harvested cells and supernatants Photosynthetic activity measurements: A pulse amplitude modulation (PAM) fluorescence analyser (Walz GmbH-PAM control WATER-ED) was used to determine
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the maximum photochemical quantum yield of photosystem II (PSII) (F v /F m ), where F v = F m − F 0 , with F 0 being autofluorescence; F m being maximum fluorescence, and F v /F m being photosynthetic efficiency). The baseline value for the system was obtained with filtered (0.2 μm) seawater. For the purposes of evaluation, three fluorescence values were averaged. F v /F m was measured directly on settled cultures that had been covered (dark adapted) for 30 min. The yield was then determined with a saturation pulse. The effect of harvesting treatments on the photosynthetic activity of the phytoplankton was measured by the chlorophyll fluorescence produced by light pulses generated from light-emitting diodes. Presence of ions – Ca2 + and Mg 2 + : The effect of magnesium or calcium compounds on the flocculation mechanism was studied. The concentration of Ca2+ and Mg2+ ions in the supernatant just before and after harvesting of algal cells with different treatment methods was monitored by atomic absorption spectroscopy (model 3110; PerkinElmer). Viscosity: After treating the culture with different pre-concentration methods, dynamic viscosities of concentrated samples were measured with a rotational viscometer (Thermo-Haake VT550) using an NV sensor at 30°C. Viscosities were measured over a range of shear rates (245–2700 s−1 ) corresponding to 45–500 L min−1 ( ∼ 3–30 m3 h−1 ) flow rates and at a temperature of 30°C. All rheological measurements were performed in triplicate. The viscosities were calculated with the average of the measurements. Kinematic viscosities of supernatants were also monitored with an Ostwald–Cannon-Fenske viscometer (at 30°C) to check for any viscosity divergence from the culture medium. 3. Results 3.1. Sedimentation by gravity No efficient sedimentation of microalgal cells was observed at the end of day 7 (Figure 1(a)). Besides, a clear appearance was observed after day 13 (Figure 2). However, from total cell density calculations and via naked eye observations, it was seen that the algae were mostly attached to the glass cylinder walls. The maximum photochemical quantum yield of PSII (Fv /F m ) measurements indicated increasing levels of stress to PSII by the sedimentation time increases. Yields decreased from 0.659 in fresh culture medium to 0.303 at the end of day 7 and 0.175 after day 13 (Figure 1(b)). 3.2. Flocculation by pH adjustment FE was investigated as a function of pH without any flocculant addition. The acidic pH adjustment treatments of C. calcitrans showed no cell flocculation. (F v /F m ) measurements at pH 3 and 5 were quite low (0.099 and 0. 121, respectively). These levels were not enough to result
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(b)
Figure 1. (a) Natural sedimentation of C. calcitrans cells. Samples were taken from the top one-third (1/3 h) of the cylinders. Data are represented with ± standard error (SE; n = 3). (b) Maximum photochemical quantum yield of PSII values for natural sedimentation samples at room temperature under daylight. Values are the average of three measurements.
Figure 2. Algal samples for natural sedimentation experiments: (a) T = 12°C at the 14th day of settling; (b) T = 12°C at the 4th day of settling; and (c) culture just before letting to settle.
in a recovery when the concentrated cells were exposed to fresh medium. The addition of NaOH to the culture medium resulted in flocculation with faster sedimentation rates. Culture pH was 8.7 before starting pH-induced flocculation. With the addition of NaOH , up to 9.51, the culture began to agglomerate; however, the obtained FEs and CFs were remarkably low (Table 2). As pH reached 9.51, harvesting efficiency (FE) was greatly increased to 86% at 10 min of settling with an SSVF value of 0.24 (Table 2). Therefore, we consider 9.51 as the pH threshold. Beyond the pH threshold, up to pH ∼ 11, increasing the alkalinity of the solution was difficult as previously experienced with Phaeodactylum tricornutum [10] and Nannochloropsis gaditana.[16] Different interfaces were also observed with different alkalinity-induced values (Figure 3). The height of the interface was recorded at regular time intervals as described earlier.[10] The sedimentation rates were compared at pHs between pHthreshold and pH 11.0 (Figure 4). The fastest sedimentation rate was 125 cm h−1 at the threshold pH (pH 9.51), whereas the slowest was 10 cm h−1 at pH 11.
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Environmental Technology Table 2.
pH
Results for harvesting of C. calcitrans by alkalinity-induced pH ((height/diameter) ratio ≈ 7).
pH-induced FE at 10 min (%)
Max. pH-induced FE at 1 h (%)
– – 86 90 – – –
10 22 87 90 90 93 98
9.00 9.30 9.51 9.56 9.61 10.54 11.0
SSVF at 10 min Max. SSVF at 1 (hf /ho ) h (hf /ho ) CF at 10 min – – 0.24 0.46 0.80 0.95 0.99
0.095 0.099 0.13 0.20 0.26 0.50 0.80
– – 3.8 1.98 0 0 0
Sedimentation rate (cm h−1 ) –b –b 125 88 29 10 6.5
Max. photochemical quantum yield of PSII-F v /F m a 0.627 0.629 0.621 0.608 0.594 0.547 0.547
photochemical quantum yield of PSII-F v /F m of fresh culture was ∼ 0.630. The F v /F m values are the average of three measurements. b Sedimentation rate could not be calculated due to partial sedimentation of the cells.
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a Maximum
Figure 3. Photographs of C. calcitrans samples showing the difference in settling at different pH values. (a, c, e) The culture at the time of the experimental study depicting no sedimentation. (b) pH induced to 9.51. Interfaces of hindered and transition settling can be observed. (d) pH induced to 10.54. Only transition settling occurs. (f) pH induced to 10.54, with an apparent compression settling.
After 2–4 h of settling, for all pH values, it was clearly seen that the culture colour changed from brownish to dark brown. However, samples checked for cell integrity under light microscopy did not present changes in cell
morphology (Figure 5). Cells were fairly sparsely embedded in a rough matrix and free cells were rarely observed. Some actively swimming cells were observed at pHs up to pHthreshold . Maximum photochemical quantum yield of
Figure 4. Graph of (hclarified water /hculture ) ratio versus sedimentation time to compare the sedimentation rate for C. calcitrans culture. Data are represented with ± SE (n = 3).
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S. S¸ irin et al.
Figure 5. Light micrographs of C. calcitrans before and after flocculation induced by pH increase or by AS addition. (a) Culture before flocculation. One cell in girdle view, with one plastid. Cells did not form chains. (b) Optimum pH for flocculation. Cells are embedded in a rough colourless matrix and do not present signs of cytoplasm shrinkage or loss of cell integrity. (c) After AS addition. Cells lying in girdle and valve view, they are clustered in dense aggregates and no matrix is visible.
PSII (F v /F m ) values of the concentrated cells also supports the microscopic results of cell integrity (Table 2). The yields were above 0.5, which indicates low levels of stress on the cells with no loss of vitality – cells were able to recover rapidly when exposed to fresh culture medium. 3.3.
Flocculation by commercial flocculants
In water treatment processes, pH is an important parameter to find out the optimum conditions for chemical flocculation. It is known that non-optimal pH causes excessive dosages. As the goal is treating water, the conditions of the organisms are not of great concern. However, in this study, the quality of the algal biomass after flocculation is important. Concentrated algae were kept in good condition (i.e. for their lipid content) to avoid any problem in further processes and final product quality after harvesting. pH adjustments were tried prior to and after the addition of commercial flocculants AS and PAC. In the trials where pH was acidified, depending on pH–flocculant dosage effect, chloroplasts deteriorated in the course of algal harvest or after some time (e.g. 2 h). In the case of NaOH solution addition to the culture, for pH adjustment, two behaviours were observed: • An alkalinity-induced flocculation started prior to flocculant addition. • After flocculant addition, turbidity of residual solution increased without cell sedimentation. Due to this reason, the PAC and AS flocculant experiments were performed without adjusting the pH. As shown in Figure 6(a), when AS was used as a chemical flocculant, FEs reached high levels ( ∼ 90%) after 30 min of settling (10–60 ppm AS). Above 60 ppm, it started to decrease due to overdose of flocculant. The FEs of 15
and 30 min settling times were almost the same along a wide concentration range of the flocculant, from 20 to 100 ppm. Only at 200 ppm of AS, the difference between the FE after 15 and 30 min settling was remarkable. Optimum concentration of AS for the studied C. calcitrans cultures could be concluded as 10 ppm with FE of 87.6% and CF of 7.10 for cost-efficient harvesting. Microscopic examination of the algal–alum sediment showed that the number of actively swimming individual cells was decreased by the increase in dose as expected. Most cells were static in the aggregates, although some flocs were observed to oscillate at a dose of 5 ppm AS. Flocculation caused no reduction in cell viability, however, as measured by photosynthetic yield at doses up to at least 70 ppm of AS (Figure 6(b)). Higher doses of the flocculant caused decrease in the pH of the culture, which might be related to the decrease in the (F v /F m ) measurements. When the culture was flocculated with PAC, the FE increased satisfactorily after 30 ppm (Figure 6(a)) for 15 min of settling. Furthermore, even at 5 ppm of PAC concentration, FE was quite good for 30 min ( ∼ 60% with CF of ∼ 6). C. calcitrans flocculated with PAC was faster to settle than the algae flocculated with AS (in high concentrations (up to 60 ppm)). PAC showed more stable behaviour of FE even at high doses, contrary to AS. Only at PAC dose of 200 ppm was the stability affected; the turbidity increased due to unstable particles and resulted in a low FE. An optimum result for PAC depends on the settling time. For 30 min of settling time, 20 ppm of PAC could be chosen as the optimum for the algal recovery range of 74%. Once again, there was no reduction in the yields of (F v /F m ) at doses up to 70 ppm of PAC (Figure 6(b)). Above concentrations of 80 ppm for both flocculants, AS and PAC, the colour of the cells changed slightly from brownish to dark brown (naked eye examination). The yields of (F v /F m ) were very small compared to that at lower doses.
Environmental Technology
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(a)
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(b)
Figure 6. (a) FE of C. calcitrans with AS and PAC. pH change after coagulant addition is also shown. Data are represented with ± SE (n = 3). (b) Maximum photochemical quantum yield of PSII values for harvested cells with PAC and AS. Values are the average of three measurements.
Analyses of presence of ions (Ca2+ and Mg2+ ) and viscosity Mg and Ca ion concentrations and also viscosities of algal cultures were studied for flocculation conditions: pHthreshold , pH = 11, PACoptimum , ASoptimum . Magnesium concentration of the supernatant was lower than the culture medium concentration (88%) for pHthreshold . Magnesium consumption increased by increasing the pH of the algal culture. For pH 11, flocculation was attempted not only with Mg2+ ions, but also with Ca2+ ions (Table 3). Supernatants of PACoptimum and ASoptimum (with the given concentrations in Table 3) did not show significant difference for Mg2+ and Ca2+ concentrations than the culture medium. The rheological characterization of algal suspensions was measured with the assumptions detailed in the earlier study.[16] The rheological behaviour of the microalgal suspensions was Newtonian where the apparent viscosity was constant when measured at different rotational speeds corresponding to different shear rates. Dynamic viscosities of 3.4.
2.2.–2.3 × 10−3 Pa s at 30°C after pre-concentration processing were recorded for measured samples (Table 3) where the dynamic viscosity of the seawater is given as 0.87 × 10−3 Pa s (temperature = 30°C; salinity = 38 ppt). The kinematic viscosity data of supernatants after harvesting of algae were also monitored and compared with that of the culture medium. No differences in viscosities were found before and after treatment (data not shown) for all samples. Similar results were obtained in [16] for P. tricornutum and N. gaditana species.
4. Discussion The typical initial step for algal harvesting is solid–liquid separation which involves concentration processes. These include coagulation, flotation, centrifugation, filtration, and gravity (natural) sedimentation. Natural sedimentation can be the first step in concentration (pre-concentration). For the studied cultures of C.
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S. S¸ irin et al. Table 3. Mg2+ –Ca2+ contents, dynamic viscosities, and maximum photochemical quantum yield of C. calcitrans samples harvested with studied methods.
Method of concentration pHthreshold pH = 11 PAC (20 ppm) AS (10 ppm)
Mg2+ contenta,b (%) 87 ± 1.35 0 97 ± 1.60 96 ± 2.30
Ca2+ contenta,b (%) 97 79 98 98
± ± ± ±
1.70 2.30 1.84 0.00
Viscosityc (Pa s) 2.2 2.2 2.2 2.3
× × × ×
10−3 10−3 10−3 10−3
R2d
Max. photochemical quantum yield of PSII-F v /F m (YII)e
0.9840 0.9840 0.9939 0.9968
0.621 0.547 0.646 0.652
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a Mg2+ and Ca2+ ion measurements were done on samples of the supernatant after harvesting. b Mg2+ and Ca2+ percentages were calculated by comparison to culture medium ion contents. c Viscosity measurements were done at 30°C on concentrated algal samples after harvesting. d R2 is the linear regression determination coefficient of viscosity. e For pH threshold and pH 11, the maximum photochemical quantum yield of PSII value of the fresh culture PAC and AS experiments it was ∼ 0.656. The F v /F m values are the average of three measurements.
calcitrans, we did not observe any efficient natural sedimentation. Cells were not in good condition at the end of the settling period either. (F v /F m ) yields were relatively low (F v /F m ≤ 0.3) after day 6 of settling, which shows relatively poor physiological state of the cells. Chlorophyll fluorescence is a good indicator of the damage to photosynthetic apparatus in the photoautotrophic cells.[17,18] Measurement of the fluorescence of chlorophyll in intact algal cells provides information on the absorption, distribution, and utilization of energy in photosynthesis. The maximal photochemical yield of PSII (F v /F m ), which is proportional to maximal photosynthetic efficiency, can change from 0.4 to 0.8 for different phytoplankton classes.[19,20] Low F v /F m values ( ≤ 0.2) point to a severe stress in the culture. The population growth rate was zero or the cell number in the population started to decrease, which means expiry of phytoplankton cells.[21,22] Natural sedimentation results of (F v /F m ) yields showed 54% of reduction compared to fresh culture (control) for 1 week of settling, whereas for 2 weeks (F v /F m ) yields were lower than 0.2. These levels were caused by a loss of vitality, which means almost no recovery possibility for the cells even when exposed to fresh medium. However, for the same algal species, Harith et al. [23] obtained sufficient harvesting results (91%) at 27°C either in the dark or daylight for 8 days with natural sedimentation. Not only at 27°C, but also at 4°C, the harvesting efficiency was promising (70%). The viability of the settled cells was also higher than 60% in all cases. The effective natural sedimentation depends primarily on the species. Nevertheless, the growth medium affects the charge and the cell density of the culture [10] as well as the biochemical composition of microalgae.[24] The sedimentation of C. calcitrans in [23] might be due to higher cell densities and different chemical composition of the culture grown in Conway medium with air and CO2 . By the consumption of reserve sources (lipids and a series of fatty acids, ß-carotene, etc.),[25,26] the cells might possibly
was ∼ 0.630, whereas for
change their densities, which let them to settle by time. Then, it becomes necessary to determine the extent to which the intracellular content varies over a period of time. pH-induced flocculation in the alkaline region using the presence of ions in the culture by the addition of NaOH was a mimicked auto-flocculation process at photosynthetically raised pH. Increasing the pH allowed the recovery of ca. 90% biomass of Dunaliella tertiolecta, [27] Anabaena marina,[28] C. calcitrans,[23] P. tricornutum,[10,29] and N. gaditana.[16] Besides González-López et al., [28] in all cases the cells were not negatively affected or damaged by the basic pHs. Our results demonstrated that effective flocculation for C. calcitrans could be attained by pH increase. Harvesting efficiency reached 86% and a CF (volume/volume (v/v)) of 4 was obtained, only after 10 min of settling with the studied cell densities. With the precipitation of magnesium hydroxide (Mg(OH)2 ), flocculation probably occurred by letting the cells enmesh into the precipitate. Rapid sedimentation rate was also achieved at optimum pH (pHthreshold ) with less precipitation resulting in low SSVF values. Beyond the pH threshold, up to pH ∼ 11, increasing the alkalinity of the solution was difficult where we consider this pH range as ‘buffer zone’.[30] Further NaOH addition initially caused extensive Mg(OH)2 precipitation and the formation of CaCO3 in the sequel (Table 3). Besides, the mean particle size of magnesium hydroxide seems to decrease as the pH value of the solution increases under particular experimental conditions,[16,31] causing a decrease in the settling rate hereby. The formation of loose flocs and sedimentation with different interfaces could be correlated with buffer zones as a result of excessive NaOH addition. The rough colourless matrix in which algal cells were embedded, observed under light microscopy, most probably corresponds to these precipitates as well (Figure 5(b)). (F v /F m ) yields were quite promising with low reduction compared to that of control. Microscopic observations and (F v /F m ) yields were also quite supportive to conclude that the cells were in good condition.
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Environmental Technology Under alkaline conditions, some chemical ions in the medium (i.e. Mg+2 and Ca+2 ) precipitate, along with the algal biomass, which both help in the harvesting process. The role played by each reaction depends on the primary particles and the ions contained in the solution.[10] Further studies are necessary to understand the optimum conditions of flocculation of different species and the same species that grow in different medium compositions. For the studied species, alkalinity-induced flocculation seems promising for the related culture medium composition and cell density even if the goal is biodiesel production. AS and PAC are widely used as flocculants for water treatment. They are used for harvesting microalgae as well, although they cause metal contamination in the harvested biomass. Therefore, the presence of flocculants further downstream after harvesting and how they affect the extraction and/or fuel conversion processes must also be well understood.[32] Additionally, if residual water is going to be recycled, the effect on culture growth and lipid production must be clarified.[10] However, besides these disadvantages, metal coagulants provide a good model system to study the interaction between flocculants and microalgal cells because their properties are well known.[33,34] PAC and AS treatment with pH adjustments at acidic pHs resulted as chloroplast deterioration in the course of algal harvest or after some time (e.g. 2 hours). For the acidic pHs, depending on pH–flocculant dosage effect, changing the pH by even a small amount is more of a sudden chemical change. The speed of chemical change might be so fast not to let the cell to adapt the new ambient environment. Thus, the response of the cell might be most probably manifest as chloroplast reaction which occurs on the membrane. From the experiments of two commercial flocculants (AS and PAC), it could be concluded that with enough time for the flocs to stabilize and collapse, efficient FEs were observed without damaging the cells,[16] up to doses of 80 ppm. PAC provided sufficient FEs and faster sedimentation rates compared to AS at lower concentrations. Optimum results for PAC and AS flocculants were obtained from a 5–20 ppm concentration. The recovery of the algae was between 60% and 80% with 7–8 CF by volume (v/v). (F v /F m ) yields indicated that at optimum doses of flocculant, no reduction in the viability was observed. Eldridge et al. [35] also investigated flocculation/coagulation of five marine algal species with commercial flocculants (AS, iron(III) sulphate, and Magnafloc 919). They also related (F v /F m ) yields of concentrated algal cells with cell viability and/or the state of the algae. Newtonian fluids are very similar to water, making both pumping and mixing relatively easy. The mixing times for Newtonian fluids are lower than those of nonNewtonian fluids. Most sludges show Newtonian fluid
9
behaviour, especially below 4% of solid concentration, and algal cultures are generally accepted to behave like Newtonian fluids.[36] The viscosities of the concentrated samples showed Newtonian behaviour regardless of the treatments. This rheological behaviour after harvesting might be expected with lower initial cell densities of the studied cultures. Soulies et al. [37] also did not observe stress dependence of the viscosity in dilute solutions of Chlorella vulgaris species. Low viscosity of Mg(OH)2 even at high concentrations might be another reason.[16] The results showed that a slight increase in viscosity after the pre-concentration process might not cause high energy losses.[38] The supernatant viscosities showed that no viscous substances such as extracellular polysaccharide were excreted by the species during the pre-concentration process due to stress.[16] 5. Conclusion For the studied C. calcitrans cultures, both alkalinityinduced flocculation and flocculation with commercial flocculants showed promising and satisfying performances with almost similar CFs. However, if the target is biodiesel production, authors’ choice would be alkalinity-induced flocculation due to no contamination risk of algae and reusability of supernatant water. The results of maximal photochemical yield of PSII (F v /F m ) of settled algal samples were compatible with microscopic observations. The settled algal cells were all in good condition for the optimum case of treatment methods. Viscosity results showed that after the flocculation step pumping and mixing are still easily attainable and that flocculation does not increase the pumping cost because the algal concentrate still follows a Newtonian behaviour. Acknowledgements The authors would like to thank Maria Pilar Rey Valera from IREC for valuable work with the algal cultures.
Disclosure statement No potential conflict of interest was reported by the authors.
Funding Funding was obtained by projects: ENE2011-22761 ‘Biorrefineria de microalgas: optimización de las etapas de cosechado y de obtención de lípidos’ funded by the Spanish Ministry of Science and Innovation and ‘Fuels from Biomass’ (research programme funded by Excma. Diputació Tarragona). The research was supported by the European Regional Development Funds (ERDF, FEDER Programa Competitividad de Catalunya 2007–2013).
Supplementary data Supplemental data for this article can be accessed at http://dx.doi.org/10.1080/09593330.2015.1015456
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[18] Silva FB, Costa AC, Alves RRP, Megguer CA. Chlorophyll fluorescence as an indicator of cellular damage by glyphosate herbicide in Raphanus sativus L. plants. Am J Plant Sci. 2014;5:2509–2519. [19] Biichel C, Wilhelm C. In vivo analysis of slow chlorophyll fluorescence induction kinetics in algae: progress, problems and perspectives. Photochem. Photobiol. 1993;58: 137–148. [20] Juneau P, Harrison PJ. Comparison by PAM fluorometry of photosynthetic activity of nine marine phytoplankton grown under identical conditions. Photochem Photobiol. 2005;81:649–653. [21] Young EB, Beardall J. Rapid ammonium- and nitrateinduced perturbations to chlorophyll a fluorescence in nitrogen-stressed Dunaliella tertiolecta (Chlorophyta). J Phycol. 2003;39:332–342. [22] Voronova EN, Il’ash LV, Pogosyana SI, Ulanova AYu, Matorin DN, Cho M-i, Rubin AB. Intrapopulation heterogeneity of the fluorescence parameters of the marine plankton alga Thalassiosira weissflogii at various nitrogen levels. Microbiology. 2009;78:419–427. [23] Harith ZT, Yusoff FM, Mohamed MS, Mohamed Din MS, Ariff AB. Effect of different flocculants on the flocculation performance of microalgae, Chaetoceros calcitrans, cells. Afr J Biotechnol. 2009;8:5971– 5978. [24] Brown MR, Dunstan GA, Norwood SJ, Miller KA. Effects of harvest stage and light on the biochemical composition of the diatom Thalassiosira pseudonana. J Phycol. 1996;32:64–73. [25] Chisti Y. Biodiesel from microalgae. Biotechnol Adv. 2007;25:294–306. [26] Molina Grima E, Belarbi E, Acién Fernández FG, Robles Medina A, Chisti Y. Recovery of algal biomass and metabolites: process options and economic. Biotechnol Adv. 2003;20:492–515. [27] Horiuchi JI, Ohba I, Tada K, Kobayashi M, Kanno T, Kishimoto M. Effective cell harvesting of the halotolerant microalga Dunaliella tertiolecta with pH control. J Biosci Bioeng. 2003;95:412–415. [28] González-López CV, Acién FG, Fernández-Sevilla JM, Sánchez-Fernández JF, Cerón MC, Molina E. Utilization of the cyanobacteria Anabaena sp. ATCC 33047 in CO2 removal processes. Bioresour Technol. 2009;100:5904– 5910. [29] Spilling K, Seppälä J, Tamminen T. Inducing autoflocculation in the diatom Phaeodactylum tricornutum through CO2 regulation. J Appl Phycol. 2011;23: 959–966. [30] Besson A, Guiraud P. High-pH-induced flocculationflotation of the hypersaline microalga Dunaliella salina. Bioresour Technol. 2013;147:464–470. [31] Xu H, Deng X. Preparation and properties of superfine Mg(OH)2 flame retardant. Trans Nonferr Met Soc China. 2006;16:488–492. [32] Borges L, Morón-Villarreyes JA, Montes D’Oca MG, Abreu PC. Effects of flocculants on lipid extraction and fatty acid composition of the microalgae Nannochloropsis oculata and Thalassiosira weissflogii. Biomass Bioenergy. 2011;35:4449–4454. [33] Wyatt NB, Gloe LM, Brady PV, Hewson JC, Grillet AM, Hankins MG, Pohl PI. Critical conditions for ferric chlorideinduced flocculation of freshwater algae. Biotechnol Bioeng. 2011;109:493–501. [34] Zhang X, Amendola P, Hewson JC, Sommerfeld M, Hu Q. Influence of growth phase on harvesting of Chlorella
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Environmental Technology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tent20
Efficient harvesting of Chaetoceros calcitrans for biodiesel production a
b
Sema Şirin , Ester Clavero & Joan Salvadó
ab
a
Departament d'Enginyeria Química, Universitat Rovira i Virgili, 43007 Tarragona, Catalonia, Spain b
Bioenergy and Biofuels Division, Institut de Recerca de l'Energia de Catalunya (IREC), C/ Marcel lí Domingo, 2, 43007 Tarragona, Catalonia, Spain Accepted author version posted online: 06 Feb 2015.Published online: 09 Mar 2015.
Click for updates To cite this article: Sema Şirin, Ester Clavero & Joan Salvadó (2015): Efficient harvesting of Chaetoceros calcitrans for biodiesel production, Environmental Technology, DOI: 10.1080/09593330.2015.1015456 To link to this article: http://dx.doi.org/10.1080/09593330.2015.1015456
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Environmental Technology, 2015 http://dx.doi.org/10.1080/09593330.2015.1015456
Efficient harvesting of Chaetoceros calcitrans for biodiesel production Sema Sirin ¸ a∗ , Ester Claverob and Joan Salvadóa,b a Departament
d’Enginyeria Química, Universitat Rovira i Virgili, 43007 Tarragona, Catalonia, Spain; b Bioenergy and Biofuels Division, Institut de Recerca de l’Energia de Catalunya (IREC), C/Marcel lí Domingo, 2, 43007 Tarragona, Catalonia, Spain
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(Received 12 November 2014; accepted 1 February 2015 ) Harvesting is one of the key challenges to determine the feasibility of producing biodiesel from algae. This paper presents experimental results for a cost-effective system to harvest Chaetoceros calcitrans, using natural sedimentation, flocculation, and inducing pH. No efficient sedimentation of microalgal cells was observed only by gravity. By alkalinity-induced flocculation, at a pH value of 9.51, 86% recovery of the cells was achieved with a sedimentation rate of 125 cm/h and a concentration factor (CF) of 4 (volume/volume (v/v)) in 10 min. The maximum photochemical quantum yield of photosystem II (F v /F m ) of concentrated cells was almost the same as fresh culture (0.621). Commercial flocculants, aluminium sulphate and poly-aluminium chloride (PAC), were also successful in harvesting the studied algal cells. Optimum concentration of aluminium sulphate (AS) could be concluded as 10 ppm with 87.6% recovery and 7.10 CF (v/v) in 30 min for cost-efficient harvesting, whereas for PAC it was 20 ppm with 74% recovery and 6.6 CF (v/v). F v /F m yields of concentrated cells with AS and PAC showed a 1% reduction compared to fresh culture. Mg+2 was the triggering ion for alkalinity-induced flocculation in the conditions studied. The rheology behaviour of the concentrated cells was Newtonian with values between 2.2 × 10−3 and 2.3 × 10−3 Pa s at 30°C. Keywords: pre-concentration; Chaetoceros calcitrans; flocculation; harvesting; microalgae
1. Introduction Microalgae have recently received considerable attention with potential uses as a sustainable energy source for biodiesel production. Although producing microalgae is a well-known process,[1] past studies have concluded that efficient harvesting is still the major challenge of commercializing biodiesel from microalgae,[2] due to 20– 30% (min.) of the total cost of biomass production being attributed to the recovery process. Generally, the biochemical composition of microalgal species depends on their growth rate and on the phase of their life cycle in which they are harvested.[3] In this study, the diatom Chaetoceros calcitrans (Paulsen) Takano was selected, which is known as a potential species for producing biodiesel,[4] with high growth rates even at low light intensities.[5] And therewithal, C. calcitrans is known to be favourable as live feed for larvae culture in aquaculture with stable cultivation and high nutritional value.[6] Several studies [7,8] have focused on the growth and cultivation conditions of C. calcitrans in order to have higher production rates and/or high lipid contents. For harvesting, from the various methods of dewatering microalgae [9] for the production of biofuel, a combination of potential methods should be considered to make the production process most economically feasible. It is essential
*Corresponding author. Email: [email protected] © 2015 Taylor & Francis
to reduce the costs and base the harvesting strategy on the lowest energy method.[10] Among the several methods (centrifugation, filtration, flocculation, bio-flocculation, etc.) that have been developed for harvesting microalgae,[9] flocculation is one of the common methods considered to be an effective and convenient process, which allows for fast treatment of large volumes.[11] Numerous chemical coagulants or flocculants have been tested in the literature.[12,13] Metal salts (aluminium sulphate (AS), ferric chloride, ferric sulphate, and others) are generally preferred in flocculation processes, because of their high harvesting efficiency. Besides, for microalgae, harvesting by changing the properties of growth medium is not a novel technique. Several studies have reported that increasing the pH value of the growth medium induced the formation of metallic hydroxide precipitates, which microalgal cells precipitated by sweeping flocculation and charge neutralization.[14] pH-induced flocculation is also a mimic of auto-flocculation, which is an alternative to high-energy consuming harvesting methods. In auto-flocculation, raised pH of the medium is promoted by prolonging the cultivation in sunlight with a limited CO2 supply, whereas in pH-induced flocculation caustic soda, lime, etc., are preferred to raise the pH levels.[15]
Ca: 11.20 ± 1.8
Mg: 55.78 ± 1.28 8.5–9.5 ∼ 38 95.33 ± 16.97
Cylindrical with two long pairs of setae
Shape of cell
4–10 (diameter)
2.20 ± 0.52 × 106
46.44 ± 5.98
Maximum photochemical quantum yield of PSII-F v /F m Mg2+ and Ca2+ ion concentration (mM) pH range Salinity (‰) Ash-free DW (mg L−1 ) Dry weight (DW) (mg L−1 ) Cell concentration (cells m L−1 )
2. Materials and methods 2.1. Cultures C. calcitrans is a roughly cylindrical alga, elliptical in valve view and rectangular in girdle view. The cells bear long cell wall prolongations (seta) at their poles which join cells together to form chains. C. calcitrans was obtained from the Institut de Recerca i Tecnologia Agroalimentàries (IRTA) in Sant Carles de la Rapita (Tarragona, Spain). Experiments were performed with aliquots of 300 L cultures obtained using a scale-up process from 200 mL to 4 L cultures. Cultures of 200 mL (conical flask) to 4 L (6 L volumetric flasks) were grown in an autoclaved Walne’s medium under the continuous illumination of daylight fluorescent lights (TDL18 W-840) at 100140 μmol photons m−2 s−1 . A stream of air was injected to provide CO2 and culture agitation. Cultures of 300 L were grown with seawater (filtered through 25, 10, 5, and 1 μm pore size filters (polyKLEAN, MICRO-KLEAN, 3M/Cuno); sterilized with bleach; and then neutralized with Na2 S2 O3 ), commercial fertilizer (0.3 mL L−1 Codafol 14.6.5, Sustainable Agro Solutions S.A., Lleida, Spain), B group vitamins (0.02 mL L−1 AquavitaB, Syva, Leon, Spain), and sodium silicate (107 μM, SigmaAldrich) in polyethylene bags at 20 ± 2°C and exposed to a continuous illumination at 100 μmol photon m−2 s−1 with cool white fluorescents. Aeration was also used to provide CO2 and agitation. The cultures were harvested at the end of exponential growth, which was after 7 days of inoculation of the diatom. Cell density (cells m L−1 ), dry weight, and ash-free dry weight (g L−1 ) protocols were described in detail by earlier studies.[10] The correlation used to estimate cell density from absorbance at 750 nm was y = 33.074*106 x + 1282020 (R2 = 0.99), where y represents the cell density (cells m L−1 ) and x is the absorbance value. Samples for absorbance measurements were fixed with formaldehyde at 0.4% final concentration. Light micrographs were taken using a Zeiss
Table 1. C. calcitrans cultures.
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The main objective of this study was to evaluate the use of different harvesting methods to concentrate the biomass of the diatom C. calcitrans. More specifically, we wanted to determine the flocculation efficiency (FE), concentration factor (CF), and settleable solids volume fraction (SSVF) of natural sedimentation; flocculation by commercial flocculants; and pH-induced flocculation methods. Sedimentation rates of the author’s chosen method are also presented. The morphological condition of the cells was examined after the application of the harvesting methods. The photosynthetic activity of the settled algal cells with chlorophyll fluorescence was studied to support the microscopic observations. Viscosity and Ca2+ and Mg2+ ion determination analyses were performed after applying different harvesting methods to elucidate the flocculation mechanism of C. calcitrans.
0.600–0.660
S. S¸ irin et al.
Mean size (μm)
2
Environmental Technology Axio Scope A1 microscope equipped with Nomarski interference contrast optics. The culture properties of C. calcitrans are detailed in Table 1. 2.2.
Experiments
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2.2.1. Sedimentation by gravity Sedimentation by gravity (natural sedimentation) in C. calcitrans cultures was observed under different light (daylight and darkness) and temperature (ambient temperatures, T = 4°C and 12°C) conditions. After harvesting, the cultures were poured into 2 L graduated cylinders (height/diameter ratio of ≈ 8) and allowed to settle. All experiments were conducted at the original pH of the culture. Experimental conditions, the calculations (FE, CF, and SSVF), and sampling heights are explained elsewhere.[10] 2.2.2. Flocculation by pH adjustment Experiments of flocculation by pH adjustment (pHinduced flocculation) were run with 250 mL volumes of culture. The target pH (3 ≤ pH ≤ 11) was achieved simply by adjusting the pH with HCl or NaOH solutions (0.1 and 1 N). In order to obtain homogeneous pH, NaOH or HCl was added to cultures under agitation (300–350 rpm), followed by slower mixing and settling. Then, the culture was poured into graduated cylinders and allowed to stand at room temperature. Sampling and calculations were performed as in natural sedimentation experiments. 2.2.3. Flocculation by commercial flocculants The effect of flocculants on harvesting was studied by using two commercial flocculants: AS and poly-aluminium chloride (PAC). The flocculants were purchased from Kemira Iberica S.A., Spain. The required concentrations of AS and PAC solutions were prepared by diluting stock solutions of flocculants to a reasonable and effective dilution ratio (e.g. considering algal cell damage and the corrosivity factor). Properties of flocculants can be found in the supplementary material (S1). Flocculation experiments were all run with smaller volumes of culture. Fifty millilitre of algal culture suspension was placed in a 100 mL beaker. Flocculant concentrations between 5 and 200 mg L−1 were added to 50 mL aliquots. The test beaker was stirred at 150 rpm for 2 min at room temperature, the contents poured into polypropylene tubes with (height/diameter) ratio of ≈ 4, and left to settle. Sampling, measurement, and calculations were performed according to Sirin et al.[10] 2.2.4. Analyses for harvested cells and supernatants Photosynthetic activity measurements: A pulse amplitude modulation (PAM) fluorescence analyser (Walz GmbH-PAM control WATER-ED) was used to determine
3
the maximum photochemical quantum yield of photosystem II (PSII) (F v /F m ), where F v = F m − F 0 , with F 0 being autofluorescence; F m being maximum fluorescence, and F v /F m being photosynthetic efficiency). The baseline value for the system was obtained with filtered (0.2 μm) seawater. For the purposes of evaluation, three fluorescence values were averaged. F v /F m was measured directly on settled cultures that had been covered (dark adapted) for 30 min. The yield was then determined with a saturation pulse. The effect of harvesting treatments on the photosynthetic activity of the phytoplankton was measured by the chlorophyll fluorescence produced by light pulses generated from light-emitting diodes. Presence of ions – Ca2 + and Mg 2 + : The effect of magnesium or calcium compounds on the flocculation mechanism was studied. The concentration of Ca2+ and Mg2+ ions in the supernatant just before and after harvesting of algal cells with different treatment methods was monitored by atomic absorption spectroscopy (model 3110; PerkinElmer). Viscosity: After treating the culture with different pre-concentration methods, dynamic viscosities of concentrated samples were measured with a rotational viscometer (Thermo-Haake VT550) using an NV sensor at 30°C. Viscosities were measured over a range of shear rates (245–2700 s−1 ) corresponding to 45–500 L min−1 ( ∼ 3–30 m3 h−1 ) flow rates and at a temperature of 30°C. All rheological measurements were performed in triplicate. The viscosities were calculated with the average of the measurements. Kinematic viscosities of supernatants were also monitored with an Ostwald–Cannon-Fenske viscometer (at 30°C) to check for any viscosity divergence from the culture medium. 3. Results 3.1. Sedimentation by gravity No efficient sedimentation of microalgal cells was observed at the end of day 7 (Figure 1(a)). Besides, a clear appearance was observed after day 13 (Figure 2). However, from total cell density calculations and via naked eye observations, it was seen that the algae were mostly attached to the glass cylinder walls. The maximum photochemical quantum yield of PSII (Fv /F m ) measurements indicated increasing levels of stress to PSII by the sedimentation time increases. Yields decreased from 0.659 in fresh culture medium to 0.303 at the end of day 7 and 0.175 after day 13 (Figure 1(b)). 3.2. Flocculation by pH adjustment FE was investigated as a function of pH without any flocculant addition. The acidic pH adjustment treatments of C. calcitrans showed no cell flocculation. (F v /F m ) measurements at pH 3 and 5 were quite low (0.099 and 0. 121, respectively). These levels were not enough to result
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(b)
Figure 1. (a) Natural sedimentation of C. calcitrans cells. Samples were taken from the top one-third (1/3 h) of the cylinders. Data are represented with ± standard error (SE; n = 3). (b) Maximum photochemical quantum yield of PSII values for natural sedimentation samples at room temperature under daylight. Values are the average of three measurements.
Figure 2. Algal samples for natural sedimentation experiments: (a) T = 12°C at the 14th day of settling; (b) T = 12°C at the 4th day of settling; and (c) culture just before letting to settle.
in a recovery when the concentrated cells were exposed to fresh medium. The addition of NaOH to the culture medium resulted in flocculation with faster sedimentation rates. Culture pH was 8.7 before starting pH-induced flocculation. With the addition of NaOH , up to 9.51, the culture began to agglomerate; however, the obtained FEs and CFs were remarkably low (Table 2). As pH reached 9.51, harvesting efficiency (FE) was greatly increased to 86% at 10 min of settling with an SSVF value of 0.24 (Table 2). Therefore, we consider 9.51 as the pH threshold. Beyond the pH threshold, up to pH ∼ 11, increasing the alkalinity of the solution was difficult as previously experienced with Phaeodactylum tricornutum [10] and Nannochloropsis gaditana.[16] Different interfaces were also observed with different alkalinity-induced values (Figure 3). The height of the interface was recorded at regular time intervals as described earlier.[10] The sedimentation rates were compared at pHs between pHthreshold and pH 11.0 (Figure 4). The fastest sedimentation rate was 125 cm h−1 at the threshold pH (pH 9.51), whereas the slowest was 10 cm h−1 at pH 11.
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Environmental Technology Table 2.
pH
Results for harvesting of C. calcitrans by alkalinity-induced pH ((height/diameter) ratio ≈ 7).
pH-induced FE at 10 min (%)
Max. pH-induced FE at 1 h (%)
– – 86 90 – – –
10 22 87 90 90 93 98
9.00 9.30 9.51 9.56 9.61 10.54 11.0
SSVF at 10 min Max. SSVF at 1 (hf /ho ) h (hf /ho ) CF at 10 min – – 0.24 0.46 0.80 0.95 0.99
0.095 0.099 0.13 0.20 0.26 0.50 0.80
– – 3.8 1.98 0 0 0
Sedimentation rate (cm h−1 ) –b –b 125 88 29 10 6.5
Max. photochemical quantum yield of PSII-F v /F m a 0.627 0.629 0.621 0.608 0.594 0.547 0.547
photochemical quantum yield of PSII-F v /F m of fresh culture was ∼ 0.630. The F v /F m values are the average of three measurements. b Sedimentation rate could not be calculated due to partial sedimentation of the cells.
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a Maximum
Figure 3. Photographs of C. calcitrans samples showing the difference in settling at different pH values. (a, c, e) The culture at the time of the experimental study depicting no sedimentation. (b) pH induced to 9.51. Interfaces of hindered and transition settling can be observed. (d) pH induced to 10.54. Only transition settling occurs. (f) pH induced to 10.54, with an apparent compression settling.
After 2–4 h of settling, for all pH values, it was clearly seen that the culture colour changed from brownish to dark brown. However, samples checked for cell integrity under light microscopy did not present changes in cell
morphology (Figure 5). Cells were fairly sparsely embedded in a rough matrix and free cells were rarely observed. Some actively swimming cells were observed at pHs up to pHthreshold . Maximum photochemical quantum yield of
Figure 4. Graph of (hclarified water /hculture ) ratio versus sedimentation time to compare the sedimentation rate for C. calcitrans culture. Data are represented with ± SE (n = 3).
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Figure 5. Light micrographs of C. calcitrans before and after flocculation induced by pH increase or by AS addition. (a) Culture before flocculation. One cell in girdle view, with one plastid. Cells did not form chains. (b) Optimum pH for flocculation. Cells are embedded in a rough colourless matrix and do not present signs of cytoplasm shrinkage or loss of cell integrity. (c) After AS addition. Cells lying in girdle and valve view, they are clustered in dense aggregates and no matrix is visible.
PSII (F v /F m ) values of the concentrated cells also supports the microscopic results of cell integrity (Table 2). The yields were above 0.5, which indicates low levels of stress on the cells with no loss of vitality – cells were able to recover rapidly when exposed to fresh culture medium. 3.3.
Flocculation by commercial flocculants
In water treatment processes, pH is an important parameter to find out the optimum conditions for chemical flocculation. It is known that non-optimal pH causes excessive dosages. As the goal is treating water, the conditions of the organisms are not of great concern. However, in this study, the quality of the algal biomass after flocculation is important. Concentrated algae were kept in good condition (i.e. for their lipid content) to avoid any problem in further processes and final product quality after harvesting. pH adjustments were tried prior to and after the addition of commercial flocculants AS and PAC. In the trials where pH was acidified, depending on pH–flocculant dosage effect, chloroplasts deteriorated in the course of algal harvest or after some time (e.g. 2 h). In the case of NaOH solution addition to the culture, for pH adjustment, two behaviours were observed: • An alkalinity-induced flocculation started prior to flocculant addition. • After flocculant addition, turbidity of residual solution increased without cell sedimentation. Due to this reason, the PAC and AS flocculant experiments were performed without adjusting the pH. As shown in Figure 6(a), when AS was used as a chemical flocculant, FEs reached high levels ( ∼ 90%) after 30 min of settling (10–60 ppm AS). Above 60 ppm, it started to decrease due to overdose of flocculant. The FEs of 15
and 30 min settling times were almost the same along a wide concentration range of the flocculant, from 20 to 100 ppm. Only at 200 ppm of AS, the difference between the FE after 15 and 30 min settling was remarkable. Optimum concentration of AS for the studied C. calcitrans cultures could be concluded as 10 ppm with FE of 87.6% and CF of 7.10 for cost-efficient harvesting. Microscopic examination of the algal–alum sediment showed that the number of actively swimming individual cells was decreased by the increase in dose as expected. Most cells were static in the aggregates, although some flocs were observed to oscillate at a dose of 5 ppm AS. Flocculation caused no reduction in cell viability, however, as measured by photosynthetic yield at doses up to at least 70 ppm of AS (Figure 6(b)). Higher doses of the flocculant caused decrease in the pH of the culture, which might be related to the decrease in the (F v /F m ) measurements. When the culture was flocculated with PAC, the FE increased satisfactorily after 30 ppm (Figure 6(a)) for 15 min of settling. Furthermore, even at 5 ppm of PAC concentration, FE was quite good for 30 min ( ∼ 60% with CF of ∼ 6). C. calcitrans flocculated with PAC was faster to settle than the algae flocculated with AS (in high concentrations (up to 60 ppm)). PAC showed more stable behaviour of FE even at high doses, contrary to AS. Only at PAC dose of 200 ppm was the stability affected; the turbidity increased due to unstable particles and resulted in a low FE. An optimum result for PAC depends on the settling time. For 30 min of settling time, 20 ppm of PAC could be chosen as the optimum for the algal recovery range of 74%. Once again, there was no reduction in the yields of (F v /F m ) at doses up to 70 ppm of PAC (Figure 6(b)). Above concentrations of 80 ppm for both flocculants, AS and PAC, the colour of the cells changed slightly from brownish to dark brown (naked eye examination). The yields of (F v /F m ) were very small compared to that at lower doses.
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(a)
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(b)
Figure 6. (a) FE of C. calcitrans with AS and PAC. pH change after coagulant addition is also shown. Data are represented with ± SE (n = 3). (b) Maximum photochemical quantum yield of PSII values for harvested cells with PAC and AS. Values are the average of three measurements.
Analyses of presence of ions (Ca2+ and Mg2+ ) and viscosity Mg and Ca ion concentrations and also viscosities of algal cultures were studied for flocculation conditions: pHthreshold , pH = 11, PACoptimum , ASoptimum . Magnesium concentration of the supernatant was lower than the culture medium concentration (88%) for pHthreshold . Magnesium consumption increased by increasing the pH of the algal culture. For pH 11, flocculation was attempted not only with Mg2+ ions, but also with Ca2+ ions (Table 3). Supernatants of PACoptimum and ASoptimum (with the given concentrations in Table 3) did not show significant difference for Mg2+ and Ca2+ concentrations than the culture medium. The rheological characterization of algal suspensions was measured with the assumptions detailed in the earlier study.[16] The rheological behaviour of the microalgal suspensions was Newtonian where the apparent viscosity was constant when measured at different rotational speeds corresponding to different shear rates. Dynamic viscosities of 3.4.
2.2.–2.3 × 10−3 Pa s at 30°C after pre-concentration processing were recorded for measured samples (Table 3) where the dynamic viscosity of the seawater is given as 0.87 × 10−3 Pa s (temperature = 30°C; salinity = 38 ppt). The kinematic viscosity data of supernatants after harvesting of algae were also monitored and compared with that of the culture medium. No differences in viscosities were found before and after treatment (data not shown) for all samples. Similar results were obtained in [16] for P. tricornutum and N. gaditana species.
4. Discussion The typical initial step for algal harvesting is solid–liquid separation which involves concentration processes. These include coagulation, flotation, centrifugation, filtration, and gravity (natural) sedimentation. Natural sedimentation can be the first step in concentration (pre-concentration). For the studied cultures of C.
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S. S¸ irin et al. Table 3. Mg2+ –Ca2+ contents, dynamic viscosities, and maximum photochemical quantum yield of C. calcitrans samples harvested with studied methods.
Method of concentration pHthreshold pH = 11 PAC (20 ppm) AS (10 ppm)
Mg2+ contenta,b (%) 87 ± 1.35 0 97 ± 1.60 96 ± 2.30
Ca2+ contenta,b (%) 97 79 98 98
± ± ± ±
1.70 2.30 1.84 0.00
Viscosityc (Pa s) 2.2 2.2 2.2 2.3
× × × ×
10−3 10−3 10−3 10−3
R2d
Max. photochemical quantum yield of PSII-F v /F m (YII)e
0.9840 0.9840 0.9939 0.9968
0.621 0.547 0.646 0.652
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a Mg2+ and Ca2+ ion measurements were done on samples of the supernatant after harvesting. b Mg2+ and Ca2+ percentages were calculated by comparison to culture medium ion contents. c Viscosity measurements were done at 30°C on concentrated algal samples after harvesting. d R2 is the linear regression determination coefficient of viscosity. e For pH threshold and pH 11, the maximum photochemical quantum yield of PSII value of the fresh culture PAC and AS experiments it was ∼ 0.656. The F v /F m values are the average of three measurements.
calcitrans, we did not observe any efficient natural sedimentation. Cells were not in good condition at the end of the settling period either. (F v /F m ) yields were relatively low (F v /F m ≤ 0.3) after day 6 of settling, which shows relatively poor physiological state of the cells. Chlorophyll fluorescence is a good indicator of the damage to photosynthetic apparatus in the photoautotrophic cells.[17,18] Measurement of the fluorescence of chlorophyll in intact algal cells provides information on the absorption, distribution, and utilization of energy in photosynthesis. The maximal photochemical yield of PSII (F v /F m ), which is proportional to maximal photosynthetic efficiency, can change from 0.4 to 0.8 for different phytoplankton classes.[19,20] Low F v /F m values ( ≤ 0.2) point to a severe stress in the culture. The population growth rate was zero or the cell number in the population started to decrease, which means expiry of phytoplankton cells.[21,22] Natural sedimentation results of (F v /F m ) yields showed 54% of reduction compared to fresh culture (control) for 1 week of settling, whereas for 2 weeks (F v /F m ) yields were lower than 0.2. These levels were caused by a loss of vitality, which means almost no recovery possibility for the cells even when exposed to fresh medium. However, for the same algal species, Harith et al. [23] obtained sufficient harvesting results (91%) at 27°C either in the dark or daylight for 8 days with natural sedimentation. Not only at 27°C, but also at 4°C, the harvesting efficiency was promising (70%). The viability of the settled cells was also higher than 60% in all cases. The effective natural sedimentation depends primarily on the species. Nevertheless, the growth medium affects the charge and the cell density of the culture [10] as well as the biochemical composition of microalgae.[24] The sedimentation of C. calcitrans in [23] might be due to higher cell densities and different chemical composition of the culture grown in Conway medium with air and CO2 . By the consumption of reserve sources (lipids and a series of fatty acids, ß-carotene, etc.),[25,26] the cells might possibly
was ∼ 0.630, whereas for
change their densities, which let them to settle by time. Then, it becomes necessary to determine the extent to which the intracellular content varies over a period of time. pH-induced flocculation in the alkaline region using the presence of ions in the culture by the addition of NaOH was a mimicked auto-flocculation process at photosynthetically raised pH. Increasing the pH allowed the recovery of ca. 90% biomass of Dunaliella tertiolecta, [27] Anabaena marina,[28] C. calcitrans,[23] P. tricornutum,[10,29] and N. gaditana.[16] Besides González-López et al., [28] in all cases the cells were not negatively affected or damaged by the basic pHs. Our results demonstrated that effective flocculation for C. calcitrans could be attained by pH increase. Harvesting efficiency reached 86% and a CF (volume/volume (v/v)) of 4 was obtained, only after 10 min of settling with the studied cell densities. With the precipitation of magnesium hydroxide (Mg(OH)2 ), flocculation probably occurred by letting the cells enmesh into the precipitate. Rapid sedimentation rate was also achieved at optimum pH (pHthreshold ) with less precipitation resulting in low SSVF values. Beyond the pH threshold, up to pH ∼ 11, increasing the alkalinity of the solution was difficult where we consider this pH range as ‘buffer zone’.[30] Further NaOH addition initially caused extensive Mg(OH)2 precipitation and the formation of CaCO3 in the sequel (Table 3). Besides, the mean particle size of magnesium hydroxide seems to decrease as the pH value of the solution increases under particular experimental conditions,[16,31] causing a decrease in the settling rate hereby. The formation of loose flocs and sedimentation with different interfaces could be correlated with buffer zones as a result of excessive NaOH addition. The rough colourless matrix in which algal cells were embedded, observed under light microscopy, most probably corresponds to these precipitates as well (Figure 5(b)). (F v /F m ) yields were quite promising with low reduction compared to that of control. Microscopic observations and (F v /F m ) yields were also quite supportive to conclude that the cells were in good condition.
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Environmental Technology Under alkaline conditions, some chemical ions in the medium (i.e. Mg+2 and Ca+2 ) precipitate, along with the algal biomass, which both help in the harvesting process. The role played by each reaction depends on the primary particles and the ions contained in the solution.[10] Further studies are necessary to understand the optimum conditions of flocculation of different species and the same species that grow in different medium compositions. For the studied species, alkalinity-induced flocculation seems promising for the related culture medium composition and cell density even if the goal is biodiesel production. AS and PAC are widely used as flocculants for water treatment. They are used for harvesting microalgae as well, although they cause metal contamination in the harvested biomass. Therefore, the presence of flocculants further downstream after harvesting and how they affect the extraction and/or fuel conversion processes must also be well understood.[32] Additionally, if residual water is going to be recycled, the effect on culture growth and lipid production must be clarified.[10] However, besides these disadvantages, metal coagulants provide a good model system to study the interaction between flocculants and microalgal cells because their properties are well known.[33,34] PAC and AS treatment with pH adjustments at acidic pHs resulted as chloroplast deterioration in the course of algal harvest or after some time (e.g. 2 hours). For the acidic pHs, depending on pH–flocculant dosage effect, changing the pH by even a small amount is more of a sudden chemical change. The speed of chemical change might be so fast not to let the cell to adapt the new ambient environment. Thus, the response of the cell might be most probably manifest as chloroplast reaction which occurs on the membrane. From the experiments of two commercial flocculants (AS and PAC), it could be concluded that with enough time for the flocs to stabilize and collapse, efficient FEs were observed without damaging the cells,[16] up to doses of 80 ppm. PAC provided sufficient FEs and faster sedimentation rates compared to AS at lower concentrations. Optimum results for PAC and AS flocculants were obtained from a 5–20 ppm concentration. The recovery of the algae was between 60% and 80% with 7–8 CF by volume (v/v). (F v /F m ) yields indicated that at optimum doses of flocculant, no reduction in the viability was observed. Eldridge et al. [35] also investigated flocculation/coagulation of five marine algal species with commercial flocculants (AS, iron(III) sulphate, and Magnafloc 919). They also related (F v /F m ) yields of concentrated algal cells with cell viability and/or the state of the algae. Newtonian fluids are very similar to water, making both pumping and mixing relatively easy. The mixing times for Newtonian fluids are lower than those of nonNewtonian fluids. Most sludges show Newtonian fluid
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behaviour, especially below 4% of solid concentration, and algal cultures are generally accepted to behave like Newtonian fluids.[36] The viscosities of the concentrated samples showed Newtonian behaviour regardless of the treatments. This rheological behaviour after harvesting might be expected with lower initial cell densities of the studied cultures. Soulies et al. [37] also did not observe stress dependence of the viscosity in dilute solutions of Chlorella vulgaris species. Low viscosity of Mg(OH)2 even at high concentrations might be another reason.[16] The results showed that a slight increase in viscosity after the pre-concentration process might not cause high energy losses.[38] The supernatant viscosities showed that no viscous substances such as extracellular polysaccharide were excreted by the species during the pre-concentration process due to stress.[16] 5. Conclusion For the studied C. calcitrans cultures, both alkalinityinduced flocculation and flocculation with commercial flocculants showed promising and satisfying performances with almost similar CFs. However, if the target is biodiesel production, authors’ choice would be alkalinity-induced flocculation due to no contamination risk of algae and reusability of supernatant water. The results of maximal photochemical yield of PSII (F v /F m ) of settled algal samples were compatible with microscopic observations. The settled algal cells were all in good condition for the optimum case of treatment methods. Viscosity results showed that after the flocculation step pumping and mixing are still easily attainable and that flocculation does not increase the pumping cost because the algal concentrate still follows a Newtonian behaviour. Acknowledgements The authors would like to thank Maria Pilar Rey Valera from IREC for valuable work with the algal cultures.
Disclosure statement No potential conflict of interest was reported by the authors.
Funding Funding was obtained by projects: ENE2011-22761 ‘Biorrefineria de microalgas: optimización de las etapas de cosechado y de obtención de lípidos’ funded by the Spanish Ministry of Science and Innovation and ‘Fuels from Biomass’ (research programme funded by Excma. Diputació Tarragona). The research was supported by the European Regional Development Funds (ERDF, FEDER Programa Competitividad de Catalunya 2007–2013).
Supplementary data Supplemental data for this article can be accessed at http://dx.doi.org/10.1080/09593330.2015.1015456
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