Evaluation of physicochemical treatment conditions for the reuse of a spent growth medium in Arthrospira platensis cultivation

Algal Research 13 (2016) 159–166

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Evaluation of physicochemical treatment conditions for the reuse of a spent growth medium in Arthrospira platensis cultivation Ana Lucía Morocho-Jácome, Guilherme Favaro Mascioli, Sunao Sato, João Carlos Monteiro de Carvalho ⁎ Department of Biochemical and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, University of São Paulo, Av. Prof. Lineu Prestes, 580, B-16, 05508-900 São Paulo, SP, Brazil

a r t i c l e

i n f o

Article history: Received 11 November 2014 Received in revised form 29 October 2015 Accepted 28 November 2015 Available online xxxx Keywords: Arthrospira platensis Biomass composition Ferric chloride Organic matter Powdered activated carbon

a b s t r a c t Arthrospira platensis is a microorganism that is produced worldwide. Its industrial cultivation requires a large quantity of alkaline medium with high salinity that could pollute the environment if discharged with no further treatment. The combined effects of the physicochemical processes of flocculation and adsorption were applied to reuse the spent modified Schlösser medium for seven days of A. platensis fed-batch cultivation using urea as nitrogen source. Different concentrations of both powdered activated carbon and ferric chloride were evaluated at different contact times to remove organic matter and pigments from the spent medium. The efficiency of the process was measured by evaluating the removal of both organic matter and pigments. The best conditions of treatment that maximized the cell growth were 24.4 mg L−1 powdered activated carbon, 20.3 mg L−1 ferric chloride and 30.4 min of contact time. Under such conditions, organic matter removal was 92.3 ± 0.6%, pigment removal was 95.3 ± 0.6% and the maximum cell concentration was (4.89 ± 0.03) × 103 mg L−1, approximately 135% higher than in the standard medium [(2.09 ± 0.05) × 103 mg L−1]. This biomass had 36.1 ± 0.6% protein content, which was also higher than those values obtained in the standard medium (25.8 ± 0.9%). © 2015 Published by Elsevier B.V.

1. Introduction Production of photosynthetic microorganisms is an important research topic that is becoming very attractive for many purposes, mainly in the food and pharmaceutical industries [1,2]. Chlorella and Arthrospira are the dominant species in the microalgae market for their primary uses as food and for health purposes. They are used to prepare powders, tablets, capsules or pastilles [3]. Arthrospira has the lowest cost of production in the nutritional market. The plant gate cost of production (not including costs such as marketing) can be estimated at about $ 5 USD kg−1 [4]. Particularly, Arthrospira platensis can be cultivated in open ponds [5] and closed photobioreactors (PBRs), mainly with a tubular configuration [6,7]. Algae can be cultivated using cheap water sources, such as the sea and saline aquifers [8,9]. Compared to wastewater, the major disadvantages of these water sources are their lower nitrogen and phosphorus contents. In fact, there are several efforts using wastewaters to produce microalgae [10–12]. An algae production operation based on wastewater also provides the benefit of wastewater treatment credits [13]. Microalgae biomass is produced for food and feed, and its molecules are used as raw materials for industrial purposes. The process requires efficient harvesting of biomass from a cultivation broth. The techniques

⁎ Corresponding author at: Av. Prof. Lineu Prestes, 580, B-16, 05508-900 São Paulo, SP, Brazil. E-mail address: [email protected] (J.C.M. de Carvalho).

http://dx.doi.org/10.1016/j.algal.2015.11.022 2211-9264/© 2015 Published by Elsevier B.V.

applied in microalgae harvesting include centrifugation, flocculation, filtration and screening, flotation, gravity sedimentation, and electrophoresis techniques [14]. The selection of the harvesting technique depends on many factors, such as microalgae characteristics, density, size and the value of the desired final products [15]. Filtration is the most common method to harvest A. platensis and produces a spent growth medium rich in organic matter (OM) and pigments (Pg), expressed as high levels of absorbance at 254 nm (absorbance up to 1.445 cm−1) and 440 nm (absorbance up to 0.420 cm− 1), respectively [16]. A conventional water treatment process utilizing coagulation/flocculation, sedimentation, and filtration, has been the most common approach for OM removal (OMR) from the natural water bodies. Recently, water and wastewater treatment technologies have developed methods with greater OMR efficiencies. The success of OMR through coagulation is strongly affected by many factors, such as the nature and properties of natural OM particles, the type and dose of coagulant, pH, ionic strength, and temperature [17]. Flocculation is the process of bringing together the micro-floc particles to form large agglomerations. Wastewater carries negative charges in aqueous solution and metal salts can be used as inorganic flocculants that hydrolyze in water at their isoelectric point to form cationic species, which are adsorbed by negatively charged colloidal particles, forming flocs [18]. Ferric chloride (FeCl3) and aluminum sulfate (Al2(SO4)3) have been used as inorganic flocculants to recover microalgae biomass [19]. As far as we know, there is no information about a flocculation process to reuse the growth medium in A. platensis cultivation. However, some aggregation mechanisms for OMR such as charge neutralization,

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entrapment, and complexation with metal ions into insoluble particulate aggregates have been recently reported [20]. Although chemical precipitation is used to remove ammonia and phosphates from sewage [21], biological processes, such as algae cultivation, are very efficient in removing nitrogen, phosphorus, and other nutrients from wastewater while producing fertilizers for agriculture [22]. Electrostatic and non-electrostatic interactions between the adsorbent surface and ionic or molecular forms of the solute are responsible for the adsorption process of organic compounds onto carbonaceous materials. Several studies have shown that activated carbon (AC) is efficient in the adsorption of numerous bio-resistant organic pollutants from aqueous systems [23]. For instance, powdered activated carbon (PAC) and granular activated carbon (GAC) were investigated for their abilities in the adsorption of N-nitrosodimethylamine precursors from blends of river water and effluents from a wastewater treatment plant, reaching a reduction of 60%–90%, with OMR of approximately 75% [24]. Water requirement and nutrient costs for the industrial production of photosynthetic microorganisms are huge in all the types of cultivation processes. Moreover, water consumption needs to be reduced through a sustainable process of algae cultivation. Relatively few attempts have tested physical and physicochemical treatments of spent growth media to diminish both production costs and environmental pollution in A. platensis cultivation [16,25]. The efficiency of an adsorption process with PAC to reuse the growth medium in batch cultivations of A. platensis was demonstrated [25]. Furthermore, a recent study demonstrated that reusing 75% of a growth medium treated with GAC in the continuous cultivation of A. platensis using tubular airlift PBRs allowed a 65% reduction in the price of the growth medium [16]. However, the study did not evaluate the simultaneous use of physicochemical processes, such as flocculation and adsorption, for reusing the A. platensis medium. A. platensis is widely cultivated in an alkaline pH (9–10). Taking into account the fact that phosphorus removal by adding metal cations is not efficient in such conditions [21], phosphorus could remain after the flocculation process and act as a nutrient for cell growth. Moreover, as commented earlier, the spent medium contains OM and Pg in its composition [16], and it is poor in nitrogen [7]. As OMR is mainly dependent on the pore size distribution of the AC, the mechanisms of OM adsorption onto AC suggest that OM competes with odor compounds in water by the active sites in the AC [26]. A recent study shows that a repeated recycling of the culture medium (without previous treatment to remove OM) in A. platensis production can accumulate OM (up to 104 mg C L−1) in the medium. This OM includes 70% of sugars (mainly rhamnose), 12% of proteins and 18% may be other organic compounds. Some organic compounds could decrease both the growth rate and its protein content [27]. Therefore, reusing the spent medium for A. platensis cultivation requires previous treatment for OMR and Pg removal (PgR), followed by the addition of sodium nitrate as a nitrogen source. The aim of this work was to evaluate the A. platensis growth in the spent medium treated with different concentrations of both PAC and ferric chloride (F), applying different contact times (T) to remove OM and Pg by the combined effects of flocculation and adsorption. 2. Materials and methods 2.1. Starting the fed-batch process in PBRs A. platensis (Spirulina platensis, UTEX 1926) was obtained from the University of Texas Culture Collection (Austin, TX, USA). It was maintained at 25 °C using a standard Schlösser medium [28]. The above medium was modified by adding urea instead of NaNO3 (named as a modified medium) and used for the starting fed-batch cultivation [7] to collect the spent modified Schlösser medium. A 3.5 L tubular airlift PBR was maintained under a continuous light intensity of 120 μmol

photons m−2 s− 1. Artificial light was supplied by fluorescent lamps and measured with a luminance meter, model LI-250A (LI-COR, Lincoln, NE, USA). The culture circulation in the PBR was set at 40 ± 1 L h− 1 using an airlift mechanism provided by an air pump (Seven Star, Guangdong, China) and a rotameter (Omel, Guarulhos, SP, Brazil) [6]. The temperature was fixed at 32 ± 1 °C [7,29] by regulating the room temperature at 28 ± 1 °C. The pH of the culture medium was controlled at 9.5 ± 0.2 by the daily addition of CO2 from a cylinder. The starting urea concentration was 1.33 mmol L−1, and the fed-batch cultivation was carried out at a constant molar flow rate of 1.16 mmol L− 1 d−1 urea for seven days until the cell concentration stabilized. Filtration with a polyester membrane (20 μm) was used to harvest A. platensis from the cell suspension and to collect the spent medium. 2.2. Medium treatment with flocculation and adsorption Two-liter batches of spent medium (Section 2.1) were treated in 4 L Erlenmeyer flasks by simple mixing in a magnetic stirrer, model 752 (Fisatom, São Paulo, Brazil), at 100 rpm with different concentrations of PAC and F at different T (Table 1, part A). Experimental design and optimization are described later in Sections 2.5.3 and 2.5.4, respectively. After optimization of the experimental conditions, the confirmation experiments (Tests 20–22), were carried out under optimal values of PAC, F, and T (Table 1, part B) estimated by mathematical models from multivariable regression analyses. The regression models were used to maximize the maximum cell concentration, Xm (mg L−1), and consequently the cell productivity, PX (mg L−1 d−1). 2.3. Reusing the treated medium of A. platensis After the treatments (see Table 1), each treated medium was settled for sedimentation for 12 h and filtered with Whatman paper no. 3. The pH value was regulated at 9.5 ± 0.2 by adding CO2 from a cylinder to restore the total carbon of the medium. As the total ammonia concentration [30] was measured and considered spent in the treated medium, sodium nitrate was added as a nitrogen source at 2.5 g L−1 according to the standard medium [28]. A. platensis was inoculated in 0.5 L Erlenmeyer flasks containing 0.2 L of each treated medium at a 50 mg L−1 starting cell concentration [31]. The culture broth was maintained in a rotator shaker at 100 rpm in the same conditions of light intensity, temperature, and pH as those in Section 2.1. Standard experiments, Tests S1–S3 (Table 1, part C), were performed with the standard medium [28]. Control experiments, Tests C1–C3 (Table 1, part C) were carried out with the spent medium (without treatment) but with 2.5 g L− 1 NaNO3. These additional tests were not considered in the multivariable regression analyses, but they were considered to compare with the results of the experimental design. 2.4. Analytical methods The efficiency of the processes (flocculation/adsorption) was determined by measuring the amount of both OM and Pg in the samples before and after the process. The OM presence was determined by measuring the UV absorbance at 254 nm [32], while the Pg presence was determined by the visible light absorbance of chlorophyll-a at 440 nm [33], both with a spectrophotometer, model 700 plμs (Femto, São Paulo, Brazil). The same spectrophotometer was used to determine the cell concentration (X) by optical density (OD) measurements at 560 nm [34] with a calibration curve (with a CV of 0.5%). The pH was determined by a potentiometer, model 2100E (Mettler Toledo, São Paulo, Brazil). The following analyses were performed in the cell-free medium samples: total carbonate concentration [35], nitrate concentration [36] and total concentration of free ammonia released by urea hydrolysis, which was determined by a potentiometer, model 710A (Orion, Beverly, MA, USA) and a selective electrode of ammonia, model 95-12 (Orion, Beverly, MA, USA) [30]. Finally, after harvesting at the end of the

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Table 1 Statistical design for the Arthrospira platensis growth in the treated medium and the experimental results. PAC

F

T

OMRd

PgRe

Xmf

PTNg

LIPh

P Xi

(mg L−1)

(mg L−1)

(min)

(%)

(%)

(×103 mg L−1)

(%)

(%)

(mg L−1 d−1)

−1 −1 −1 −1 1 1 1 1 0 0 0 0 −1.687 1.687 0 0 0 0 0

30.0 50.0 30.0 50.0 30.0 50.0 30.0 50.0 23.1 56.9 40.0 40.0 40.0 40.0 40.0 40.0 40.0 40.0 40.0

6.0 6.0 14.0 14.0 6.0 6.0 14.0 14.0 10.0 10.0 3.3 16.7 10.0 10.0 10.0 10.0 10.0 10.0 10.0

20.0 20.0 20.0 20.0 40.0 40.0 40.0 40.0 30.0 30.0 30.0 30.0 13.1 46.9 30.0 30.0 30.0 30.0 30.0

81.6 83.1 82.4 81.1 86.7 90.4 91.5 90.7 90.1 89.9 88.3 92.7 73.6 91.2 91.3 91.0 91.2 91.1 91.0

86.2 86.8 84.8 85.3 91.2 93.5 95.9 95.0 94.5 94.3 92.0 96.6 78.4 95.2 94.8 94.5 95.3 94.9 94.8

4.01 4.11 3.70 3.76 4.43 4.63 4.97 4.73 4.76 4.64 4.51 5.01 3.33 4.71 4.73 4.75 4.72 4.70 4.77

27.6 28.5 27.1 27.1 29.9 32.6 34.6 32.3 31.8 29.7 31.7 36.2 25.1 33.3 31.7 33.1 31.2 32.3 32.6

7.4 6.7 8.2 7.1 8.0 6.5 7.4 7.3 6.9 7.5 6.6 6.9 8.4 8.0 7.3 7.9 7.3 7.5 7.4

573 587 529 537 633 661 710 676 680 663 644 716 476 673 676 679 674 671 681

Part B: optimization confirmation 20 −1.56 2.58 21 −1.56 2.58 22 −1.56 2.58

0.04 0.04 0.04

24.4 24.4 24.4

20.3 20.3 20.3

30.4 30.4 30.4

93.4 91.8 91.5

95.7 95.4 95.3

4.93 4.88 4.87

35.7 36.8 35.8

7.8 7.7 7.9

704 697 696

Part C: additional tests – S1j – S2j j – S3 – C1 k – C2 k – C3 k

– – – – – –

– – – – – –

– – – – – –

– – – – – –

– – – – – –

– – – – – –

2.13 2.09 2.04 2.15 2.34 2.03

26.8 25.7 25.0 24.4 24.3 25.1

6.5 7.0 7.0 8.0 8.0 8.0

304 299 291 307 334 290

Test

X1a

X2 b

Part A: statistical design 1 −1 2 1 3 −1 4 1 5 −1 6 1 7 −1 8 1 9 −1.687 10 1.687 11 0 12 0 13 0 14 0 15 0 16 0 17 0 18 0 19 0

a b c d e f g h i j k

−1 −1 1 1 −1 −1 1 1 0 0 −1.687 1.687 0 0 0 0 0 0 0

– – – – – –

X3 c

X1 = codified values of powered activated carbon concentration (PAC). X2 = codified values of ferric chloride concentration (F). X3 = codified values of contact time (T). OMR = organic matter removal. PgR = pigment removal. Xm = maximum cell concentration. PTN = total proteins content of dry biomass. LIP = total lipid content of dry biomass. Px = cell productivity. S1–S3 = standard tests performed with the standard medium (Schlösser [28]) with 2.5 g L−1 NaNO3. C1–C3 = control tests performed with the spent medium without treatment, but with 2.5 g L−1 NaNO3.

cultivations procedures, the recovered cells were filtered, washed with distilled water to remove all the adsorbed salts, and dried at 55 °C for 12 h using a forced-air circulation system. The total protein content of the dry biomass (PTN) was determined by the Kjeldahl method [37]. The total lipid content of the dry biomass (LIP) was determined by extraction with a solution of 2:1 (v/v) chloroform–methanol in a Soxhlet apparatus [38].

2.5.2. Kinetic parameter The Xm values are the maximum cell concentrations of each culture. Xo is the starting cell concentration (50 mg L−1). The cell productivity (Px) was calculated as the ratio between the total amount of cells produced per unit volume and the cultivation time (Tc), in accordance with the equation: PX ¼

2.5. Data elaboration 2.5.1. Organic matter and pigments removal OMR and PgR were measured by absorbance decreases at 254 and 440 nm, respectively. OMR and PgR were defined as the ratio between the absorbance decrease and the starting absorbance according to the equation:

OMR or PgR ¼


 AAT −ABT  100 %; ABT

ð1Þ

where ABT = medium absorbance before treatment and AAT = medium absorbance after treatment, for each wavelength.

ðX m −X o Þ : Tc

ð2Þ

2.5.3. Experimental design The experiments were planned using a 23 central composite design (CCD). The central point was repeated five times (Table 1, part A) to verify the reproducibility of the results. The design combines the vertices of the hypercube and star points [38], and the inclusion of the later points permits the estimation of the quadratic coefficients of the independent variables. It evaluated the effect of the three independent variables: PAC, F and T on the response variables: OMR, PgR, Xm, PTN and PX. The codified values of the experimental design were proportional to the actual values of the three independent variables. The codified and actual values are correlated according to the equation: xi ¼

ðX i −X o Þ ; ΔX i

ð3Þ

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where xi corresponds to the codified values of the independent variables, Xi represents the actual value of each independent variable, Xo is the actual value of the independent variable at the central point and ΔXi is the step change of Xi [39]. Each unit of codified value corresponded to the actual value of 10.0 mg L− 1 PAC, 4.0 mg L− 1 F, and 10 min. The central point was represented by PAC = 40.0 mg L−1, F = 10.0 mg L−1, and T = 30 min (Table 1) [25]. 2.5.4. Analysis of results and response surface methodology (RSM) A second order model describes the correlation between the response variables (Yi) and the independent variables, according to the general equation: Y i ¼ ai þ

X

b X j ij j

þ

X

c X2 j ij j

þ

X

d 0X X 0 j ij j j j

ð4Þ

where Y1 = OMR, Y2 = PgR, Y3 = Xm, Y4 = PTN, and Y5 = Px were derived from varying the codified values (X1, X2, and X3) of the independent variables PAC, F, and T, respectively. The dependent and independent variables are i and j, respectively, while j′, indicates their interactions; ai is the intercept, bij identifies the linear coefficients, cij represents the quadratic coefficients, and dijj′ corresponds to the interactive ones. Multivariable regression analyses were used to calculate the correlation coefficients estimated by quadratic Eq. (4), using the values of the experimental design (Table 1, part A) to optimize Xm. The analyses of variance for the regressions were performed considering an error of 5% at most (P b 0.05). The multivariable regression analyses as well as the analyses of variance were done utilizing the statistical package S-PLUS 2000. The codified levels and the experimental values of the independent variables were used for regression [40]. The relationship between many measurable response variables and a number of independent variables was evaluated using RSM [41]. 3. Results and discussion The starting cultivation of A. platensis in PBRs (Section 2.1) produced the spent medium to be treated according to Section 2.2. Almost the total amount of the starting growth medium was collected (~3.5 L). 3.1. Experimental evaluation of the combined effects of PAC, F and T on OMR and PgR The association of flocculation with F with adsorption using PAC as a simultaneous treatment process has not yet been reported for reusing the medium in A. platensis cultivation. A sustainable alternative in the large scale production of A. platensis must remove not only OM but also Pg from the spent medium to be reused in new cultures. Table 1 shows slightly higher values of PgR (78%–97%) than OMR (74%–93%). The highest average values of OMR and PgR in the tests for confirmation optimization were 92.3 ± 0.6% and 95.3 ± 0.6%, respectively (Table 1, part B). The physicochemical properties of OM could influence OMR. The combined effects of flocculation and adsorption lead to high removal of not only small but also large hydrophobic OM [42,43]. Due to the high values of both OMR and PgR (Table 1), we suggest that OM in the spent medium of A. platensis cultivation could be mainly of a hydrophobic nature, that is, mainly lipids and lipopolysaccharides [44] from cell lysis. However, another study that focuses on OM characterization in the spent medium from A. platensis cultivation may be developed. The high values of OMR (Table 1) suggest that the combined effects of flocculation and adsorption were better than the use of each process separately [43,45]. The RSM applied to the values of OMR and PgR (Fig. 1A and B, respectively) had a good mathematical model (P b 0.001 in both cases). The regression fit for OMR shows negative values of the quadratic coefficients

Fig. 1. Response surfaces of A. Organic matter removal (OMR, %) and B. pigment removal (PgR, %) estimated as functions of the codified values of both powdered activated carbon (X1) and ferric chloride (X2) maintaining the codified value of the contact time (X3) at the intermediate level (X3 = 0).

c11, c12, and c13 (Table 2) corresponding to the values of X1, X2, and X3, respectively. The adjusted model shows that the adjusted R2 = 0.93 and a low P value, thus confirming that it is statistically significant. The PgR fitting (adjusted R2 = 0.92) follows the same behavior as OMR (Table 2). Fig. 1A and B illustrate the response surfaces of OMR and PgR, respectively, as a function of X1 and X2, keeping X3 at the central level (T = 30.0 min). Particularly, the optimal conditions of OMR are at a low X1 (codified PAC value) and a high X2 (codified F value) in Fig. 1A. Table 2 Correlation coefficients estimated by Eq. (4) for prediction of organic matter removal, OMR; pigment removal, PgR; maximum cell concentration, Xm; total protein content, PTN and cell productivity, PX. Parameter

OMR

PgR

Xm

PTN

PX

yi ai bij

i=1 91.17

i=2 94.92

i=3 4741

i=4 32.55

i=5 677.1

j=1 j=2 j=3

0.194 0.834 4.433

0.158 0.808 4.444

−6.02 60.15 402.3

−0.164 0.737 2.405

−0.926 8.725 57.43

j=1 j=2 j=3

−0.709 −0.533 −3.379

−0.548 −0.583 −3.218

−51.55 −30.47 −290.5

−0.774 0.349 −1.319

−7.324 −4.337 −41.41

−0.925 0.35 0.775 27.52 0.93 b0.001

−0.413 0.038 1.138 23.35 0.92 b0.001

−60.00 −25.00 162.5 16.65 0.89 b0.001

−0.738 −0.063 0.788 8.59 0.79 b0.001

−8.50 −3.50 23.25 16.62 0.89 b0.001

cij

dijj’ jj′ = 1,2 jj′ = 1,3 jj′ = 2,3 Fcalc Adjusted R2 P value

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3.2. Experimental evaluation of the combined effects of PAC, F and T on A. platensis growth in the reused medium A. platensis is an alkalophilic microorganism that grows at a pH range of 9 and 10 in the standard medium [5]. To avoid any lack of carbon source in all the tests, the pH value was maintained at 9.5 ± 0.2 by the daily addition of CO2 from a cylinder. Thus, the CO2 consumed by the cell growth would be restored in the medium by CO2 addition. The carbon source would be fixed in the form of NaHCO3 and Na2CO3 due to the medium pH. The inorganic carbon was measured as total carbonate concentration just before CO2 addition to demonstrate that the level of carbon was maintained during the cultivation. The total carbonate values (10.5–11.6 g L−1) are almost equal to the value (12.0 g L−1) in the standard medium [28]. These carbonate values may deter the cell growth limitation because of the carbon limitation. Considering that each treated medium was reused after the addition of the same amount of NaNO3 (2.5 g L−1), the treatment conditions of Table 1 may influence the maximum cell concentration (Xm). The different final chemical composition of each treated medium that includes residuals of ferric salts, Pg, and OM can influence the cell growth. The lowest Xm value was obtained in Test 13, which corresponds to the lowest OMR and PgR values. This lowest cell growth was a consequence of OM and Pg remaining in the treated medium, which could contribute to the shadow effect already caused by cells, thus decreasing the bioavailability of light in the cultivation. Fig. 2 shows that the tests of the optimization confirmation (Tests 20–22) have the highest X m average values [(4.89 ± 0.03) × 10 3 mg L − 1 ; Table 1, part B]. These Xm average values are 135% higher than those obtained in the standard experiments (Table 1, part C) and 125% higher than those in the control experiments (Table 1, part C). In a general way, the high values of both OMR and PgR allow a high Xm in the reused medium. This high cell growth could be explained by the benefits of the treatment methodology for not only OMR and PgR, but also for improving A. platensis growth in the treated medium. Boyd et al. [46] reported that phytoplankton biomass could be increased with incremental additions in iron supply. Furthermore, the different physiological responses to iron bioavailability could reflect the nitrogen-fixation strategies, cellular size, unicellular, and/or colonial organization that characterize each cyanobacteria species [47]. Considering that the cell growth in each treated medium was higher than in the standard medium (Table 1), which could be a consequence of ferric salts' influence on the cell growth, such a standard medium may require

Fig. 2. Cell concentration (X, mg L−1) and cultivation time (Tc, d) of Arthrospira platensis in the treated medium. Tests 20–22 (□): confirmation tests under optimal conditions of treatment (powdered activated carbon, PAC = 24.4 mg L−1; ferric chloride, F = 20.3 mg L−1 and contact time, T = 30.4 min); Tests 23–25 (○): tests using the standard medium; and Tests 26–28 (◊): tests using the spent medium without treatment; error bars correspond to the standard deviation.

163

some additional ferric salts in this particular cyanobacterium strain. Thus, additional experiments were performed comparing Xm in the standard medium [28] and using such a medium with the addition of 20.0 mg L− 1 FeCl3, evidencing an increase in the Xm value of 29.6%. However, as the biologically available concentrations of Fe depend on the chemical speciation of Fe (III) in the presence of EDTA [47], a complementary study could be performed to confirm this hypothesis for A. platensis cultivation. Furthermore, regarding the spent medium, ferric salts can also precipitate in such a medium and could be adsorbed by PAC. As a consequence, it is not possible to establish a direct relationship between the initial and final concentrations of ferric salts in each treated medium. The periodic microscopic examination of the culture broth did not evince any appreciable bacterial growth. The high alkalinity and the salinity of the medium were sufficient to prevent bacterial contamination [30]. The final nitrate concentrations were low (0.04–0.08 g L−1) at the end of the cultivations as was expected in cultivations with no carbon limitation. Xm and PX have the same behavior because of their relationship in Eq. (2) and the same cultivation time (Tc). The values of PX from this research using the treated medium are higher than the PX values in the standard medium. However, there is evidence that the productivity of Chlorella vulgaris was unaffected neither by the presence nor by the accumulation of OM during medium recycling with no further treatment [48]. The treatment efficiency was also evaluated using the mathematical models to predict A. platensis growth. The multiple regression for the Xm data (Table 1, part A) (adjusted R2 = 0.89, P b 0.001) shows that Xm is a

Fig. 3. Response surfaces of A. maximum cell concentration (Xm, mg L−1) and B. cell productivity (PX, mg L−1 d−1) estimated as functions of the codified values of both powdered activated carbon (X1) and ferric chloride (X2) maintaining the codified value of the contact time (X3) at the intermediate level (X3 = 0).

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quadratic function of X1, X2, and X3. Fig. 3A illustrates the response surface of Xm as a function of X1 and X2, keeping X3 at an intermediate level (T = 30.0 min). PX fitting (adjusted R2 = 0.89, P b 0.001) has the same analyses as Xm. Therefore, Fig. 3A shows the same profile as Fig. 3B, as was explained before. The mathematical model of Xm is similar to the models of both OMR and PgR. The slopes of the lines in Fig. 4 show that the increase in OMR and PgR leads to the same effect on cell growth. Although Xm could also be induced by ferric salts in the standard medium as was explained earlier, the influence of OMR and PgR on Xm could be higher than ferric salts in the treated medium (Fig. 4). Nevertheless, as the relationship between OMR, PgR, and the remaining ferric salts in each treated medium was not evident, we can suggest that proper OMR and PgR have an important effect on the cell growth in the treated medium. Additionally, there is a negative interaction between X1 (PAC) and X2 (F) that reflects the negatives values of the coefficients of the quadratic model for Xm (Table 2). Fig. 3A shows that Xm increased proportionally to F at low PAC values, but Xm decreases with F after a threshold at high PAC values. In addition, an incremental increase in PAC also induces high Xm until the maximum value of this variable at low F values, which suggests that even PAC could contribute to Xm incremental increases. It is difficult to explain the sole influence of each variable on cell growth because the treatment process proposed in this study involves complex physicochemical phenomena that could modify the biodisponibility of not only ferric salts but also other nutrients in the cells. The differentiation of Eq. (4) to maximize Xm in the treated medium led to optimal conditions of treatment: PAC = 24.4 mg L− 1, F = 20.3 mg L−1, and T = 30.4 min. The model's validity was demonstrated because the Xm average value at optimization confirmation tests [(4.89 ± 0.03) × 103 mg L−1] is 1% higher than the value estimated by the second order model (4.85 × 103 mg L−1). 3.3. Experimental evaluation of the combined effects of PAC, F and T on A. platensis biomass composition 3.3.1. Lipid content Total lipid content (LIP) on cyanobacteria biomass can be influenced by the environmental conditions, such as the temperature, light intensity, cell concentration and nitrogen source. Tantichareon et al. [49] reported that Arthrospira sp. lipids and fatty acids depend on the strain and the environmental conditions of the culture. LIP values in the overall experiments of this work (Table 1) practically did not vary. In fact, LIP values in A. platensis are almost unvaried by the experimental conditions applied by other researchers [7,50]. LIP values in Table 1 are almost

Fig. 4. Maximum cell concentration (Xm) resulting from organic matter removal (OMR, ♦) and pigment removal (PgR, ●). Linear regressions show relationship for OMR (- - -) and PgR (—).

equal to the LIP value (7.2%) reported by Rafiqul et al. [29]¸ who investigated the optimal conditions of temperature, light intensity, and pH for Spirulina cultivation in a closed PBR. 3.3.2. Protein content The total protein content of dry biomass (PTN) in terms of cyanobacteria is affected by the environmental and nutritional conditions of the culture medium [38]. The PTN values in A. platensis biomass increased with the rate of incremental increase of both the nitrogen [51] and ferric salts [52] supply. Table 1 (part A) shows that PTN can vary according to the experimental conditions of treatment in this study (25.1–36.2%). Particularly, Test 13 reached the lowest PTN value (25.1%), and Test 12 had the highest PTN value (36.2%), probably due to the extreme treatment conditions (the lowest T and the highest F, respectively). Furthermore, the PTN values have a direct correlation with the OM, PgR, and Xm values (Table 1, part A). Under optimized growth conditions, the PTN average value (36.1 ± 0.6%) is higher than not only the standard tests (Tests S1–S3, 25.8 ± 0.9%) but also the control tests (Tests C1–C3, 24.6 ± 0.5%). The PTN average value in the treated medium is also higher than the values reached by Ferreira et al. [51] for A. platensis biomass (PTN = 28%–38%) cultivated by a fed-batch process with ammonium sulfate in a tubular PBR. The PTN regression (adjusted R2 = 0.79, P b 0.001) had negative values for the quadratic coefficients c41 and c43 (Table 2), indicating that the decreasing profile of the curve is the same irrespective of the independent variable levels. Fig. 5 illustrates the three dimensional response surface plots of PTN, which show the effects of the two independent variables named X1 and X2 (PAC and F, respectively), keeping X3 at an intermediate level (T = 30.0 min). Table 3 compares the experimental and estimated values from each mathematical model for all the variables evaluated in this research (OMR, PgR, Xm, PTN and PX). There are no differences higher than 5% between these two values (the experimental and the estimated ones), thus justifying the mathematical model's validity. The results of the RSM of this study let us suggest that the removal of both OM and Pg and the presence of the remaining ferric salts after these treatments could not only sustain A. platensis growth but also improve biomass quality in the treated medium. 4. Conclusion Removal of both OM and Pg (OMR and PgR, respectively) by the combination of flocculation and adsorption can contribute in reusing the spent medium from A. platensis cultivation. The best conditions to treat the spent medium of A. platensis cultivation were as follows:

Fig. 5. Response surface of the total protein content of the dry biomass (PTN, %) estimated as a function of the codified values of both powdered activated carbon (X1) and ferric chloride (X2) maintaining the codified value of the contact time (X3) at the intermediate level (X3 = 0).

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165

Table 3 Comparison of the values of OMR, PgR, Xm, PTN and PX experimentally obtained and the estimated ones from the RSM. Test

X1a

X2b

Part A: experimental design 1 −1 −1 2 1 −1 3 −1 1 4 1 1 5 −1 −1 6 1 −1 7 −1 1 8 1 1 9 −1.687 0 10 1.687 0 11 0 −1.687 12 0 1.687 13 0 0 14 0 0 15 0 0 16 0 0 17 0 0 18 0 0 19 0 0

X3c

−1 −1 −1 −1 1 1 1 1 0 0 0 0 −1.687 1.687 0 0 0 0 0

Part B: optimization confirmation 20 −1.56 2.58 0.04 21 −1.56 2.58 0.04 22 −1.56 2.58 0.04 a b c

OMR

OMR est

PgR

PgR est

Xm

Xm est

PTN

PTN est

Px

Px est

(%)

(%)

(%)

(%)

(×103 mg L−1)

(×103 mg L−1)

(%)

(%)

(mg L−1 d−1)

(mg L−1 d−1)

81.6 83.1 82.4 81.1 86.7 90.4 91.5 90.7 90.1 89.9 88.3 92.7 73.6 91.2 91.3 91.0 91.2 91.1 91.0

81.3 82.8 83.3 81.1 87.9 90.8 93.0 92.2 88.8 89.5 88.2 91.1 74.1 89.0 91.2 91.2 91.2 91.2 91.2

86.2 86.8 84.8 85.3 91.2 93.5 95.9 95.0 94.5 94.3 92.0 96.6 78.4 95.2 94.8 94.5 95.3 94.9 94.8

85.9 87.0 86.1 85.5 92.5 93.7 97.2 96.7 93.1 93.6 91.9 94.6 78.3 93.3 94.9 94.9 94.9 94.9 94.9

4.01 4.11 3.70 3.76 4.43 4.63 4.97 4.73 4.76 4.64 4.51 5.01 3.33 4.71 4.73 4.75 4.72 4.70 4.77

3.99 4.15 3.91 3.82 4.52 4.58 5.08 4.90 4.60 4.58 4.55 4.76 3.24 4.59 4.74 4.74 4.74 4.74 4.74

27.6 28.5 27.1 27.1 29.9 32.6 34.6 32.3 31.8 29.7 31.7 36.2 25.1 33.3 31.7 33.1 31.2 32.3 32.6

27.8 29.1 29.2 27.5 31.2 32.2 35.7 33.7 30.6 30.1 32.3 34.8 24.7 32.9 32.6 32.6 32.6 32.6 32.6

573 587 529 537 633 661 710 676 680 663 644 716 476 673 676 679 674 671 681

570 592 558 546 645 654 726 701 658 655 650 679 462 656 677 677 677 677 677

93.4 91.8 91.5

91.7 91.7 91.7

95.7 95.4 95.3

93.5 93.5 93.5

4.93 4.88 4.87

4.85 4.85 4.85

35.7 36.8 35.8

38.3 38.3 38.3

704 697 696

693 693 693

X1 = codified values of powered activated-carbon concentration (PAC). X2 = codified values of ferric chloride concentration (F). X3 = codified values of contact time (T). est = values estimated by mathematical equation.

PAC = 24.4 mg L−1, F = 20.3 mg L−1, and T = 30.4 min. Under such optimal conditions, where OMR = 92.3 ± 0.6% and PgR = 95.3 ± 0.6% led to the highest maximum cell concentration, Xm = (4.89 ± 0.03) × 103 mg L−1. This Xm average value was higher than not only the standard medium [(2.09 ± 0.05) × 103 mg L−1] but also the spent medium without treatment [(2.18 ± 0.16) × 103 mg L−1]. This research suggests that Xm was a linear function of both OMR and PgR. The PTN were increased according to the cell growth. The biomass cultivated in the treated medium under optimal conditions had PTN = 36.1 ± 0.6%, which was higher than both the standard medium (25.8 ± 0.9%) and the spent medium without treatment (24.6 ± 0.9%). Finally, the combined effects of flocculation and adsorption processes represent a promising alternative for A. platensis cultivation in the treated medium to produce biomass with high protein contents, when compared with the standard medium and/or the spent medium without treatment. Conflict of interest The authors have no conflict of interest to declare. Acknowledgments The authors acknowledge the support of “Fundação de Amparo à Pesquisa do Estado de São Paulo” (FAPESP) (processes 2010/52073-3 and 2011/52028-0), São Paulo, Brazil. References [1] A. Parikh, D. Madamwar, Partial characterization of extracellular polysaccharides from cyanobacteria, Bioresour. Technol. 97 (2006) 1822–1827. [2] N. Thajuddin, G. Subramaniam, Cyanobacterial biodiversity and potential applications in biotechnology, Curr. Sci. 29 (2005) 47–57. [3] O. Pulz, W. Gross, Valuable products from biotechnology of microalgae, Appl. Microbiol. Biotechnol. 65 (2004) 635–648. [4] J.R. Benemann, Opportunities and Challenges in Algae Biofuels Production. A Position Paper in Line With Algae World, Algae World 2008, 2008 (Available via FAO. http://www.fao.org/uploads/media/algae_positionpaper.pdf. Accessed 21 July 2013).

[5] M.S. Rodrigues, L.S. Ferreira, A. Converti, S. Sato, J.C.M. Carvalho, Fed-batch cultivation of Arthrospira (Spirulina) platensis: potassium nitrate and ammonium chloride as simultaneous nitrogen sources, Bioresour. Technol. 101 (2010) 4491–4498. [6] L.S. Ferreira, M.S. Rodrigues, A. Converti, S. Sato, J.C.M. Carvalho, Arthrospira (Spirulina) platensis cultivation in tubular photobioreactor: use of no-cost CO2 from ethanol fermentation, Appl. Energy 92 (2012) 379–385. [7] A.L. Morocho-Jácome, A. Converti, S. Sato, J.C.M. Carvalho, Kinetic and thermodynamic investigation on Arthrospira (Spirulina) platensis fed-batch cultivation in tubular photobioreactor using urea as nitrogen source, J. Chem. Technol. Biotechnol. 87 (2012) 1574–1583. [8] Q. Hu, M. Sommerfeld, E. Jarvis, M. Ghirardi, M. Posewitz, M. Seibert, Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances, Plant J. 54 (2008) 621–639. [9] P.M. Schenk, S.R. Thomas-Hall, E. Stephens, U.C. Marx, J.H. Mussgnug, C. Posten, O. Kruse, B. Hankamer, Second generation biofuels: high-efficiency microalgae for biodiesel production, Bioenergy Res. 1 (2008) 20–43. [10] M.K. Ji, H.S. Yun, S. Park, H. Lee, Y.T. Park, S. Bae, J. Ham, J. Choi, Effect of food wastewater on biomass production by a green microalga Scenedesmus obliquus for bioenergy generation, Bioresour. Technol. 179 (2015) 624–628. [11] E. Posadas, M.M. Morales, C. Gomez, F.G. Acién, R. Muñoz, Influence of pH and CO 2 source on the performance of microalgae-based secondary domestic wastewater treatment in outdoors pilot raceways, Chem. Eng. J. 265 (2015) 239–248. [12] Â.P. Matos, W.B. Ferreira, R.C.O. Torres, L.R.I. Morioka, M.H.M. Canella, J. Rotta, T. Silva, E.H.S. Moecke, E.S. Sant'Anna, Optimization of biomass production of Chlorella vulgaris grown in desalination concentrate, J. Appl. Phycol. (2014) 1–11. [13] T.J. Lundquist, I.C. Woertz, N.W.T. Quinn, J.R. Benemann, A Realistic Technology and Engineering Assessment of Algae Biofuel Production, Energy Biosciences Institute, 2010 (Available via Energy Biosciences Institute. http://www.energybiosciencesinstitute. org/sites/default/files/media/AlgaeReportFINAL.pdf. Accessed 21 May 2013). [14] 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. [15] L. Brennan, P. Owende, Biofuels from microalgae — a review of technologies for production, processing, and extractions of biofuels and co-products, Renew. Sust. Energ. Rev. 14 (2010) 557–577. [16] A.L. Morocho-Jácome, G.F. Mascioli, S. Sato, J.C.M. Carvalho, Continuous cultivation of Arthrospira platensis using exhausted medium treated with granular activated carbon, J. Hydrol. 522 (2015) 467–474. [17] M. Kabsch-Korbutowicz, Effect of Al coagulant type on natural organic matter removal efficiency in coagulation/ultrafiltration process, Desalination 185 (2005) 327–333. [18] T. Suopajärvi, H. Liimatainen, O. Hormi, J. Niinimäki, Coagulation–flocculation treatment of municipal wastewater based on anionized nanocelluloses, Chem. Eng. J. 231 (2013) 59–67. [19] A. Papazi, P. Makridis, P. Divanach, Harvesting Chlorella minutissima using cell coagulants, J. Appl. Phycol. 22 (2010) 349–355.

166

A.L. Morocho-Jácome et al. / Algal Research 13 (2016) 159–166

[20] A. Matilainen, M. Vepsäläinen, M. Sillanpää, Natural organic matter removal by coagulation during drinking water treatment: a review, Adv. Colloid Interf. Sci. 159 (2010) 189–197. [21] A.P. Sincero, G.A. Sincero, Physical–Chemical Treatment of Water and Wastewater, CRC Press, 2002. [22] S.A. Razzak, M.M. Hossain, R.A. Lucky, A.S. Bassi, Integrated CO2 capture, wastewater treatment and biofuel production by microalgae culturing—a review, Renew. Sust. Energ. Rev. 27 (2013) 622–653. [23] S.D. Faust, O.M. Aly, Adsorption Process for Water Treatment, Butterworths Publishers, Stoneham, 1987. [24] D. Hanigan, J. Zhang, P. Herckes, Adsorption of N-nitrosodimethylamine precursors by powdered and granular activated carbon, Environ. Sci. Technol. 46 (2012) 12630–12639. [25] J.C.M. Carvalho, S. Sato, A.L. Morocho-Jácome, Método de reaproveitamento de efluente a partir do cultivo de microrganismos fotossintetizantes, usos do método de reaproveitamento e usos do material orgânico reaproveitado. Br Patent PI (2010) 1.003.465–0 A2, RPI 2192.65. [26] G. Newcombe, J. Morrison, C. Hepplewhite, Simultaneous adsorption of MIB and NOM onto activated carbon. I. Characterisation of the system and NOM adsorption, Carbon 40 (2002) 2135–2146. [27] O. Depraetere, G. Pierre, W. Noppe, D. Vandamme, I. Foubert, P. Michaud, K. Muylaert, Influence of culture medium recycling on the performance of Arthrospira platensis cultures, Algal Res. 10 (2015) 48–54. [28] U.G. Schlösser, Sammlung von algenkulturen, Ber. Deut. Bot. Ges. 95 (1982) 181–276. [29] I.M. Rafiqul, K.C.A. Jalal, M.Z. Alam, Environmental factors for optimization of Spirulina biomass in laboratory culture, Biotechnology 4 (2005) 19–22. [30] J.C.M. Carvalho, F.R. Francisco, K.A. Almeida, S. Sato, A. Converti, Cultivation of Arthrospira (Spirulina) platensis by fed-batch addition of ammonium chloride at exponentially-increasing feeding rate, J. Phycol. 40 (2004) 589–597. [31] E.A. Cezare, Contribução ao estudo da produção de biomassa de Spirulina platensis empregando ureia como fonte de nitrogênio por meio de processo descontínuo alimentado(Master Thesis) University of São Paulo, São Paulo, 1998. [32] J.K. Edzwald, W.C. Becker, K.L. Wattier, Surrogate parameters for monitoring organic matter and THM precursors, J. Am. Water Works Assoc. 77 (1985) 122–132. [33] G.M. Ferrari, S. Tassan, A method using chemical oxidation to remove light absorption by phytoplankton pigments, J. Phycol. 35 (1999) 1090–1098. [34] A. Leduy, N. Therien, An improved method for optical density measurement of the semicroscopic blue algae Spirulina maxima, Biotechnol. Bioeng. 19 (1977) 1219–1224. [35] W.C. Pierce, E.L. Haenisch, Quantitative Analysis, third ed. John Willey and Sons, New York, USA, 1948. [36] A.I. Vogel, Análise química quantitativa, first ed. Livros Técnicos e Científicos, Rio de Janeiro, 2002. [37] Association of Official Analytical Chemistry, in: W. Horwitz, G. Latimer (Eds.), Official Methods of Analysis, 18th edn, Rev 2AOAC International, Arlington-VA, 2007. [38] E.J. Olguín, S. Galicia, E. Hernández, O. Angulo, The effect of low light flux and nitrogen deficiency on the chemical composition of Spirulina sp. growth on pig waste, Bioresour. Technol. 77 (2001) 19–24.

[39] A.N. Amenaghawon, A.A. Balogun, E.E. Agbonghae, S.E. Ogbeide, C.O. Okieimen, Statistical optimisation of dilute acid pre-treatment of corn stover using response surface methodology, J. E. 2 (2013) 34–40. [40] I.E. Nikerel, E. Toksoy, B. Kırdar, R. Yıldırım, Optimizing medium composition for TaqI endonuclease production by recombinant Escherichia coli cells using response surface methodology, Process Biochem. 40 (2005) 1633–1639. [41] R.H. Myers, D.C. Montgomery, Response Surface Methodology. Process and Product Optimization Using Designed Experiments, second ed. Ed Wiley, New York, 2002. [42] S. Kim, J. Cho, H.H. Ngo, The effect of pre-treatment to ultrafiltration of biologically treated sewage effluent: a detailed effluent organic matter (EfOM) characterization, Water Res. 38 (2004) 1933–1939. [43] L. Joseph, J.R.V. Flora, Y.G. Park, M. Badawy, H. Saleh, Removal of natural organic matter from potential drinking water sources by combined coagulation and adsorption using carbon nanomaterials, Sep. Purif. Technol. 95 (2012) 64–72. [44] Z. Cohen, The Chemicals of Spirulina, in: A. Vonshak (Ed.), Spirulina platensis (Arthrospira): Physiology, Cell-biology and Biotechnology, Taylor and Francis, London 1997, pp. 175–202. [45] H.K. Shon, S. Vigneswaran, I.S. Kim, J. Cho, H.H. Ngo, The effect of pretreatment to ultrafiltration of biologically treated sewage effluent: a detailed effluent organic matter (EfOM) characterization, Water Res. 38 (2004) 1933–1939. [46] P.W. Boyd, A.J. Watson, C.S. Law, E.R. Abraham, T. Trull, R. Murdoch, D.C. Bakker, A.R. Bowie, K.O. Buesseler, H. Chang, M. Charette, P. Croot, K. Downing, R. Frew, M. Gall, M. Hadfield, J. Hall, M. Harvey, G. Jameson, E.J. Laroche, M. Liddicoat, R. Ling, M.T. Maldonado, R.M. Mckay, S. Nodder, S. Pickmere, R. Pridmore, S. Rintoul, K. Safi, P. Sutton, R. Strzepek, K. Tanneberger, S. Turner, A. Waite, J. Zeldis, A mesoscale phytoplankton bloom in the polar Southern ocean stimulated by iron fertilization, Nature 407 (2000) 695–702. [47] I. Berman-Frank, A. Quigg, Z.V. Finkel, A.J. Irwin, L. Haramaty, Nitrogen-fixation strategies and Fe requirements in cyanobacteria, Limnol. Oceanogr. 52 (2007) 2260–2269. [48] F. Hadj-Romdhane, X. Zheng, P. Jaouen, J. Pruvost, D. Grizeau, J.P. Croué, P. Bourseau, The culture of Chlorella vulgaris in a recycled supernatant: effects on biomass production and medium quality, Bioresour. Technol. 132 (2013) 285–292. [49] M. Tanticharoen, M. Reungjitchachawali, B. Boonag, P. Vonktaveesuk, A. Vonshak, Z. Cohen, Optimization of γ-linolenic acid (GLA) production in Spirulina platensis, J. Appl. Phycol. 6 (1994) 295–300. [50] C.E.N. Sassano, L.A. Gioielli, L.S. Ferreira, M.S. Rodrigues, S. Sato, A. Converti, J.C.M. Carvalho, Evaluation of the composition of continuously-cultivated Arthrospira (Spirulina) platensis using ammonium chloride as nitrogen source, Biomass Bioenergy 34 (2010) 1732–1738. [51] L.S. Ferreira, M.S. Rodrigues, A. Converti, S. Sato, J.C.M. Carvalho, A new approach to ammonium sulphate feeding for fed-batch Arthrospira (Spirulina) platensis cultivation in tubular photobioreactor, Biotechnol. Prog. 26 (2010) 1271–1277. [52] A.L. Morocho-Jácome, J.C.M. Carvalho, Ferric Salts Could Influence on Arthrospira platensis Growth and Biomass Composition, 7th International Algae Congress, Hamburg-Netherlands. On Line, 2013.