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Continuous cultivation of Chlorella minutissima 26a in a tube-cylinder internal-loop airlift photobioreactor to support 3G biorefineries
Renewable Energy 130 (2019) 439e445
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Renewable Energy journal homepage: www.elsevier.com/locate/renene
Continuous cultivation of Chlorella minutissima 26a in a tube-cylinder internal-loop airlift photobioreactor to support 3G biorefineries lcio Jose Iza rio Filho b, Anuj Kumar Chandel a, Geronimo Virginio Tagliaferro a, *, He sar dos Santos a rio da Silva a, Messias Borges Silva b, c, Júlio Ce Silvio Silve ~o Paulo, CEP 12602-810, Lorena, SP, Brazil Department of Biotechnology, Engineering School of Lorena, University of Sa ~o Paulo, CEP 12602-810, Lorena, SP, Brazil Department of Chemical Engineering, Engineering School of Lorena, University of Sa c , Sa ~o Paulo State University, CEP 12516-410 - Guaratingueta , SP, Brazil Department of Production, Engineering School of Guaratingueta a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 August 2017 Received in revised form 11 April 2018 Accepted 12 June 2018 Available online 14 June 2018
Microalgae Chlorella minutissima 26a was cultivated in a tube-cylinder internal-loop airlift photobioreactor under continuous cultivation conditions. The goal was to investigate the influence of different nitrate levels on the growth and composition of microalgae. Three nitrate concentrations (75, 150 and 225 mg L1) were assessed under a fixed flow rate and the outlet flow was analyzed for concentration of biomass, lipid, carbohydrate and protein. Nitrate concentration at higher level (225 mg L1) in the medium promoted biomass growth (188.6 mg L1 d1) and lipid production (92.8 mg L1 d1), and decreased carbohydrate amount (29.1 mg L1 d1) without any change in protein content (37.7 mg L1 d1). Use of tube-cylinder internal-loop airlift photobioreactor in continuous mode could be a promising approach in algal biorefineries so called 3G biorefineries, resulting in high biomass productivity in a simple cultivation system. © 2018 Published by Elsevier Ltd.
Keywords: Chlorella minutissima Continuous microalgae cultivation Tube-cylinder internal-loop airlift photobioreactor 3G biorefineries Nitrogen repletion effect
1. Introduction Emission of carbon dioxide and other gases into the atmosphere by excessive burning of fossil fuels has considerably impacted on the greenhouse effect, thus promoting global warming and undesirable climate changes [1]. Strategic actions to reduce the greenhouse gas emissions and their harmful effect on the environment are urgent and mandatory. Gas emissions caused by burning of renewable fuels can be sequestered through photosynthesis during the biomass growth [2,3]. In this context, microalgae have been considered as one of the most promising alternative sources for biofuel production because of their fast growth rate and high biomass productivity thriving on cheap and renewable carbon sources. Microalgae biomass can be employed as a raw material for fuels production as it comprised of lipids and carbohydrates which are considered as promising feedstock for biodiesel, bioethanol and biochemicals production. Cultivation of microalgae require small physical spaces as compared to the terrestrial crops, and significantly contribute to the greenhouse gases reduction by CO2 capture
* Corresponding author. E-mail address: [email protected] (G.V. Tagliaferro). https://doi.org/10.1016/j.renene.2018.06.041 0960-1481/© 2018 Published by Elsevier Ltd.
[4e6]. In addition, microalgae can also be harnessed for the production of large number of bioactive compounds for different applications in food supplements, cosmetics, pharmaceuticals, among others [7]. Thus, a flexible multiproduct industry can be envisioned through integrated installations of 3G biorefinery by harnessing the potential of microalgae [8e10]. However, there are several obstacles that must be overcome in order to take maximum advantage from microalgae in large-scale processes such as production cost reduction, biomass yield and productivity maximization, increase in the productivity of lipids, carbohydrates and other interesting compounds [11]. In order to increase biomass production and to modify its composition, appropriate strategies for example - evaluation of the effect of nitrogen source concentration in the medium [12,13], promotion of intense interaction of microalgae with the cultivation medium, supply of optimized light and carbon source during cultivation, and the choice of an alternative photobioreactor and operation mode are necessary to adopt [14,15]. For the appropriate yields from bench-scale microalgal cultivation, more studies are requires that allow to measure, evaluate and control environmental and nutritional factors affecting growth and composition of biomass production [16]. These benchmark studies
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will pave the outcome for acquisition of necessary information for large-scale microalgae propagation. In such a case, photobioreactors enable cellular photosynthetic metabolism and offer several unique advantages, such as low possibility of contamination, better control of gas-liquid mass transfer rate, higher biomass density, greater exposure to light, high productivity per area and low cost of biomass harvesting. However, the main disadvantage of photobioreactors is the low penetration of light due to the increased cell density in the medium during the cultivation time [15]. The later needs careful evaluation of reactor designing, internal geometry of reactor, appropriate air flow rate and other operating conditions, enabling ideal amount of oxygen transfer and removal, mixing, recirculation time and light exposure frequency for adequate microalgae growth [15,17e19]. In continuous cultivation of microalgae, high biomass productivity can be achieved by reducing non-productive time, employing a uniform and optimized culture medium which facilitates the design of downstream steps [20]. Despite their advantages, airlift photobioreactors have been scarcely reported in literature for microalgae cultivation under continuous mode [16,21,22], with no previous report on Chlorella minutissima cultivation in a tube-cylinder internal-loop airlift photobioreactor. Chlorella sp. have been reported as a promising candidate, as they have a fast and easy growth [23,24], with lipid content ranging from 10.0 to 48.0% (wt) [25,26], carbohydrate content from 12.0 to 17.0% (wt) and protein content from 40.0 to 60.0% (wt) [27,28] depending on cultivation conditions. This study reports the effect of different nitrogen concentration on growth and composition of C. minutissima 26a in an autotrophic culture by using a tube-cylinder internal-loop airlift photobioreactor. The cultivation was operated in a continuous mode, and biomass production and chemical composition was assessed in terms of lipids, carbohydrates, protein and ash content. 2. Methods 2.1. Microalgae strain, storage and seed culture The microalgae C. minutissima 26a was provided by the Seaweed Culture Collection of the Oceanographic Institute at the University of S~ ao Paulo (S~ ao Paulo, SP, Brazil). Microalgae were stored and kept in artificial seawater f/2 medium [29], composed of: 33.3 g L1 NaCl, 75.0 mg L1 NaNO3, 5.0 mg L1 NaH2PO4$H2O, 3.15 mg L1 FeCl3$6H2O, 22.2 mg L1 ZnSO4$7H2O, 180 mg L1 MnCl2, 6.3 mg L1 Na2MoO4$2H2O, 10 mg L1 CoCl2$6H2O, 9.8 mg L1 CuSO2$5H2O, 100 mg L1 Thiamine (B1), 0.5 mg L1 Cyanocobalamin (B12) and 0.5 mg L1 Biotin (B7), in 250 mL Erlenmeyer flasks kept at 20 C under illuminating conditions (photon flux of 80e90 mmol m2 s1), and sub-cultured every six weeks. Seed cultures were prepared in the same f/2 medium (900 mL) with the addition of a stock culture (100 mL). Thus, autotrophic cultivation was carried out during 10 days by using a batch tube photobioreactor which was consisted of a transparent plastic bottle cylinder of 12.0 mm of diameter and 28.0 mm of height, and aerated at 0.24 vvm. Temperature was kept at 30 ± 2 C with continuous light supply at photon flux of 130 ± 2 mmol m2 s1.
light penetration. The cylindrical tube was located concentrically towards the outer column with 30 mm space between them, aiming to promote an upstream flow towards the central tube and downstream flow towards the outer tube. Compressed air was pumped through a sterile filter and supplied to the reactor via porous stones placed centrally at the base of the draft tube at the flow rate of 0.24 vvm. For all experiments, temperature was kept at 30 ± 2 C and continuous light supply was provided via fluorescent lamps with photon flux density of 125e130 mmol m2 s1. A peristaltic pump was used to feed the photobioreactor during the continuous mode operation. 2.3. Batch cultivation in airlift photobioreactor Batch experiments were carried out to determine the maximum specific growth rate (mmax), with the estimated feed flow rate during the continuous cultivation. Culture medium was consisted of synthetic seawater f/2 with 150 mg L1 NaNO3, and the seed culture (as described in section 2.1) was added at a volumetric fraction of 10%. Samples were taken periodically taken for dry cell weight (DCW) measurements. Specific growth rate was estimated by the slope of regression in the exponential phase (linear region) obtained from the curve of natural logarithm of dry cell weight as a function of time. The hydraulic residence time (HRT) was calculated as a function of the dilution ratio (“D”) the by following equation: HRT ¼ 1/D where D ¼ 0.8 mmax. 2.4. Continuous cultivation in the airlift photobioreactor In all experiments, seed culture was prepared as described in section 2.1, was added into the medium at a volumetric fraction of 10%. For continuous cultivation, firstly, the airlift photobioreactor was operated in batch mode until the microalgae growth reached to the exponential phase. Thereafter, the airlift photobioreactor was fed with the culture medium at a flow rate of 0.4 mL min1, corresponding to a hydraulic residence time (HRT) of 155 h. The harvesting was performed daily (approximately after 24 h), and the culture solution was centrifuged at 1800 g and dried in an oven at 60 C for 24 h.
2.2. Tube-cylinder internal-loop airlift photobioreactor The tube-cylinder airlift photobioreactor used in this study was having following dimensions: 550 mm in height and 120 mm in diameter with the concentric tube (one tube is placed inside another) of 340 mm by 60 mm, with a working volume of 3.8 L. The bioreactor lay out is shown in Fig. 1. The tubes were made of transparent acrylic polymer for the visual observation of effective
Fig. 1. Schematic diagram of continuous cultivation system used for the cultivation of microalga C. minutissima.
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2.5. Analytical methods Cell growth was monitored by measuring the dry cell weight (DCW). Aliquots of 10 mL were taken from the medium, centrifuged at 1800 g for 10 min, dried in an oven at 60 C for 24 h, and weighed on an analytical balance. Humidity was determined by using a semi-analytical infrared balance (model MB25, OHAUS, Pine Brook, USA) with a heating program to get the final temperature of 135 C. Nitrate concentrations in the medium were determined according to the standard method of APHA 4500-NO-3 [30]. Total intracellular lipids were extracted by following the procedure proposed by Bligh and Dyer [31], and oil yield of microalgae (%) was calculated taking into consideration the dry mass of the sample. Carbohydrates in the algal biomass was determined by the method of Moxley and Zang [32], using phenol-sulfuric method for total sugar determination [33] and high performance liquid chromatography (HPLC) for measurement of different monomeric sugars present in biomass hydrolysate. HPLC Agilent 1200 series (Agilent Technologies, Inc., USA) was equipped with a HPX-87H (300 7.8 mm) column (Bio-Rad, USA) and the analysis conditions were as following: 45 C column temperature, 0.01 N H2SO4 as the mobile phase at 0.6 mL min1 flow rate, 20 mL injection volume and refractive index detector RID-6A. Crude protein content was estimated according to Becker [34] “protein concentration ¼ nitrogen content 6.25”, with the total nitrogen concentration measured by Kjeldahl method [35]. Ash content was determined according to the analytical method of Wychen and Laurens [36]. All analyses were performed in triplicate. 3. Results and discussion 3.1. Batch growth profile of C. minutissima 26a in a tube-cylinder internal-loop airlift photobioreactor Considering the microalgae growth profile in batch run, specific growth rate (mmax) was found as 0.008 h1 (0.19 day1), which is close to the value reported by Cabello et al. [37] for the microalgae Scenedesmus obtusiusculus when cultivated in a 20.0 L tubular airlift photobioreactor (mmax, 0.005 h1). However, mmax values can vary widely, as shown in the work of Olivieri et al. [19] who reported different values from 0.1 to 0.8 day1 for different species of Chlorella. This variation in mmax is due to the differences in cultivation systems, growth conditions and culture medium used to cultivate Chlorella spp. Although different nitrogen source concentrations were used in continuous cultivation could result in variation of mmax values, the value calculated during the batch growth experiment was selected to have an understanding on the dilution rate to be used in continuous experiments. Thus, a dilution rate (D) was 20% lower than mmax value (D ¼ 0.006 h1) was considered with the purpose of avoiding washout in reactor. This dilution corresponded to the HRT of 155 h. A reactor of 3.8 L working volume was used and the feed was supplemented with the flow rate of 0.40 mL min1. 3.2. Effect of nitrate concentration on biomass growth in airlift photobioreactor continuous cultivation Continuous cultivation was started as a batch mode and after 155 h of cultivation, the feed was initiated in a continuous mode. The continuous operation was operated for 720 h. The results obtained for biomass growth and remaining nitrate concentration in the medium are shown in Fig. 2. Two main cultivation phases i.e. initial batch and continuous operation were presented. Continuous operation was presented by considering hydraulics residences times: HRT1, HRT2 and HRT3.
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As Fig. 2 showed, microalgae biomass increased concomitantly by increasing nitrogen source concentration in the medium. DCW values (Fig. 2a) in the batch phase was 0.25 g L1, 0.40 g L1 and 0.6 g L1 for nitrate initial concentrations of 75 mg L1, 150 mg L1 and 225 mg L1, respectively, during 155 h of culture time. Increase in biomass concentration was also observed using higher nitrogen source concentrations after starting the continuous feed into the reactor at 155 h. Fig. 2a shows that, during the first hydraulic residence time of cultivation, HRT1 (155e310 h), the growth profile was unstable, probably due to the transition between batch and continuous operation modes. This was expected, as HRT value represents the time required to exchange the entire reactor volume for the feed flow. Fig. 2 shows that, after first HRT, continuous cultivation reached into steady state with the DCW values (average ± standard deviation) of 0.70 ± 0.04 g L1, 0.96 ± 0.09 g L1 and 1.31 ± 0.08 g L1 for experiments where nitrate solution was fed with the feeding rate of 75 mg L1, 150 mg L1 and 225 mg L1, respectively, at the beginning of HRT2 phase. Fig. 2b shows the remaining nitrate concentration in the reactor during the cultivation process. Depletion in nitrate concentration in the reactor was evident during the batch phase, which decreased up to about 10 mg L1 from the beginning of feeding flow. During the continuous phase, only small differences could be observed for experiments with feeding of different nitrate concentrations. These differences were more evident at the beginning of region HRT3, with nitrate concentrations of about 14 mg L1, 25 mg L1 and 37 mg L1 inside the reactor for the tests with high nitrate concentration at feeding flow of 75 mg L1, 150 mg L1 and 225 mg L1, respectively. A complete consumption of the nitrogen source was n-Oropeza et al. [38] in the batch cultivation of observed by Milla the microalga Nannochloropsis oculata after a period of 6, 8 and 8 days in cultures with initial nitrate concentrations of 150, 200 and 250 mg L1, respectively. In the present study, nitrate was added in reactor continuously before the complete depletion of nitrogen. However, initial nitrogen source concentration had an effect on total produced biomass, and on microalgae composition. 3.3. Effect of nitrate concentration on biomass composition in airlift photobioreactor continuous cultivation Besides biomass production, nitrogen source concentration has been regarded as a decisive factor in microalgae biomass composition [13,38]. Fig. 3 shows biomass composition in terms of its main components as a function of NaNO3 concentration in airlift photobioreactor continuous cultivation by considering three hydraulic residence times from starting the feeding flow. During first hydraulic residence time (155e310 h), initial nitrate concentration in the feeding medium (in the feeding flowrate), showed a slight difference in the biomass chemical composition as compared with the biomass composition obtained from the stationary state (hydraulics residences times HRT2 and HRT3, 310e620 h). However, for both of them, transition time or stationary state, there was a similar behavior regarding compositional changes at different nitrate concentrations. Protein content was not influenced by changes in initial nitrate concentration in the medium within the evaluated range. Protein content remained of about 20% in all cases. Although there are some reports in literature which indicate change in protein content due to variation in initial nitrogen source concentration [23,39]. However, protein content depends on the evaluated range of nitrate concentration. Kim et al. [13], found higher protein content when nitrate concentration was increased from 75 mg L1 to 375 mg L1. Pancha et al. [40] did not observe any decrease in crude protein content in microalgae Scenedesmus sp. CCNM 1077, when nitrate
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Fig. 2. Profile of dry biomass (a), and nitrate concentration (b) left-over nitrate concentration in the airlift photobioreactor during the process carried out by using different nitrate concentrations in the medium. DCW: dry cell weight; HRT: hydraulic residence time.
concentration in the growth medium was decreased from 247 to 61.75 mg L1. In the present work, changes in nitrate concentration showed main change in lipid and carbohydrate content (Fig. 3), but only when nitrate concentration increased from 75 to 150 mg L1. There were no considerable changes found when nitrate concentration increased from 150 to 225 mg L1. In the steady state, lipid content increased from about 45% to about 49% when initial nitrate concentration was changed from 75 to 150 mg L1. On the other hand, carbohydrate content decreased from about 21% to 16% for nitrate concentration ranging from 75 to 150 mg L1, and showed a slight decrease when the nitrate concentration changed to 225 mg L1. Similar behavior was reported by Feng et al. [41] using Isochrysis zhangjiangensis cultured in batch experiments with nitrate addition at regular intervals. In that work, experiments were carried out by varying nitrate concentration at a large range (0.075e9.0 g L1). The authors observed that increased nitrogen concentration in the medium resulted in higher lipid content in the cells and lower carbohydrate concentrations with no major changes in protein content. Lipid accumulation in microalgae was promoted by increased nitrate concentration in the culture medium (Fig. 3). This is the opposite of the observed behavior for some Chlorella species in some studies [42,43]. However, in the present study, even at the increased nitrogen concentration in the feed medium, remaining nitrate was found low (Fig. 2b) causing nitrogen stress conditions for cells. The role of nitrogen in lipid accumulation in microalgae is a complex topic and must be carefully evaluated, resulting in different lipid accumulation by different species. Type of nitrogen sources and feeding pattern also influences the lipid accumulation in microalgae. As discussed by Feng et al. [41], metabolic changes that favor lipid accumulation can occur both by stress caused by
nitrogen source depletion in the medium or by its excess availnchez-García et al. [44] observed that C. minutissima ability. Sa when grown in the medium in which initial nitrate concentration increased from 57 to 113 mg L1, lipid content in the cells increased from 23 to 37%, keeping this value when nitrate concentration was increased to 225 mg L1. Table 1 shows the composition of carbohydrate fraction of biomass cultured in different nitrate concentrations on steady state, during 3rd hydraulic residence time (HRT3). Xylose, galactose and mannose sugars were determined together because they were coeluted in HPLC column, the same behavior was observed by Cheng et al. [45]. Biomass cultivated with 75 mg L1 of nitrate concentration in the feeding medium comprised of xylose, galactose, and mannose in higher quantity (10.85 ± 0.55%), representing 50.2% of the total sugars, while glucose presented the second value (5.94 ± 0.3%), representing 27.5% of the total sugars. However, for the biomass cultivated using 225 mg L1 of nitrate concentration, glucose was the main sugar with higher mass fraction value (8.18 ± 0.28%), representing 53.8% of the total sugars, while total monomeric sugars (xylose þ galactose þ mannose) represented only 33.6% of total value. In all cases, arabinose was found in lower quantity (~4%), with a decreased mass fraction value when initial nitrate concentration was increased. Increasing in the initial nitrate concentration in the culture medium from 75 to 225 mg L1 promoted an increase in glucose mass fraction and decreased the total sugars (xylose þ galactose þ mannose þ arabinose) mass fraction in biomass. Carbohydrates represent a group of reducing sugars and polysaccharides such as starch and cellulose. Among them, starch is the most abundant polysaccharide in Chlorella sps. Ortiz-Tena et al. [46] grown Chlorella vulgaris in BG-11 medium in autotrophic batch cultivation and observed that 46.5% of the biomass carbohydrates were composed of starch followed by 26.4% of monomeric sugars and only 6% of structural polysaccharides (cell wall). This study showed the rhamnose (29.1%), galactose (21.2%), glucose (16.2%), xylose and arabinose (11.4%), besides glucosamine (7.9%), mannose (7.4%) and glucuronic acid (6.8%). In the present study, sugars present in cell wall were not measured. However, glucose was present in small quantities when 75 mg L1 of nitrate was used in the feeding medium. C. minutissima showed very low amount of carbohydrate in form of starch when grown in the feeding medium composed of 75 mg L1 of nitrate. When nitrate concentration was increased to 225 mg L1, the increase in glucose content can indicate changes in cell wall or starch storage. Increase in nitrate concentration (>225 mg L1) may lead the high starch accumulation in Chlorella microalgae [47]. The effect of nitrogen source concentration in the continuous process must be considered from a global viewpoint and thus productivity is an important parameter. Table 2 shows the effect of different nitrogen concentration in the medium on biomass, lipid, protein, and carbohydrate productivity during steady state. As shown in Fig. 3, increase in nitrate concentration in the medium resulted in higher productivity values. When nitrate concentration was increased from 75 to 225 mg L1, biomass, lipid, protein and carbohydrate productivities were 88%, 106%, 81% and 37% higher, respectively. Although carbohydrate content in the biomass decreased with increasing nitrate concentration in the medium (Fig. 3), but productivity increased, reaching a maximum value of 29.1 ± 1.7 mg L1 d1 for a concentration of 225 mg L1 (Table 2), due to high biomass growth. Values in Table 2 are indicative of the potential of continuous internal-loop airlift photobioreactor. Maximum values were reached for biomass (188.6 ± 11.2 mg L1 d1) and lipid productivity (92.8 ± 5.5 mg L1 d1). These values are higher than those observed in other continuous autotrophic cultivation processes
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Fig. 3. Biochemical composition of Chlorella minutissima 26a cultured in media with different nitrate concentrations in continuous internal-loop airlift photobioreactor. Data are correspondent to average composition at different hydraulic residence time (HRT).
Table 1 Carbohydrates composition after acid hydrolysis of microalgae biomass cultured at steady state (HRT3) under different nitrate concentration in the medium. Sugars
Mass fraction (%) Nitrate concentration (mg L1) 75
Total carbohydrate Glucose Xyl þ Gal þ Man Arabinose Others
21.6 ± 2.10 5.94 ± 0.31 10.85 ± 0.55 3.60 ± 0.62 1.21 ± 0.42
150 16.1 ± 1.98 6.07 ± 0.52 7.12 ± 0.43 1.51 ± 0.32 1.40 ± 0.61
Table 2 Influence of nitrate concentration on the biomass productivity, lipids, protein and carbohydrate amount in biomass during the continuous cultivation in airlift photobioreactor at steady state (average ± standard deviation) with D ¼ 0.006 h1. Nitrate concentration (mg L1)
Productivity (mg L1 d1) Biomass
Lipid
Protein
Carbohydrate
75 150 225
100.2 ± 6.1 138.8 ± 12.3 188.6 ± 11.2
45.1 ± 2.7 67.9 ± 6.0 92.8 ± 5.5
20.8 ± 1.3 27.8 ± 2.5 37.7 ± 2.2
21.3 ± 1.3 22.6 ± 2.0 29.1 ± 1.7
225 15.2 ± 1.56 8.18 ± 0.28 5.11 ± 0.48 1.12 ± 0.41 0.79 ± 0.39
HRT3 e 3rd hydraulic residence time after starting continuous process (from 465 h to 620 h of cultivation).
using C. minutissima as reported by Tang et al. [48]. In this work, when microalgae were cultured in a continuous stirred tank
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photobioreactor, maximum biomass and lipid productivities were about 140 mg L1 d1 and 50 mg L1 d1, respectively. Although further studies are necessary to optimize continuous autotrophic cultivation (use of CO2 instead of air) and investigating other options (heterotrophic or mixotrophic), the airlift photobioreactor used in this study seems to be an interesting alternative. Future studies using other possibilities of using this reactor, such as control in the photoperiod may provide interesting information. In the photobioreactor, there are two cultivation zones, a light region between the external and draft tube, and a dark region inside draft tube (Fig. 1). In the light region, cells receive artificial light along the reactor length to for enable photosynthesis. In the dark zone, there is air mixing and the cells are kept without light exposure. Thus, the photoperiod covered by the airlift photobioreactor enables to avoid the effect of photo-inhibition that could reduce biomass productivity [49]. 4. Conclusion Nitrogen source concentration in the medium has strongly influenced the growth and composition of C. minutissima when cultured in tube-cylinder internal-loop airlift photobioreactor. By increasing the nitrate concentration in growth medium, higher biomass production and lipid content in the cells were observed, with lower carbohydrate content. At steady state, biomass productivity was 188.6 mg L1 d1 using 225 mg L1 of nitrate concentration. Biomass was consisted of 49.2% of lipids, 20% of proteins and 15.4% of carbohydrate. Tube-cylinder internal-loop airlift photobioreactor demonstrated its potential and simplicity for the growth of C. minutissima. Notes
[10]
[11]
[12]
[13]
[14]
[15] [16]
[17]
[18]
[19]
[20] [21]
[22]
[23]
The authors declare no competing financial interest. Acknowledgements Authors would like to acknowledge Jaime Alves Capucho (EEL/ USP) for the technical design of airlift photobioreactor and the Seaweed culture collection, Department of Biological Oceanog~o Paulo (USP) for kindly donating the raphy, University of Sa microalgae Chlorella minutissima 26a. Authors also acknowledge financial support from CAPES.
[24]
[25]
[26]
[27]
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Continuous cultivation of Chlorella minutissima 26a in a tube-cylinder internal-loop airlift photobioreactor to support 3G biorefineries lcio Jose Iza rio Filho b, Anuj Kumar Chandel a, Geronimo Virginio Tagliaferro a, *, He sar dos Santos a rio da Silva a, Messias Borges Silva b, c, Júlio Ce Silvio Silve ~o Paulo, CEP 12602-810, Lorena, SP, Brazil Department of Biotechnology, Engineering School of Lorena, University of Sa ~o Paulo, CEP 12602-810, Lorena, SP, Brazil Department of Chemical Engineering, Engineering School of Lorena, University of Sa c , Sa ~o Paulo State University, CEP 12516-410 - Guaratingueta , SP, Brazil Department of Production, Engineering School of Guaratingueta a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 August 2017 Received in revised form 11 April 2018 Accepted 12 June 2018 Available online 14 June 2018
Microalgae Chlorella minutissima 26a was cultivated in a tube-cylinder internal-loop airlift photobioreactor under continuous cultivation conditions. The goal was to investigate the influence of different nitrate levels on the growth and composition of microalgae. Three nitrate concentrations (75, 150 and 225 mg L1) were assessed under a fixed flow rate and the outlet flow was analyzed for concentration of biomass, lipid, carbohydrate and protein. Nitrate concentration at higher level (225 mg L1) in the medium promoted biomass growth (188.6 mg L1 d1) and lipid production (92.8 mg L1 d1), and decreased carbohydrate amount (29.1 mg L1 d1) without any change in protein content (37.7 mg L1 d1). Use of tube-cylinder internal-loop airlift photobioreactor in continuous mode could be a promising approach in algal biorefineries so called 3G biorefineries, resulting in high biomass productivity in a simple cultivation system. © 2018 Published by Elsevier Ltd.
Keywords: Chlorella minutissima Continuous microalgae cultivation Tube-cylinder internal-loop airlift photobioreactor 3G biorefineries Nitrogen repletion effect
1. Introduction Emission of carbon dioxide and other gases into the atmosphere by excessive burning of fossil fuels has considerably impacted on the greenhouse effect, thus promoting global warming and undesirable climate changes [1]. Strategic actions to reduce the greenhouse gas emissions and their harmful effect on the environment are urgent and mandatory. Gas emissions caused by burning of renewable fuels can be sequestered through photosynthesis during the biomass growth [2,3]. In this context, microalgae have been considered as one of the most promising alternative sources for biofuel production because of their fast growth rate and high biomass productivity thriving on cheap and renewable carbon sources. Microalgae biomass can be employed as a raw material for fuels production as it comprised of lipids and carbohydrates which are considered as promising feedstock for biodiesel, bioethanol and biochemicals production. Cultivation of microalgae require small physical spaces as compared to the terrestrial crops, and significantly contribute to the greenhouse gases reduction by CO2 capture
* Corresponding author. E-mail address: [email protected] (G.V. Tagliaferro). https://doi.org/10.1016/j.renene.2018.06.041 0960-1481/© 2018 Published by Elsevier Ltd.
[4e6]. In addition, microalgae can also be harnessed for the production of large number of bioactive compounds for different applications in food supplements, cosmetics, pharmaceuticals, among others [7]. Thus, a flexible multiproduct industry can be envisioned through integrated installations of 3G biorefinery by harnessing the potential of microalgae [8e10]. However, there are several obstacles that must be overcome in order to take maximum advantage from microalgae in large-scale processes such as production cost reduction, biomass yield and productivity maximization, increase in the productivity of lipids, carbohydrates and other interesting compounds [11]. In order to increase biomass production and to modify its composition, appropriate strategies for example - evaluation of the effect of nitrogen source concentration in the medium [12,13], promotion of intense interaction of microalgae with the cultivation medium, supply of optimized light and carbon source during cultivation, and the choice of an alternative photobioreactor and operation mode are necessary to adopt [14,15]. For the appropriate yields from bench-scale microalgal cultivation, more studies are requires that allow to measure, evaluate and control environmental and nutritional factors affecting growth and composition of biomass production [16]. These benchmark studies
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will pave the outcome for acquisition of necessary information for large-scale microalgae propagation. In such a case, photobioreactors enable cellular photosynthetic metabolism and offer several unique advantages, such as low possibility of contamination, better control of gas-liquid mass transfer rate, higher biomass density, greater exposure to light, high productivity per area and low cost of biomass harvesting. However, the main disadvantage of photobioreactors is the low penetration of light due to the increased cell density in the medium during the cultivation time [15]. The later needs careful evaluation of reactor designing, internal geometry of reactor, appropriate air flow rate and other operating conditions, enabling ideal amount of oxygen transfer and removal, mixing, recirculation time and light exposure frequency for adequate microalgae growth [15,17e19]. In continuous cultivation of microalgae, high biomass productivity can be achieved by reducing non-productive time, employing a uniform and optimized culture medium which facilitates the design of downstream steps [20]. Despite their advantages, airlift photobioreactors have been scarcely reported in literature for microalgae cultivation under continuous mode [16,21,22], with no previous report on Chlorella minutissima cultivation in a tube-cylinder internal-loop airlift photobioreactor. Chlorella sp. have been reported as a promising candidate, as they have a fast and easy growth [23,24], with lipid content ranging from 10.0 to 48.0% (wt) [25,26], carbohydrate content from 12.0 to 17.0% (wt) and protein content from 40.0 to 60.0% (wt) [27,28] depending on cultivation conditions. This study reports the effect of different nitrogen concentration on growth and composition of C. minutissima 26a in an autotrophic culture by using a tube-cylinder internal-loop airlift photobioreactor. The cultivation was operated in a continuous mode, and biomass production and chemical composition was assessed in terms of lipids, carbohydrates, protein and ash content. 2. Methods 2.1. Microalgae strain, storage and seed culture The microalgae C. minutissima 26a was provided by the Seaweed Culture Collection of the Oceanographic Institute at the University of S~ ao Paulo (S~ ao Paulo, SP, Brazil). Microalgae were stored and kept in artificial seawater f/2 medium [29], composed of: 33.3 g L1 NaCl, 75.0 mg L1 NaNO3, 5.0 mg L1 NaH2PO4$H2O, 3.15 mg L1 FeCl3$6H2O, 22.2 mg L1 ZnSO4$7H2O, 180 mg L1 MnCl2, 6.3 mg L1 Na2MoO4$2H2O, 10 mg L1 CoCl2$6H2O, 9.8 mg L1 CuSO2$5H2O, 100 mg L1 Thiamine (B1), 0.5 mg L1 Cyanocobalamin (B12) and 0.5 mg L1 Biotin (B7), in 250 mL Erlenmeyer flasks kept at 20 C under illuminating conditions (photon flux of 80e90 mmol m2 s1), and sub-cultured every six weeks. Seed cultures were prepared in the same f/2 medium (900 mL) with the addition of a stock culture (100 mL). Thus, autotrophic cultivation was carried out during 10 days by using a batch tube photobioreactor which was consisted of a transparent plastic bottle cylinder of 12.0 mm of diameter and 28.0 mm of height, and aerated at 0.24 vvm. Temperature was kept at 30 ± 2 C with continuous light supply at photon flux of 130 ± 2 mmol m2 s1.
light penetration. The cylindrical tube was located concentrically towards the outer column with 30 mm space between them, aiming to promote an upstream flow towards the central tube and downstream flow towards the outer tube. Compressed air was pumped through a sterile filter and supplied to the reactor via porous stones placed centrally at the base of the draft tube at the flow rate of 0.24 vvm. For all experiments, temperature was kept at 30 ± 2 C and continuous light supply was provided via fluorescent lamps with photon flux density of 125e130 mmol m2 s1. A peristaltic pump was used to feed the photobioreactor during the continuous mode operation. 2.3. Batch cultivation in airlift photobioreactor Batch experiments were carried out to determine the maximum specific growth rate (mmax), with the estimated feed flow rate during the continuous cultivation. Culture medium was consisted of synthetic seawater f/2 with 150 mg L1 NaNO3, and the seed culture (as described in section 2.1) was added at a volumetric fraction of 10%. Samples were taken periodically taken for dry cell weight (DCW) measurements. Specific growth rate was estimated by the slope of regression in the exponential phase (linear region) obtained from the curve of natural logarithm of dry cell weight as a function of time. The hydraulic residence time (HRT) was calculated as a function of the dilution ratio (“D”) the by following equation: HRT ¼ 1/D where D ¼ 0.8 mmax. 2.4. Continuous cultivation in the airlift photobioreactor In all experiments, seed culture was prepared as described in section 2.1, was added into the medium at a volumetric fraction of 10%. For continuous cultivation, firstly, the airlift photobioreactor was operated in batch mode until the microalgae growth reached to the exponential phase. Thereafter, the airlift photobioreactor was fed with the culture medium at a flow rate of 0.4 mL min1, corresponding to a hydraulic residence time (HRT) of 155 h. The harvesting was performed daily (approximately after 24 h), and the culture solution was centrifuged at 1800 g and dried in an oven at 60 C for 24 h.
2.2. Tube-cylinder internal-loop airlift photobioreactor The tube-cylinder airlift photobioreactor used in this study was having following dimensions: 550 mm in height and 120 mm in diameter with the concentric tube (one tube is placed inside another) of 340 mm by 60 mm, with a working volume of 3.8 L. The bioreactor lay out is shown in Fig. 1. The tubes were made of transparent acrylic polymer for the visual observation of effective
Fig. 1. Schematic diagram of continuous cultivation system used for the cultivation of microalga C. minutissima.
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2.5. Analytical methods Cell growth was monitored by measuring the dry cell weight (DCW). Aliquots of 10 mL were taken from the medium, centrifuged at 1800 g for 10 min, dried in an oven at 60 C for 24 h, and weighed on an analytical balance. Humidity was determined by using a semi-analytical infrared balance (model MB25, OHAUS, Pine Brook, USA) with a heating program to get the final temperature of 135 C. Nitrate concentrations in the medium were determined according to the standard method of APHA 4500-NO-3 [30]. Total intracellular lipids were extracted by following the procedure proposed by Bligh and Dyer [31], and oil yield of microalgae (%) was calculated taking into consideration the dry mass of the sample. Carbohydrates in the algal biomass was determined by the method of Moxley and Zang [32], using phenol-sulfuric method for total sugar determination [33] and high performance liquid chromatography (HPLC) for measurement of different monomeric sugars present in biomass hydrolysate. HPLC Agilent 1200 series (Agilent Technologies, Inc., USA) was equipped with a HPX-87H (300 7.8 mm) column (Bio-Rad, USA) and the analysis conditions were as following: 45 C column temperature, 0.01 N H2SO4 as the mobile phase at 0.6 mL min1 flow rate, 20 mL injection volume and refractive index detector RID-6A. Crude protein content was estimated according to Becker [34] “protein concentration ¼ nitrogen content 6.25”, with the total nitrogen concentration measured by Kjeldahl method [35]. Ash content was determined according to the analytical method of Wychen and Laurens [36]. All analyses were performed in triplicate. 3. Results and discussion 3.1. Batch growth profile of C. minutissima 26a in a tube-cylinder internal-loop airlift photobioreactor Considering the microalgae growth profile in batch run, specific growth rate (mmax) was found as 0.008 h1 (0.19 day1), which is close to the value reported by Cabello et al. [37] for the microalgae Scenedesmus obtusiusculus when cultivated in a 20.0 L tubular airlift photobioreactor (mmax, 0.005 h1). However, mmax values can vary widely, as shown in the work of Olivieri et al. [19] who reported different values from 0.1 to 0.8 day1 for different species of Chlorella. This variation in mmax is due to the differences in cultivation systems, growth conditions and culture medium used to cultivate Chlorella spp. Although different nitrogen source concentrations were used in continuous cultivation could result in variation of mmax values, the value calculated during the batch growth experiment was selected to have an understanding on the dilution rate to be used in continuous experiments. Thus, a dilution rate (D) was 20% lower than mmax value (D ¼ 0.006 h1) was considered with the purpose of avoiding washout in reactor. This dilution corresponded to the HRT of 155 h. A reactor of 3.8 L working volume was used and the feed was supplemented with the flow rate of 0.40 mL min1. 3.2. Effect of nitrate concentration on biomass growth in airlift photobioreactor continuous cultivation Continuous cultivation was started as a batch mode and after 155 h of cultivation, the feed was initiated in a continuous mode. The continuous operation was operated for 720 h. The results obtained for biomass growth and remaining nitrate concentration in the medium are shown in Fig. 2. Two main cultivation phases i.e. initial batch and continuous operation were presented. Continuous operation was presented by considering hydraulics residences times: HRT1, HRT2 and HRT3.
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As Fig. 2 showed, microalgae biomass increased concomitantly by increasing nitrogen source concentration in the medium. DCW values (Fig. 2a) in the batch phase was 0.25 g L1, 0.40 g L1 and 0.6 g L1 for nitrate initial concentrations of 75 mg L1, 150 mg L1 and 225 mg L1, respectively, during 155 h of culture time. Increase in biomass concentration was also observed using higher nitrogen source concentrations after starting the continuous feed into the reactor at 155 h. Fig. 2a shows that, during the first hydraulic residence time of cultivation, HRT1 (155e310 h), the growth profile was unstable, probably due to the transition between batch and continuous operation modes. This was expected, as HRT value represents the time required to exchange the entire reactor volume for the feed flow. Fig. 2 shows that, after first HRT, continuous cultivation reached into steady state with the DCW values (average ± standard deviation) of 0.70 ± 0.04 g L1, 0.96 ± 0.09 g L1 and 1.31 ± 0.08 g L1 for experiments where nitrate solution was fed with the feeding rate of 75 mg L1, 150 mg L1 and 225 mg L1, respectively, at the beginning of HRT2 phase. Fig. 2b shows the remaining nitrate concentration in the reactor during the cultivation process. Depletion in nitrate concentration in the reactor was evident during the batch phase, which decreased up to about 10 mg L1 from the beginning of feeding flow. During the continuous phase, only small differences could be observed for experiments with feeding of different nitrate concentrations. These differences were more evident at the beginning of region HRT3, with nitrate concentrations of about 14 mg L1, 25 mg L1 and 37 mg L1 inside the reactor for the tests with high nitrate concentration at feeding flow of 75 mg L1, 150 mg L1 and 225 mg L1, respectively. A complete consumption of the nitrogen source was n-Oropeza et al. [38] in the batch cultivation of observed by Milla the microalga Nannochloropsis oculata after a period of 6, 8 and 8 days in cultures with initial nitrate concentrations of 150, 200 and 250 mg L1, respectively. In the present study, nitrate was added in reactor continuously before the complete depletion of nitrogen. However, initial nitrogen source concentration had an effect on total produced biomass, and on microalgae composition. 3.3. Effect of nitrate concentration on biomass composition in airlift photobioreactor continuous cultivation Besides biomass production, nitrogen source concentration has been regarded as a decisive factor in microalgae biomass composition [13,38]. Fig. 3 shows biomass composition in terms of its main components as a function of NaNO3 concentration in airlift photobioreactor continuous cultivation by considering three hydraulic residence times from starting the feeding flow. During first hydraulic residence time (155e310 h), initial nitrate concentration in the feeding medium (in the feeding flowrate), showed a slight difference in the biomass chemical composition as compared with the biomass composition obtained from the stationary state (hydraulics residences times HRT2 and HRT3, 310e620 h). However, for both of them, transition time or stationary state, there was a similar behavior regarding compositional changes at different nitrate concentrations. Protein content was not influenced by changes in initial nitrate concentration in the medium within the evaluated range. Protein content remained of about 20% in all cases. Although there are some reports in literature which indicate change in protein content due to variation in initial nitrogen source concentration [23,39]. However, protein content depends on the evaluated range of nitrate concentration. Kim et al. [13], found higher protein content when nitrate concentration was increased from 75 mg L1 to 375 mg L1. Pancha et al. [40] did not observe any decrease in crude protein content in microalgae Scenedesmus sp. CCNM 1077, when nitrate
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Fig. 2. Profile of dry biomass (a), and nitrate concentration (b) left-over nitrate concentration in the airlift photobioreactor during the process carried out by using different nitrate concentrations in the medium. DCW: dry cell weight; HRT: hydraulic residence time.
concentration in the growth medium was decreased from 247 to 61.75 mg L1. In the present work, changes in nitrate concentration showed main change in lipid and carbohydrate content (Fig. 3), but only when nitrate concentration increased from 75 to 150 mg L1. There were no considerable changes found when nitrate concentration increased from 150 to 225 mg L1. In the steady state, lipid content increased from about 45% to about 49% when initial nitrate concentration was changed from 75 to 150 mg L1. On the other hand, carbohydrate content decreased from about 21% to 16% for nitrate concentration ranging from 75 to 150 mg L1, and showed a slight decrease when the nitrate concentration changed to 225 mg L1. Similar behavior was reported by Feng et al. [41] using Isochrysis zhangjiangensis cultured in batch experiments with nitrate addition at regular intervals. In that work, experiments were carried out by varying nitrate concentration at a large range (0.075e9.0 g L1). The authors observed that increased nitrogen concentration in the medium resulted in higher lipid content in the cells and lower carbohydrate concentrations with no major changes in protein content. Lipid accumulation in microalgae was promoted by increased nitrate concentration in the culture medium (Fig. 3). This is the opposite of the observed behavior for some Chlorella species in some studies [42,43]. However, in the present study, even at the increased nitrogen concentration in the feed medium, remaining nitrate was found low (Fig. 2b) causing nitrogen stress conditions for cells. The role of nitrogen in lipid accumulation in microalgae is a complex topic and must be carefully evaluated, resulting in different lipid accumulation by different species. Type of nitrogen sources and feeding pattern also influences the lipid accumulation in microalgae. As discussed by Feng et al. [41], metabolic changes that favor lipid accumulation can occur both by stress caused by
nitrogen source depletion in the medium or by its excess availnchez-García et al. [44] observed that C. minutissima ability. Sa when grown in the medium in which initial nitrate concentration increased from 57 to 113 mg L1, lipid content in the cells increased from 23 to 37%, keeping this value when nitrate concentration was increased to 225 mg L1. Table 1 shows the composition of carbohydrate fraction of biomass cultured in different nitrate concentrations on steady state, during 3rd hydraulic residence time (HRT3). Xylose, galactose and mannose sugars were determined together because they were coeluted in HPLC column, the same behavior was observed by Cheng et al. [45]. Biomass cultivated with 75 mg L1 of nitrate concentration in the feeding medium comprised of xylose, galactose, and mannose in higher quantity (10.85 ± 0.55%), representing 50.2% of the total sugars, while glucose presented the second value (5.94 ± 0.3%), representing 27.5% of the total sugars. However, for the biomass cultivated using 225 mg L1 of nitrate concentration, glucose was the main sugar with higher mass fraction value (8.18 ± 0.28%), representing 53.8% of the total sugars, while total monomeric sugars (xylose þ galactose þ mannose) represented only 33.6% of total value. In all cases, arabinose was found in lower quantity (~4%), with a decreased mass fraction value when initial nitrate concentration was increased. Increasing in the initial nitrate concentration in the culture medium from 75 to 225 mg L1 promoted an increase in glucose mass fraction and decreased the total sugars (xylose þ galactose þ mannose þ arabinose) mass fraction in biomass. Carbohydrates represent a group of reducing sugars and polysaccharides such as starch and cellulose. Among them, starch is the most abundant polysaccharide in Chlorella sps. Ortiz-Tena et al. [46] grown Chlorella vulgaris in BG-11 medium in autotrophic batch cultivation and observed that 46.5% of the biomass carbohydrates were composed of starch followed by 26.4% of monomeric sugars and only 6% of structural polysaccharides (cell wall). This study showed the rhamnose (29.1%), galactose (21.2%), glucose (16.2%), xylose and arabinose (11.4%), besides glucosamine (7.9%), mannose (7.4%) and glucuronic acid (6.8%). In the present study, sugars present in cell wall were not measured. However, glucose was present in small quantities when 75 mg L1 of nitrate was used in the feeding medium. C. minutissima showed very low amount of carbohydrate in form of starch when grown in the feeding medium composed of 75 mg L1 of nitrate. When nitrate concentration was increased to 225 mg L1, the increase in glucose content can indicate changes in cell wall or starch storage. Increase in nitrate concentration (>225 mg L1) may lead the high starch accumulation in Chlorella microalgae [47]. The effect of nitrogen source concentration in the continuous process must be considered from a global viewpoint and thus productivity is an important parameter. Table 2 shows the effect of different nitrogen concentration in the medium on biomass, lipid, protein, and carbohydrate productivity during steady state. As shown in Fig. 3, increase in nitrate concentration in the medium resulted in higher productivity values. When nitrate concentration was increased from 75 to 225 mg L1, biomass, lipid, protein and carbohydrate productivities were 88%, 106%, 81% and 37% higher, respectively. Although carbohydrate content in the biomass decreased with increasing nitrate concentration in the medium (Fig. 3), but productivity increased, reaching a maximum value of 29.1 ± 1.7 mg L1 d1 for a concentration of 225 mg L1 (Table 2), due to high biomass growth. Values in Table 2 are indicative of the potential of continuous internal-loop airlift photobioreactor. Maximum values were reached for biomass (188.6 ± 11.2 mg L1 d1) and lipid productivity (92.8 ± 5.5 mg L1 d1). These values are higher than those observed in other continuous autotrophic cultivation processes
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Fig. 3. Biochemical composition of Chlorella minutissima 26a cultured in media with different nitrate concentrations in continuous internal-loop airlift photobioreactor. Data are correspondent to average composition at different hydraulic residence time (HRT).
Table 1 Carbohydrates composition after acid hydrolysis of microalgae biomass cultured at steady state (HRT3) under different nitrate concentration in the medium. Sugars
Mass fraction (%) Nitrate concentration (mg L1) 75
Total carbohydrate Glucose Xyl þ Gal þ Man Arabinose Others
21.6 ± 2.10 5.94 ± 0.31 10.85 ± 0.55 3.60 ± 0.62 1.21 ± 0.42
150 16.1 ± 1.98 6.07 ± 0.52 7.12 ± 0.43 1.51 ± 0.32 1.40 ± 0.61
Table 2 Influence of nitrate concentration on the biomass productivity, lipids, protein and carbohydrate amount in biomass during the continuous cultivation in airlift photobioreactor at steady state (average ± standard deviation) with D ¼ 0.006 h1. Nitrate concentration (mg L1)
Productivity (mg L1 d1) Biomass
Lipid
Protein
Carbohydrate
75 150 225
100.2 ± 6.1 138.8 ± 12.3 188.6 ± 11.2
45.1 ± 2.7 67.9 ± 6.0 92.8 ± 5.5
20.8 ± 1.3 27.8 ± 2.5 37.7 ± 2.2
21.3 ± 1.3 22.6 ± 2.0 29.1 ± 1.7
225 15.2 ± 1.56 8.18 ± 0.28 5.11 ± 0.48 1.12 ± 0.41 0.79 ± 0.39
HRT3 e 3rd hydraulic residence time after starting continuous process (from 465 h to 620 h of cultivation).
using C. minutissima as reported by Tang et al. [48]. In this work, when microalgae were cultured in a continuous stirred tank
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photobioreactor, maximum biomass and lipid productivities were about 140 mg L1 d1 and 50 mg L1 d1, respectively. Although further studies are necessary to optimize continuous autotrophic cultivation (use of CO2 instead of air) and investigating other options (heterotrophic or mixotrophic), the airlift photobioreactor used in this study seems to be an interesting alternative. Future studies using other possibilities of using this reactor, such as control in the photoperiod may provide interesting information. In the photobioreactor, there are two cultivation zones, a light region between the external and draft tube, and a dark region inside draft tube (Fig. 1). In the light region, cells receive artificial light along the reactor length to for enable photosynthesis. In the dark zone, there is air mixing and the cells are kept without light exposure. Thus, the photoperiod covered by the airlift photobioreactor enables to avoid the effect of photo-inhibition that could reduce biomass productivity [49]. 4. Conclusion Nitrogen source concentration in the medium has strongly influenced the growth and composition of C. minutissima when cultured in tube-cylinder internal-loop airlift photobioreactor. By increasing the nitrate concentration in growth medium, higher biomass production and lipid content in the cells were observed, with lower carbohydrate content. At steady state, biomass productivity was 188.6 mg L1 d1 using 225 mg L1 of nitrate concentration. Biomass was consisted of 49.2% of lipids, 20% of proteins and 15.4% of carbohydrate. Tube-cylinder internal-loop airlift photobioreactor demonstrated its potential and simplicity for the growth of C. minutissima. Notes
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[13]
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The authors declare no competing financial interest. Acknowledgements Authors would like to acknowledge Jaime Alves Capucho (EEL/ USP) for the technical design of airlift photobioreactor and the Seaweed culture collection, Department of Biological Oceanog~o Paulo (USP) for kindly donating the raphy, University of Sa microalgae Chlorella minutissima 26a. Authors also acknowledge financial support from CAPES.
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