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Struvite as alternative nutrient source for cultivation of microalgae Chlorella vulgaris
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Journal of the Taiwan Institute of Chemical Engineers 000 (2015) 1–4
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Journal of the Taiwan Institute of Chemical Engineers journal homepage: www.elsevier.com/locate/jtice
Struvite as alternative nutrient source for cultivation of microalgae Chlorella vulgaris Niels M. Moed a, Duu-Jong Lee a,b,∗, Jo-Shu Chang c a
Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 106, Taiwan Department of Chemical Engineering, National Taiwan University, Taipei 106, Taiwan c Resaerch Center for Energy Technology and Strategy, National Cheng Kung University, Tainan 701, Taiwan b
a r t i c l e
i n f o
Article history: Received 8 February 2015 Revised 10 April 2015 Accepted 18 April 2015 Available online xxx Keywords: Struvite Lipids Phosphorous Nitrogen Magnesium Bold’s Basal Media
a b s t r a c t Cultivating microalgae for derived biofuels acquires high nutrient costs. The use of struvite as an alternative nutrient for cultivation of microalgae (Chlorella vulgaris) was tested. Recipes were designed and adopted to supplement the struvite, using Bold’s Basal Media (BBM) as a reference nutrient source and enriched CO2 gas (10% v/v) or air as gas source. The C. vulgaris cells could grow well with most of the tested recipes. The lipids (C15–C18) contents for cultivated microalgae ranged 1.01–3.98 g/L. Struvite, a cheaper nutrient source than BBM, has been experimentally confirmed a feasible nutrient source for the growth of C. vulgaris. © 2015 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
1. Introduction Global phosphorus deposits are running out [1]. One viable source of phosphates is municipal wastewater. Struvite is a waste product at low cost with a chemical formula of NH4 MgPO4 6H2 O, which could be formed by adding magnesium to wastewaters containing ammonia and phosphate [2–5]. A practical example presented in Nepal showed an affordable installation which precipitated urine as struvite using nylon filters and low dosage of magnesium [6]. Algae derived biofuel is considered a promising alternative to fossil fuels [7–9]. However, cultivation of algae acquires high nutrient costs with commercial fertilizers [10]. Significant (>50%) cost reductions are said to be obtainable in biofuel production from algae if CO2 , nutrients and water can be obtained in an affordable way [11]. Renewable energy sources that require biomass are dependent on phosphorus as a nutrient. Sewage or urine is enriched in phosphorus [12,13], while efforts were made to cultivate microalgae in urines [14]. Zhang et al. [15] noted that Chlorella sorokiniana can grow in undiluted urine with 84% of nitrogen and 100% of phosphorus being consumed in batch tests; however, these authors found bacterial contamination (in urine) to the algal broth. Restated, due to the effect of challenging microorganisms, directly feeding algae with wastewater can be problematic [16–19]. One possible solution is using struvite
∗
Corresponding author. Tel.: +886 2 23625632; fax: +886 2 23623040. E-mail address: [email protected], [email protected] (D.-J. Lee).
precipitation instead of wastewaters (with challenging bacteria) as nitrogen and phosphate source. No papers were available on using struvite as a nutrient source for microalgae growth. This paper for the first time used struvite as a nutrient source for the cultivation of microalgae (Chlorella vulgaris). The Bold’s Basal Media (BBM) was used as the reference nutrient source. Contents of lipids from cultivated microalgae cells suitable for biodiesel production were measured for specific samples. 2. Materials and methods 2.1. Production of struvite The method for generating the struvite was described by Kurtulus and Tas [20]. 20.331 g MgCl2 6H2 O (Acros Organics, Geel, Belgium) in 200 mL of distilled water and 13.206 g (NH4 )2 HPO4 (Riedel-de-Haën, Nanover, Germany) in 750 mL of distilled water were intensively mixed for 2 h. After this the precipitate was filtered and washed with distilled water. Afterward the collected precipitate was dried at 37 °C. The particle size of the formed struvite was around 100 μm. 2.2. Algae cultivation The BBM has the following compositions (mg per liter: K2 HPO4 , 75; KH2 PO4 , 175; MgSO4 7H2 O, 75; NaNO3 , 250; CaCl2 2H2 O, 25; NaCl, 25; EDTA-Na4 salt, 50; KOH, 30; FeSO4 7H2 O, 5; H3 BO3 , 11.4; ZnSO4 7H2 O, 1.41; MnCl2 4H2 O, 2.9; CuSO4 5H2 O, 0.25 g;
http://dx.doi.org/10.1016/j.jtice.2015.04.027 1876-1070/© 2015 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Please cite this article as: N.M. Moed et al., Struvite as alternative nutrient source for cultivation of microalgae Chlorella vulgaris, Journal of the Taiwan Institute of Chemical Engineers (2015), http://dx.doi.org/10.1016/j.jtice.2015.04.027
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N.M. Moed et al. / Journal of the Taiwan Institute of Chemical Engineers 000 (2015) 1–4 Table 1 Receipts for the tests. All units are in mg/L. Chemical
R1
R2
R3
R4
R5
R6
R7
BBM×1
BBM×2
Str×1
Str×2
Str×3
K2 HPO4 KH2 PO4 MgSO4 7H2 O NaNO3 CaCl2 2H2 O NaCl EDTA-Na4 KOH FeSO4 7H2 O H2 SO4 H3 BO3 ZnSO4 7H2 O MnCl2 4H2 O CuSO4 5H2 O Co(NO3 )2 6H2 O Na2 MoO4 2H2 O Struvite KCl K2 SO4 NaCl
0 0 0 0 25 25 50 31 4.98 1.84 11.42 1.412 0.232 0.252 0.08 0.192 721 0 0 0
0 0 0 0 25 25 50 31 4.98 1.84 11.42 1.412 0.232 0.252 0.08 0.192 721 10 0 0
0 0 0 0 25 25 50 31 4.98 1.84 11.42 1.412 0.232 0.252 0.08 0.192 721 7.2 2 0
0 0 0 0 25 25 50 31 4.98 1.84 11.42 1.412 0.232 0.252 0.08 0.192 721 0 14.2 0
0 0 0 0 25 25 50 31 4.98 1.84 11.42 1.412 0.232 0.252 0.08 0.192 721 0 14.2 0
0 0 75 0 25 25 50 31 4.98 1.84 11.42 1.412 0.232 0.252 0.08 0.192 721 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 721 10 0 0
75 175 75 250 25 25 50 31 4.98 1.84 11.42 1.412 0.232 0.252 0.08 0.192 0 0 0 0
150 350 150 500 25 25 50 31 4.98 1.84 11.42 1.412 0.232 0.252 0.08 0.192 0 0 0 0
0 0 0 0 25 25 50 31 4.98 1.84 11.42 1.412 0.232 0.252 0.08 0.192 721 143.4 40 400
0 0 0 0 25 25 50 31 4.98 1.84 11.42 1.412 0.232 0.252 0.08 0.192 1442 143.4 40 400
0 0 0 0 25 25 50 31 4.98 1.84 11.42 1.412 0.232 0.252 0.08 0.192 2163 143.4 40 400
2.3. Chemical analysis The pH of suspension was measured using a pH meter (Multi 3430, WTW, Weilheim, Germany). The pH meter was standardized routinely using standard pH buffers. The algae samples were quantified by OD using an optical density meter (DR 2700, Hach, Loveland, Colorado, USA) at a wavelength of 685 nm. The total suspended solids of algae suspensions were quantified by filtration and drying at 110 °C for 24 h. The particle size distributions of algae suspensions were measured using an automated analyzer (HYDRO 2000MU, Malvern, Malvern, UK). The collected cells were washed and fractured, with the intracellular lipids being extracted using the methods by Nakanishi et al. [22]. The lipids in extract were converted to fatty acid methyl esters (FAMEs), which were purified by gas chromatography [23]. The fatty acids C15–C18 were counted as the part useful for biofuel production.
1.8 1.6
1 2 3 4 5 6 7 8
1.4 1.2 1.0
OD
Co(NO3 )2 6H2 O, 0.08 g; Na2 MoO4 2H2 O, 0.192). The struvite was used to replace the first four ingredients in BBM. In feasibility tests, 721 mg/L struvite was used to replace the nitrogen, phosphorus and magnesium sources in BBM. In recipes 1–5, struvite was added with BBM without K2 HPO4 , KH2 PO4 , MgSO4 7H2 O and NaNO3 . In recipes 2–5, equivalent amounts of KCl, K2 SO4 + KCl, K2 SO4 , K2 SO4 + KCl + NaCl were respectively added to make the same quantities of elements in the feed. Recipe 5 has the most similar compositions as in the BBM. In recipe 6, struvite was added with BBM without MgSO4 7H2 O. Recipe 7 had only struvite and KCl in water. Recipe 8 was the control (the BBM). Further tests with the recipe 5 but with 1442 or 2163 mg/L struvite were conducted, termed as struvite 2× and 3× test, respectively. In certain tests the concentrations of BBM were doubled (2×). All recipes are listed in Table 1. The gas fed was the enriched CO2 gas (10% v/v). In specific tests with recipe 5, ambient air was also used to demonstrate the effects of CO2 concentration on cell growth. Before algae cell cultivation, all chemicals were sterilized. The microalgae Chlorella vulgaris cultivated based on protocol based on Cheng et al. [21]. The algae cells were first cultivated in BBM for seven days and were diluted to optical density (OD) 0.2 as seed. Gas (10% CO2 + 90% air or ambient air) flow was fed into each bottle via diffuser at bottom at a rate of 100 mL/min. Fluorescent lamps provided uniform light of intensity of 25 μmol/m2 s onto each cultivation bottle. The light was supplied with no dark periods.
0.8 0.6 0.4 0.2 0.0 0
2
4
6
8
Time (d) Fig. 1. Optical densities of algae suspensions with recipe 1–8.
3. Results and discussion 3.1. Feasibility tests Except for recipe 7, the OD of cultivated algae suspensions grew with time (Fig. 1). Correspondingly, the suspension pH of recipes 1–7 was decreased from around 8.7 to 7.2 (data not shown). The two worst (recipes 7 and 8) and the two best (recipes 5 and 6) growth performances were noted. Recipe 7 was expected to be poor, because the only ingredients are struvite and potassium chloride. The best growth was spotted for recipe 5, which stayed on top after reaching that position within 2 days. Recipe 6 also did better than most others. Recipes 1 and 4 were only slightly better than the other recipes at the end of the run. These results confirm that appropriate recipe could be applied with struvite-based medium for cultivation of C. vulgaris. 3.2. Growth with recipe 5 The growth tests with recipe 5 were further conducted. Algae growth using enriched CO2 was clearly faster than using air (Fig. 2). The corresponding suspension pH dropped from above 8 to around 7 in all tests (data not shown). The enriched CO2 gas consisted for 10% of CO2 , while air only has 0.039% of CO2 , a big difference in CO2 contents; however, the corresponding growth rate difference was not that significant as expected. This observation indicated that most CO2
Please cite this article as: N.M. Moed et al., Struvite as alternative nutrient source for cultivation of microalgae Chlorella vulgaris, Journal of the Taiwan Institute of Chemical Engineers (2015), http://dx.doi.org/10.1016/j.jtice.2015.04.027
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N.M. Moed et al. / Journal of the Taiwan Institute of Chemical Engineers 000 (2015) 1–4 Table 2 Experimental results after 7 day growth tests with BBM at different concentrations or with different quantities of struvite.
2.2 2.0
BBM+CO2
1.8
BBM+air 5+CO2
1.6
5+air
1.4
OD
3
1.2 1.0 0.8
Parameter
OD
TSS
Usable lipid
Usable lipid
Unit BBM 1× BBM 2× Struvite 1× Struvite 2×
– 2.36 2.65 2.66 2.58
g/L 19.9 11.5 17.1 17.2
% 20.0 8.8 16.6 10.8
g/L 3.98 1.01 2.85 1.85
0.6 0.4 0.2 0.0
0
2
4
6
8
Time (d) Fig. 2. Optical densities of algae suspensions with recipe 5 and enriched CO2 or air feed.
5
4
OD
3
3.3. Use of struvite for algae cell growth
2 1x struvite 2x struvite 3x struvite
1
0
pended solids (VSS) measurement. Nonetheless, we can still conclude from the experimental observation that algae cells could grow on double and triple amounts of struvite at longer period than with original recipe 5: the OD peaked on day 8 for struvite 2× and on day 11 for struvite 3×. If the cultivated algae are to be used for biodiesel production, the lipid profiles of the cultivated algal cells has to be considered. The total and lipid contents (C15–C18) of four collected samples are shown in Fig. 4. The BBM 1× had the highest lipid content. The algae grown on struvite 2× were a close follow up. The BBM 2× actually grew less well than the struvite 2×. The C15–C18 lipid quantities for the samples followed (Table 2): BBM 1× > struvite 1× > struvite 2× > BBM 2×. This result is welcomed since the recipe 5 without excess chemicals could produce sufficient lipids for biodiesel production.
0
2
4
6
8
10
12
14
Time (d) Fig. 3. Optical densities of algae suspensions with recipe 5 at different quantities of struvite.
in the enriched gas feed was wasted to the discharge. Additionally, to cultivate microalgae using ambient air is welcomed since no external CO2 source such as fossil fuel power plant has to be settled close to the cultivation site. The growth tests with different quantities of struvite revealed high OD (Fig. 3), likely attributable to the suspended solids from the dosed struvite. The volatile biomass could not be accurately measured since the struvite would decompose at the temperature for volatile sus-
To make a financial comparison, prices of all chemicals were found by online suppliers on Alibaba (December 2014). Excessively high prices were sometimes found for food grade ingredients, these prices were not used in the calculations. The first four chemical costs for making 1 m3 BBM are as follows (€): K2 HPO4 , 6.98–12.2; KH2 PO4 , 11.4–21.3; MgSO4 7H2 O, 1.71–5.49; NaNO3 , 1.32–2.03. Struvite is used mainly for replacing the first four chemicals in the BBM recipe. The struvite can be obtained from wastewaters, which has a very cost (est. to be € 0.11 per m3 of struvite 1× media), much lower than € 31.2 for the BBM used. Restated, the use of struvite, a waste from wastewater treatment, can be significantly cheaper than the use of BBM for algae cultivation. 4. Conclusions This study for the first time demonstrated the feasibility of using struvite as an alternative phosphorous, nitrogen and magnesium source for cultivation of C. vulgaris. Effects of CO2 contents in gas feed
Fig. 4. Lipid contents for samples from BBM or from recipe 5. Usable lipids are fatty acids of C15–C18.
Please cite this article as: N.M. Moed et al., Struvite as alternative nutrient source for cultivation of microalgae Chlorella vulgaris, Journal of the Taiwan Institute of Chemical Engineers (2015), http://dx.doi.org/10.1016/j.jtice.2015.04.027
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N.M. Moed et al. / Journal of the Taiwan Institute of Chemical Engineers 000 (2015) 1–4
and those of chemical concentrations in liquid medium were tested. The BBM medium with 721 mg/L struvite as replacement can produce 2.85 g/L lipids suitable for biodiesel production, comparable to the use of whole BBM as medium. Use of struvite as alternative nutrient can significantly reduce algae cultivation cost. Acknowledgments This work was financially supported by National Science Council and National Taiwan University of Science and Technology. References [1] Wallan P, Davidsson S, Johansson S, Höök M. Phosphate rock production and depletion: regional disaggregated modeling and global implications. Resour Conser Recycl 2014;93:178–87. [2] Huang H, Yang J, Li D. Recovery and removal of ammonia-nitrogen and phosphate from swine wastewater by internal recycling chlorination product. Bioresour Technol 2014;172:253–9. [3] Su CC, Abarca RRM, de Luna MDG, Lu MC. Phosphate recovery from fluidizedbed wastewater by struvite crystallization technology. J Taiwan Inst Chem Eng 2014;45:2395–402. [4] Bi W, Li YY, Hu YY. Recovery of phosphorus and nitrogen from alkaline hydrolysis supernatant of excess sludge by magnesium ammonium phosphate. Bioresour Technol 2014;166:1–8. [5] Nguyen TAH, Ngo HH, Guo WS, Zhang J, Liang S, Lee DJ, et al. Modification of agricultural waste/by-products for enhanced phosphate removal and recovery: potential and obstacles. Bioresour Technol 2014;169:750–62. [6] Etter B, Tilley E, Khadka R, Udert KM. Low-cost struvite production using sourceseparated urine in Nepal. Water Res 2011;45:852–62. [7] Shu CH, Tsai CC, Chen KY, Liao WH, Huang HC. Enhancing high quality oil accumulation and carbon dioxide fixation by a mixed culture of Chlorella sp. and Saccharomyces cerevisiae. J Taiwan Inst Chem Eng 2013;44:936–42. [8] Daghrir R, Igounet L, Brar SK, Drogui P. Novel electrochemical method for the recovery of lipids from microalgae for biodiesel production. J Taiwan Inst Chem Eng 2014;45:153–62.
[9] Lee DJ, Chen GY, Chang YR, Lee KR. Harvesting of chitosan coagulated Chlorella vulgaris using cyclic membrane filtration-cleaning. J Taiwan Inst Chem Eng 2012;43:948–52. [10] Ho SH, Ye XT, Hasunuma T, Chang JS, Kondo A. Perspectives on engineering strategies for improving biofuel production from microalgae-A critical review. Biotechnol Adv 2014;32:1448–59. [11] Slade R, Bauenm A. Micro-algae cultivation for biofuels: cost, energy balance, environmental impacts and future prospects. Biomass Bioenergy 2013;53:29–38. [12] Liu Z, Zhao QL, Lee DJ, Yang N. Enhancing phosphorus recovery by a new internal recycle seeding MAP reactor. Bioresour Technol 2008;99:6488–93. [13] Liu Y, Kumar S, Kwag JH, Ra C. Magnesium ammonium phosphate formation, recovery and its application as valuable resources: a review. J Chem Technol Biotechnol 2013;88:181–9. [14] Adamsson M. Potential use of human urine by greenhouse culturing of microalgae (Scenedesmus acuminatus), zooplankton (Daphnia magna) and tomatoes (Lycopersicon). Ecol Eng 2000;16:243–54. [15] Zhang S, Lim CY, Chen CL, Liu H, Wang JY. Urban nutrient recovery from fresh human urine through cultivation of Chlorella sorokiniana. J Environ Manage 2014;145:129–36. [16] Wang L, Min M, Li YC, Chen P, Chen YF, Liu YH, et al. Cultivation of green algae Chlorella sp. in different wastewaters from municipal wastewater treatment plant. Appl Biochem Biotechnol 2010;162:1174–86. [17] He PJ, Mao B, Lü F, Shao LM, Lee DJ, Chang JS. The combined effect of bacteria and Chlorella vulgaris on the treatment of municipal wastewaters. Bioresour Technol 2013;146:562–8. [18] He PJ, Mao B, Shen CM, Shao LM, Lee DJ, Chang JS. Cultivation of Chlorella vulgaris on wastewater containing high levels of ammonia for biodiesel production. Bioresour Technol 2013;129:177–81. [19] Ma XC, Zhou WG, Fu ZQ, Cheng YL, Min M, Liu YH, et al. Effect of wastewaterborne bacteria on algal growth and nutrients removal in wastewater-based algae cultivation systems. Bioresour Technol 2014;167:8–13. [20] Kurtulus G, Tas AC. Transformation of neat and heated struvite (MgNH4 PO4 6H2 O). Mater Lett 2011;65:2883–6. [21] Cheng YL, Juang YC, Liao GY, Ho SH, Yeh KL, Chen CY, et al. Dispersed ozone flotation of Chlorella vulgaris. Bioresour Technol 2010;101:9092–6. [22] Nakanishi A, Aikawa S, Ho SH, Chen CY, Chang JS, Hasunuma T, et al. Development of lipid productivities under different CO2 conditions of marine microalgae Chlamydomonas sp. JSC4. Bioresour Technol 2014;152:147–52. [23] Tran DT, Le BH, Lee DJ, Chen CL, Wang HY, Chang JS. Microalgae harvesting and subsequent biodiesel conversion. Bioresour Technol 2013;140:179–86.
Please cite this article as: N.M. Moed et al., Struvite as alternative nutrient source for cultivation of microalgae Chlorella vulgaris, Journal of the Taiwan Institute of Chemical Engineers (2015), http://dx.doi.org/10.1016/j.jtice.2015.04.027
JID: JTICE
[m5G;April 30, 2015;21:16]
Journal of the Taiwan Institute of Chemical Engineers 000 (2015) 1–4
Contents lists available at ScienceDirect
Journal of the Taiwan Institute of Chemical Engineers journal homepage: www.elsevier.com/locate/jtice
Struvite as alternative nutrient source for cultivation of microalgae Chlorella vulgaris Niels M. Moed a, Duu-Jong Lee a,b,∗, Jo-Shu Chang c a
Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 106, Taiwan Department of Chemical Engineering, National Taiwan University, Taipei 106, Taiwan c Resaerch Center for Energy Technology and Strategy, National Cheng Kung University, Tainan 701, Taiwan b
a r t i c l e
i n f o
Article history: Received 8 February 2015 Revised 10 April 2015 Accepted 18 April 2015 Available online xxx Keywords: Struvite Lipids Phosphorous Nitrogen Magnesium Bold’s Basal Media
a b s t r a c t Cultivating microalgae for derived biofuels acquires high nutrient costs. The use of struvite as an alternative nutrient for cultivation of microalgae (Chlorella vulgaris) was tested. Recipes were designed and adopted to supplement the struvite, using Bold’s Basal Media (BBM) as a reference nutrient source and enriched CO2 gas (10% v/v) or air as gas source. The C. vulgaris cells could grow well with most of the tested recipes. The lipids (C15–C18) contents for cultivated microalgae ranged 1.01–3.98 g/L. Struvite, a cheaper nutrient source than BBM, has been experimentally confirmed a feasible nutrient source for the growth of C. vulgaris. © 2015 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
1. Introduction Global phosphorus deposits are running out [1]. One viable source of phosphates is municipal wastewater. Struvite is a waste product at low cost with a chemical formula of NH4 MgPO4 6H2 O, which could be formed by adding magnesium to wastewaters containing ammonia and phosphate [2–5]. A practical example presented in Nepal showed an affordable installation which precipitated urine as struvite using nylon filters and low dosage of magnesium [6]. Algae derived biofuel is considered a promising alternative to fossil fuels [7–9]. However, cultivation of algae acquires high nutrient costs with commercial fertilizers [10]. Significant (>50%) cost reductions are said to be obtainable in biofuel production from algae if CO2 , nutrients and water can be obtained in an affordable way [11]. Renewable energy sources that require biomass are dependent on phosphorus as a nutrient. Sewage or urine is enriched in phosphorus [12,13], while efforts were made to cultivate microalgae in urines [14]. Zhang et al. [15] noted that Chlorella sorokiniana can grow in undiluted urine with 84% of nitrogen and 100% of phosphorus being consumed in batch tests; however, these authors found bacterial contamination (in urine) to the algal broth. Restated, due to the effect of challenging microorganisms, directly feeding algae with wastewater can be problematic [16–19]. One possible solution is using struvite
∗
Corresponding author. Tel.: +886 2 23625632; fax: +886 2 23623040. E-mail address: [email protected], [email protected] (D.-J. Lee).
precipitation instead of wastewaters (with challenging bacteria) as nitrogen and phosphate source. No papers were available on using struvite as a nutrient source for microalgae growth. This paper for the first time used struvite as a nutrient source for the cultivation of microalgae (Chlorella vulgaris). The Bold’s Basal Media (BBM) was used as the reference nutrient source. Contents of lipids from cultivated microalgae cells suitable for biodiesel production were measured for specific samples. 2. Materials and methods 2.1. Production of struvite The method for generating the struvite was described by Kurtulus and Tas [20]. 20.331 g MgCl2 6H2 O (Acros Organics, Geel, Belgium) in 200 mL of distilled water and 13.206 g (NH4 )2 HPO4 (Riedel-de-Haën, Nanover, Germany) in 750 mL of distilled water were intensively mixed for 2 h. After this the precipitate was filtered and washed with distilled water. Afterward the collected precipitate was dried at 37 °C. The particle size of the formed struvite was around 100 μm. 2.2. Algae cultivation The BBM has the following compositions (mg per liter: K2 HPO4 , 75; KH2 PO4 , 175; MgSO4 7H2 O, 75; NaNO3 , 250; CaCl2 2H2 O, 25; NaCl, 25; EDTA-Na4 salt, 50; KOH, 30; FeSO4 7H2 O, 5; H3 BO3 , 11.4; ZnSO4 7H2 O, 1.41; MnCl2 4H2 O, 2.9; CuSO4 5H2 O, 0.25 g;
http://dx.doi.org/10.1016/j.jtice.2015.04.027 1876-1070/© 2015 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Please cite this article as: N.M. Moed et al., Struvite as alternative nutrient source for cultivation of microalgae Chlorella vulgaris, Journal of the Taiwan Institute of Chemical Engineers (2015), http://dx.doi.org/10.1016/j.jtice.2015.04.027
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N.M. Moed et al. / Journal of the Taiwan Institute of Chemical Engineers 000 (2015) 1–4 Table 1 Receipts for the tests. All units are in mg/L. Chemical
R1
R2
R3
R4
R5
R6
R7
BBM×1
BBM×2
Str×1
Str×2
Str×3
K2 HPO4 KH2 PO4 MgSO4 7H2 O NaNO3 CaCl2 2H2 O NaCl EDTA-Na4 KOH FeSO4 7H2 O H2 SO4 H3 BO3 ZnSO4 7H2 O MnCl2 4H2 O CuSO4 5H2 O Co(NO3 )2 6H2 O Na2 MoO4 2H2 O Struvite KCl K2 SO4 NaCl
0 0 0 0 25 25 50 31 4.98 1.84 11.42 1.412 0.232 0.252 0.08 0.192 721 0 0 0
0 0 0 0 25 25 50 31 4.98 1.84 11.42 1.412 0.232 0.252 0.08 0.192 721 10 0 0
0 0 0 0 25 25 50 31 4.98 1.84 11.42 1.412 0.232 0.252 0.08 0.192 721 7.2 2 0
0 0 0 0 25 25 50 31 4.98 1.84 11.42 1.412 0.232 0.252 0.08 0.192 721 0 14.2 0
0 0 0 0 25 25 50 31 4.98 1.84 11.42 1.412 0.232 0.252 0.08 0.192 721 0 14.2 0
0 0 75 0 25 25 50 31 4.98 1.84 11.42 1.412 0.232 0.252 0.08 0.192 721 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 721 10 0 0
75 175 75 250 25 25 50 31 4.98 1.84 11.42 1.412 0.232 0.252 0.08 0.192 0 0 0 0
150 350 150 500 25 25 50 31 4.98 1.84 11.42 1.412 0.232 0.252 0.08 0.192 0 0 0 0
0 0 0 0 25 25 50 31 4.98 1.84 11.42 1.412 0.232 0.252 0.08 0.192 721 143.4 40 400
0 0 0 0 25 25 50 31 4.98 1.84 11.42 1.412 0.232 0.252 0.08 0.192 1442 143.4 40 400
0 0 0 0 25 25 50 31 4.98 1.84 11.42 1.412 0.232 0.252 0.08 0.192 2163 143.4 40 400
2.3. Chemical analysis The pH of suspension was measured using a pH meter (Multi 3430, WTW, Weilheim, Germany). The pH meter was standardized routinely using standard pH buffers. The algae samples were quantified by OD using an optical density meter (DR 2700, Hach, Loveland, Colorado, USA) at a wavelength of 685 nm. The total suspended solids of algae suspensions were quantified by filtration and drying at 110 °C for 24 h. The particle size distributions of algae suspensions were measured using an automated analyzer (HYDRO 2000MU, Malvern, Malvern, UK). The collected cells were washed and fractured, with the intracellular lipids being extracted using the methods by Nakanishi et al. [22]. The lipids in extract were converted to fatty acid methyl esters (FAMEs), which were purified by gas chromatography [23]. The fatty acids C15–C18 were counted as the part useful for biofuel production.
1.8 1.6
1 2 3 4 5 6 7 8
1.4 1.2 1.0
OD
Co(NO3 )2 6H2 O, 0.08 g; Na2 MoO4 2H2 O, 0.192). The struvite was used to replace the first four ingredients in BBM. In feasibility tests, 721 mg/L struvite was used to replace the nitrogen, phosphorus and magnesium sources in BBM. In recipes 1–5, struvite was added with BBM without K2 HPO4 , KH2 PO4 , MgSO4 7H2 O and NaNO3 . In recipes 2–5, equivalent amounts of KCl, K2 SO4 + KCl, K2 SO4 , K2 SO4 + KCl + NaCl were respectively added to make the same quantities of elements in the feed. Recipe 5 has the most similar compositions as in the BBM. In recipe 6, struvite was added with BBM without MgSO4 7H2 O. Recipe 7 had only struvite and KCl in water. Recipe 8 was the control (the BBM). Further tests with the recipe 5 but with 1442 or 2163 mg/L struvite were conducted, termed as struvite 2× and 3× test, respectively. In certain tests the concentrations of BBM were doubled (2×). All recipes are listed in Table 1. The gas fed was the enriched CO2 gas (10% v/v). In specific tests with recipe 5, ambient air was also used to demonstrate the effects of CO2 concentration on cell growth. Before algae cell cultivation, all chemicals were sterilized. The microalgae Chlorella vulgaris cultivated based on protocol based on Cheng et al. [21]. The algae cells were first cultivated in BBM for seven days and were diluted to optical density (OD) 0.2 as seed. Gas (10% CO2 + 90% air or ambient air) flow was fed into each bottle via diffuser at bottom at a rate of 100 mL/min. Fluorescent lamps provided uniform light of intensity of 25 μmol/m2 s onto each cultivation bottle. The light was supplied with no dark periods.
0.8 0.6 0.4 0.2 0.0 0
2
4
6
8
Time (d) Fig. 1. Optical densities of algae suspensions with recipe 1–8.
3. Results and discussion 3.1. Feasibility tests Except for recipe 7, the OD of cultivated algae suspensions grew with time (Fig. 1). Correspondingly, the suspension pH of recipes 1–7 was decreased from around 8.7 to 7.2 (data not shown). The two worst (recipes 7 and 8) and the two best (recipes 5 and 6) growth performances were noted. Recipe 7 was expected to be poor, because the only ingredients are struvite and potassium chloride. The best growth was spotted for recipe 5, which stayed on top after reaching that position within 2 days. Recipe 6 also did better than most others. Recipes 1 and 4 were only slightly better than the other recipes at the end of the run. These results confirm that appropriate recipe could be applied with struvite-based medium for cultivation of C. vulgaris. 3.2. Growth with recipe 5 The growth tests with recipe 5 were further conducted. Algae growth using enriched CO2 was clearly faster than using air (Fig. 2). The corresponding suspension pH dropped from above 8 to around 7 in all tests (data not shown). The enriched CO2 gas consisted for 10% of CO2 , while air only has 0.039% of CO2 , a big difference in CO2 contents; however, the corresponding growth rate difference was not that significant as expected. This observation indicated that most CO2
Please cite this article as: N.M. Moed et al., Struvite as alternative nutrient source for cultivation of microalgae Chlorella vulgaris, Journal of the Taiwan Institute of Chemical Engineers (2015), http://dx.doi.org/10.1016/j.jtice.2015.04.027
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N.M. Moed et al. / Journal of the Taiwan Institute of Chemical Engineers 000 (2015) 1–4 Table 2 Experimental results after 7 day growth tests with BBM at different concentrations or with different quantities of struvite.
2.2 2.0
BBM+CO2
1.8
BBM+air 5+CO2
1.6
5+air
1.4
OD
3
1.2 1.0 0.8
Parameter
OD
TSS
Usable lipid
Usable lipid
Unit BBM 1× BBM 2× Struvite 1× Struvite 2×
– 2.36 2.65 2.66 2.58
g/L 19.9 11.5 17.1 17.2
% 20.0 8.8 16.6 10.8
g/L 3.98 1.01 2.85 1.85
0.6 0.4 0.2 0.0
0
2
4
6
8
Time (d) Fig. 2. Optical densities of algae suspensions with recipe 5 and enriched CO2 or air feed.
5
4
OD
3
3.3. Use of struvite for algae cell growth
2 1x struvite 2x struvite 3x struvite
1
0
pended solids (VSS) measurement. Nonetheless, we can still conclude from the experimental observation that algae cells could grow on double and triple amounts of struvite at longer period than with original recipe 5: the OD peaked on day 8 for struvite 2× and on day 11 for struvite 3×. If the cultivated algae are to be used for biodiesel production, the lipid profiles of the cultivated algal cells has to be considered. The total and lipid contents (C15–C18) of four collected samples are shown in Fig. 4. The BBM 1× had the highest lipid content. The algae grown on struvite 2× were a close follow up. The BBM 2× actually grew less well than the struvite 2×. The C15–C18 lipid quantities for the samples followed (Table 2): BBM 1× > struvite 1× > struvite 2× > BBM 2×. This result is welcomed since the recipe 5 without excess chemicals could produce sufficient lipids for biodiesel production.
0
2
4
6
8
10
12
14
Time (d) Fig. 3. Optical densities of algae suspensions with recipe 5 at different quantities of struvite.
in the enriched gas feed was wasted to the discharge. Additionally, to cultivate microalgae using ambient air is welcomed since no external CO2 source such as fossil fuel power plant has to be settled close to the cultivation site. The growth tests with different quantities of struvite revealed high OD (Fig. 3), likely attributable to the suspended solids from the dosed struvite. The volatile biomass could not be accurately measured since the struvite would decompose at the temperature for volatile sus-
To make a financial comparison, prices of all chemicals were found by online suppliers on Alibaba (December 2014). Excessively high prices were sometimes found for food grade ingredients, these prices were not used in the calculations. The first four chemical costs for making 1 m3 BBM are as follows (€): K2 HPO4 , 6.98–12.2; KH2 PO4 , 11.4–21.3; MgSO4 7H2 O, 1.71–5.49; NaNO3 , 1.32–2.03. Struvite is used mainly for replacing the first four chemicals in the BBM recipe. The struvite can be obtained from wastewaters, which has a very cost (est. to be € 0.11 per m3 of struvite 1× media), much lower than € 31.2 for the BBM used. Restated, the use of struvite, a waste from wastewater treatment, can be significantly cheaper than the use of BBM for algae cultivation. 4. Conclusions This study for the first time demonstrated the feasibility of using struvite as an alternative phosphorous, nitrogen and magnesium source for cultivation of C. vulgaris. Effects of CO2 contents in gas feed
Fig. 4. Lipid contents for samples from BBM or from recipe 5. Usable lipids are fatty acids of C15–C18.
Please cite this article as: N.M. Moed et al., Struvite as alternative nutrient source for cultivation of microalgae Chlorella vulgaris, Journal of the Taiwan Institute of Chemical Engineers (2015), http://dx.doi.org/10.1016/j.jtice.2015.04.027
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and those of chemical concentrations in liquid medium were tested. The BBM medium with 721 mg/L struvite as replacement can produce 2.85 g/L lipids suitable for biodiesel production, comparable to the use of whole BBM as medium. Use of struvite as alternative nutrient can significantly reduce algae cultivation cost. Acknowledgments This work was financially supported by National Science Council and National Taiwan University of Science and Technology. References [1] Wallan P, Davidsson S, Johansson S, Höök M. Phosphate rock production and depletion: regional disaggregated modeling and global implications. Resour Conser Recycl 2014;93:178–87. [2] Huang H, Yang J, Li D. Recovery and removal of ammonia-nitrogen and phosphate from swine wastewater by internal recycling chlorination product. Bioresour Technol 2014;172:253–9. [3] Su CC, Abarca RRM, de Luna MDG, Lu MC. Phosphate recovery from fluidizedbed wastewater by struvite crystallization technology. J Taiwan Inst Chem Eng 2014;45:2395–402. [4] Bi W, Li YY, Hu YY. Recovery of phosphorus and nitrogen from alkaline hydrolysis supernatant of excess sludge by magnesium ammonium phosphate. Bioresour Technol 2014;166:1–8. [5] Nguyen TAH, Ngo HH, Guo WS, Zhang J, Liang S, Lee DJ, et al. Modification of agricultural waste/by-products for enhanced phosphate removal and recovery: potential and obstacles. Bioresour Technol 2014;169:750–62. [6] Etter B, Tilley E, Khadka R, Udert KM. Low-cost struvite production using sourceseparated urine in Nepal. Water Res 2011;45:852–62. [7] Shu CH, Tsai CC, Chen KY, Liao WH, Huang HC. Enhancing high quality oil accumulation and carbon dioxide fixation by a mixed culture of Chlorella sp. and Saccharomyces cerevisiae. J Taiwan Inst Chem Eng 2013;44:936–42. [8] Daghrir R, Igounet L, Brar SK, Drogui P. Novel electrochemical method for the recovery of lipids from microalgae for biodiesel production. J Taiwan Inst Chem Eng 2014;45:153–62.
[9] Lee DJ, Chen GY, Chang YR, Lee KR. Harvesting of chitosan coagulated Chlorella vulgaris using cyclic membrane filtration-cleaning. J Taiwan Inst Chem Eng 2012;43:948–52. [10] Ho SH, Ye XT, Hasunuma T, Chang JS, Kondo A. Perspectives on engineering strategies for improving biofuel production from microalgae-A critical review. Biotechnol Adv 2014;32:1448–59. [11] Slade R, Bauenm A. Micro-algae cultivation for biofuels: cost, energy balance, environmental impacts and future prospects. Biomass Bioenergy 2013;53:29–38. [12] Liu Z, Zhao QL, Lee DJ, Yang N. Enhancing phosphorus recovery by a new internal recycle seeding MAP reactor. Bioresour Technol 2008;99:6488–93. [13] Liu Y, Kumar S, Kwag JH, Ra C. Magnesium ammonium phosphate formation, recovery and its application as valuable resources: a review. J Chem Technol Biotechnol 2013;88:181–9. [14] Adamsson M. Potential use of human urine by greenhouse culturing of microalgae (Scenedesmus acuminatus), zooplankton (Daphnia magna) and tomatoes (Lycopersicon). Ecol Eng 2000;16:243–54. [15] Zhang S, Lim CY, Chen CL, Liu H, Wang JY. Urban nutrient recovery from fresh human urine through cultivation of Chlorella sorokiniana. J Environ Manage 2014;145:129–36. [16] Wang L, Min M, Li YC, Chen P, Chen YF, Liu YH, et al. Cultivation of green algae Chlorella sp. in different wastewaters from municipal wastewater treatment plant. Appl Biochem Biotechnol 2010;162:1174–86. [17] He PJ, Mao B, Lü F, Shao LM, Lee DJ, Chang JS. The combined effect of bacteria and Chlorella vulgaris on the treatment of municipal wastewaters. Bioresour Technol 2013;146:562–8. [18] He PJ, Mao B, Shen CM, Shao LM, Lee DJ, Chang JS. Cultivation of Chlorella vulgaris on wastewater containing high levels of ammonia for biodiesel production. Bioresour Technol 2013;129:177–81. [19] Ma XC, Zhou WG, Fu ZQ, Cheng YL, Min M, Liu YH, et al. Effect of wastewaterborne bacteria on algal growth and nutrients removal in wastewater-based algae cultivation systems. Bioresour Technol 2014;167:8–13. [20] Kurtulus G, Tas AC. Transformation of neat and heated struvite (MgNH4 PO4 6H2 O). Mater Lett 2011;65:2883–6. [21] Cheng YL, Juang YC, Liao GY, Ho SH, Yeh KL, Chen CY, et al. Dispersed ozone flotation of Chlorella vulgaris. Bioresour Technol 2010;101:9092–6. [22] Nakanishi A, Aikawa S, Ho SH, Chen CY, Chang JS, Hasunuma T, et al. Development of lipid productivities under different CO2 conditions of marine microalgae Chlamydomonas sp. JSC4. Bioresour Technol 2014;152:147–52. [23] Tran DT, Le BH, Lee DJ, Chen CL, Wang HY, Chang JS. Microalgae harvesting and subsequent biodiesel conversion. Bioresour Technol 2013;140:179–86.
Please cite this article as: N.M. Moed et al., Struvite as alternative nutrient source for cultivation of microalgae Chlorella vulgaris, Journal of the Taiwan Institute of Chemical Engineers (2015), http://dx.doi.org/10.1016/j.jtice.2015.04.027