CHEMICAL ENGINEERING TRANSACTIONS VOL. 78, 2020 A publication of The Italian Association of Chemical Engineering Online at www.cetjournal.it Guest Editors: Jeng Shiun Lim, Nor Alafiza Yunus, Jiří Jaromír Klemeš Copyright © 2020, AIDIC Servizi S.r.l. ISBN 978-88-95608-76-1; ISSN 2283-9216 Minimising the Cost of Spirulina platensis Culture Medium using Vinh Hao Natural Mineral Water Dung V. Trinh*, Phuong T. H. Nguyen Ho Chi Minh City University of Technology, Vietnam National University Ho Chi Minh City, 268 Ly Thuong Kiet Street, District 10, Ho Chi Minh City, Vietnam trinhdung@hcmut.edu.vn This paper describes the use of a linear programming method to construct a formula for preparing culture medium for Spirulina platensis (S. platensis) algae using Vinh Hao natural mineral water (Binh Thuan Province, Vietnam) with a low cost. The results show that the cost of preparing the culture medium using Vinh Hao mineral water is less than 252.49 VND/L, which is approximately 13.05 % less than the cost of Zarrouk medium. To verify the optimised medium composition, S. platensis algae was cultured in the two media under the same conditions (25 to 40 °C; pH 8.5 to 10.2). The algae biomass concentration reached 1.0 g/L after 7 d of culturing. The experimental growth rates of S. platensis algae in Vinh Hao culture medium and Zarrouk medium had an average difference of 1.49 %. This study provides a basis for implementing S. platensis algae production on an industrial scale using media formed from Vinh Hao natural mineral water from Binh Thuan Province, Vietnam. 1. Introduction The biomass of the Cyanophyta spirulina (S. platensis) has high nutritional value and is widely used for food and medicine (Le, 1999). In the presence of sufficient nutrients, including macro-minerals and micro-minerals, S. platensis algae synthesise biomass from carbon nutrient sources in the form of bicarbonate ionised HCO using sunlight energy (Gorbunova, 2013). Previous research has shown that S. platensis algae grows well in Zarrouk medium (Kedik et al., 2006). However, S. platensis is costly to culture on an industrial scale when using Zarrouk medium due to the cost of the necessary components used to prepare this nutrient medium (Henrikson R., 2010). Seawater enriched with a commercial compound fertilizer (NPK), NaHCO and FeSO was used as less expensive culture medium for S. platensis (Wu et at., 1993). Costa et at. (2004) used water from Mangueira Lagoon (Rio Grande do Sul State, Brazil) as a culture medium for S. platensis was low cost. Additionally, the use of wastewater materials (Kamyab et al., 2019) or the effluent from anaerobic digestion (Chaiklahan et al., 2010) for manufacturing algae products. However, these products have implications for food and pharmaceutical safety (Amha, 2008). To overcome these disadvantages, it is necessary to find a cheaper and cleaner algae culture medium by taking advantage of clean and cheap natural mineral water sources in Vietnam (Trinh et al., 2017). By applying a linear programming method, a formula for preparing an S. platensis culture medium with the lowest cost was developed using Vinh Hao natural mineral water. The obtained medium was also assessed by comparing the growth rate of S. platensis algae cultured in Vinh Hao medium with that in Zarrouk medium under the same conditions. 2. Experimental methods 2.1 Minimising the cost of preparing the culture medium by using Vinh Hao natural mineral water Vinh Hao natural mineral water was acquired from the natural water source at Vinh Hao commune, Tuy Phong District, Binh Thuan Province, Vietnam. The water mine code was 711 (depth of 71 m). The water composition is described in Table 1 (Vo, 1998). Zarrouk medium (Zarrouk, 1996) with the component substrates, macronutrients and micronutrients for S. platensis is shown in Table 2 and Table 3. DOI: 10.3303/CET2078004 Paper Received: 21/03/2019; Revised: 26/07/2019; Accepted: 02/11/2019 Please cite this article as: Trinh D.V., Nguyen P.T.H., 2020, Minimising the Cost of Spirulina platensis Culture Medium using Vinh Hao Natural Mineral Water, Chemical Engineering Transactions, 78, 19-24 DOI:10.3303/CET2078004 19 The data in Table 1 and Table 2 indicate that many minerals are needed to compose S. platensis algae media using Vinh Hao natural mineral water so that it will have the same mineral content as Zarrouk medium, such as: HCO , NO , SO , Cl ,  PO , Na , K , Ca , Mg  and Fe . The content of these ions in the mineral water is less than in Zarrouk medium; therefore, supplementation is required. Table 1: Composition of Vinh Hao natural mineral water Anion mg/L mge/L Cation mg/L mge/L HCO3 - 2445.64 40.080 Li+ 0.74 0.107 Cl- 31.45 0.890 Na+ 905.0 393.65 F- 8.00 0.421 K+ 32.1 0.821 SO4 2- 1.44 0.030 Ca2+ 14.83 0.740 NO3 - 0.08 0.001 Mg2+ 6.08 0.500 PO4 3- 0.21 0.04 Fe2+ 0.20 0.007 SiO3 2- - - NH4 + 0.01 0.001 mge/L = milligrams of ion per litre Table 2: Composition of Zarrouk medium Chemical formula Zarrouk, g/L NaHCO3 16.8 NaNO3 2.5 NaCl 1.0 K2SO4 1.0 K2HPO4 0.5 MgSO4.7H2O 0.2 CaCl2.2H2O 0.04 FeSO4.7H2O 0.01 EDTA 0.08 Micronutrient solution 1 1.0 mg/L Micronutrient solution 2 1.0 mg/L Table 3: Composition of micronutrient solution Chemical formula Micronutrient solution 1, mg/L Chemical formula Micronutrient solution 2, mg/L H3BO3 2.86 NH4VO3 0.023 MnCl2.4H2O 1.81 NiSO4.7H2O 0.048 ZnSO4.7H2O 0.22 Na2WO4 0.018 CuSO4.5H2O 0.08 Ti2(SO4)3 0.040 MoO3 0.01 Co(NO3)2.6H2O 0.044 The problem of minimising the cost of the culture medium using Vinh Hao mineral water is set out as follows: the mineral content of the culture medium prepared with the natural mineral water must be equivalent to Zarrouk medium but at a lower cost. The mathematical model of the problem is described as Eq(1): ( ) = ( + + ⋯ ) = ∑ cixi (1) where xi corresponds to the weight of 8 substances to be added (g/L): NaHCO , NaNO , NaCl, K SO , K HPO ,MgSO .7H O, FeSO .7H O and CaCl .6H O, and ci are the prices of these substances (VND/g). The amount of each mineral in the medium prepared from Vinh Hao natural mineral water cannot be smaller than the amount available in Zarrouk medium; this condition is shown as Eq(2): ≥≥ 0  = 1 − 8 (2) where aji is the ratio between the molecular weight of ion j (Mj) and the molecular weight of compound i (Mi), and the amount is added xi (shown in Table 4 and Table 5); bj is the amount to be added to Vinh Hao mineral 20 water and is described as Eq(3). Therefore, the culture medium has an amount of mineral j that not less than that in Zarrouk medium. = ( .) − ( . ) (3) The ratio of aji mineral composition of 10 minerals (HCO , NO , Na , K , HPO ,SO , Cl ,Ca ,Mg , Fe ) from 8 basic compounds, which are shown in Table 4, is determined as Eq(4): = (4) As shown in Table 6: = 61;  = 84;  = = = 0.7262 mj(Zar.) is the content of mineral j in Zarrouk medium and is calculated as Eq(5): ( .) = ∑ . (5) mj(V.H) is the content of mineral j in Vinh Hao natural mineral water and is calculated as Eq(6): ( . ) = ∑ . (6) Table 4: Molecular weights of substances used in modelling and experimentation Chemical formula NaHCO3 NaNO3 NaCl K2HPO4.3H2O K2SO4 CaCl2.6H2O MgSO4.7H2O FeSO4.7H2O Mi. g/mol 84 85 58.5 228 174 219 246 278 Table 5: Molecular weight of minerals used in modelling and experimentation Chemical formula HCO3 - NO3 - Na+ K+ HPO4 2- SO4 2- Cl− Ca2+ Mg2+ Fe2+ Mj. g/mol 61 62 23 39 96 96 35.5 40 24 56 2.2 Experimental verification with optimised medium To evaluate the results of the mathematical model, two similar samples of S. platensis algae were experimentally cultivated in the optimised medium and in Zarrouk medium. The experiment was conducted with the following tools: precision balance; Erlenmeyer flasks (500 mL and 250 mL); volumetric flasks (500 mL and 100 mL); measuring pipettes (10 mL and 2 mL); measuring cylinders (250 mL and 10 mL). Other tools: alcohol thermometer, measure scale 0 - 100 °C (France); pH meter (Hanna, Model HI98172); Beckman Coulter DU 750 spectrophotometer (USA), measuring at 750 nm. The concentration of S. platensis algae is calculated with Eq(7): = . (7) where k is the conversion factor for S. platensis; k = 0.73. The S. platensis algae used in the experiment was purchased from the experimental biology room, Research Institute for Aquaculture No. 2, No. 116 Nguyen Dinh Chieu St., Da Kao Ward, District 1, Ho Chi Minh City. The experimental verification for the optimal Vinh Hao natural mineral water media was carried out as follows. Two parallel experimental samples of nutrient medium were prepared according to the optimised results using Vinh Hao natural mineral water (S1 and S2) and a sample using Zarrouk medium (Table 6). Every hour throughout the day, the following parameters were measured: temperature of the air and of the algae culture medium, pH of the medium, and the optical density using a spectrophotometer at a wavelength of 750 nm (OD750). The results were analysed by analysis of variance (ANOVA) with a confidence probability greater than 95% (p <0.05). Table 6: S. platensis algae cultures for comparison of the optimised medium and Zarrouk medium Medium S1 S2 Zarrouk Vinh Hao medium, mL 150 150 0 Zarrouk medium, mL 0 0 150 S. platensis (1 g/L), mL 15 15 15 21 3. Results and discussion 3.1 Minimising the cost of preparing the algae culture medium To minimise the cost of the algae medium, (Eq(1) was applied, and the coefficients aji and bj were calculated according to Eq(3) to Eq(6) using the data in Table 4 andTable 5. The results are presented in Table 7. Table 7: Calculation results of aji matrix coefficient components, vector bj Substance NaHCO3 NaNO3 K2HPO4.3H2O K2SO4 NaCl MgSO4.7H2O FeSO4.7H2O CaCl2.6H2O bj aj1 aj2 aj3 aj4 aj5 aj6 aj7 aj8 HCO3− 0.7262 0 0 0 0 0 0 0 9.7544 NO3− 0 0.7294 0 0 0 0 0 0 1.8234 Na+ 0.2738 0.2706 0 0 0.3932 0 0 0 4.7646 K+ 0 0 0.3421 0.4483 0 0 0 0 0.5872 HPO4 2− 0 0 0.4211 0 0 0 0 0 0.2103 SO4 2− 0 0 0 0.5517 0 0.3902 0.0007 0 0.6283 Cl− 0 0 0 0 0.6068 0 0 0.3242 0.5882 Ca2+ 0 0 0 0 0 0 0 0.1826 0.0148 Mg2+ 0 0 0 0 0 0.0976 0 0 0.0134 Fe2+ 0 0 0 0 0 0 0.0397 0 0.0002 Cost minimisation was achieved through a simplex algorithm, calculated in Microsoft Excel (Trinh, 2016), shown in Table 8. The prices of the components were provided by South Chemical Import Export Joint Stock Company (SOUTHCHIMEX JSC; Vietnam). The results in Table 8 show that preparing the medium with Vinh Hao natural mineral water will achieve a cost reduction of 252.59 VND (13.05 %) compared with Zarrouk medium. Table 8: Minimising the cost of preparing S. platensis culture medium using Vinh Hao natural mineral water Chemical formula NaHCO3 NaNO3 NaCl K2HPO4.3H2O K2SO4 MgSO4.7H2O FeSO4.7H2O CaCl2.6H2O ΣcixiMin xi, g/L 13.43 2.50 1.04 0.50 0.93 0.30 0.00 0.08 1,682.71 Zarrouk, g/L 16.80 2.50 1.00 0.50 1.00 0.20 0.01 0.04 1,935.3 Price ci, VND/g 76 150 62 104 148 90 70 70 Δ=252.59 3.2 Changes in temperature and pH of the culture medium The temperature of the S. platensis culture medium was taken hourly, from 5:00 am (at sunrise) to 18:00 (at sunset). The amount of sun received was highly dependent on the weather, which affected the temperature. The average temperatures of the culture medium are shown in Table 9 and included 1 rainy day, 5 sunny days and 2 shady days. Table 9: Change in the average temperature of the culture medium (Binh Thanh District, HCM City) Day τ (h) 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Rain T (oC) 25.0 27.0 33.0 34.0 34.0 35.0 33.0 34.5 35.5 38.0 39.0 40.0 37.5 28.0 Shade 24.0 26.0 28.0 32.0 36.0 37.0 37.5 37.0 35.0 36.0 34.5 31.0 31.5 29.0 Sun 25.0 30.0 35.5 37.0 36.5 40.0 39.0 38.5 40.0 42.0 37.5 38.0 36.0 30.0 The temperature increased rapidly from 6:00 am to 8:00 am, while from 8:00 am to 16:00 the temperature changed more slowly. On sunny days (and even rainy days), the temperature of the medium exceeded the optimal temperature range for S. platensis algae (30 to 38 °C) and reached dangerous temperatures (above 40 °C), but only for a short time. Changes in the pH of two parallel experimental samples (S1 and S2) and a sample with Zarrouk medium are described in Table 10.These results show that the pH of all three samples varied similarly. They gradually increased from 8.5 to 10.2, which is within the suitable pH range for S. platensis; the photosynthesis of S. platensis produces ions that raise the pH, described as Eq(8): . + . + ℎ. → ( ) + . + . (8) 22 where n is the number of reacted ions, h is the Planck constant and is the light frequency. Table 10: pH changes in the culture medium Day S1 S2 Zarrouk 1 8.50 8.55 8.50 2 8.70 8.80 8.70 3 9.20 9.30 9.30 4 9.45 9.50 9.60 5 9.60 9.65 9.70 6 9.70 9.80 9.80 7 9.90 9.95 9.90 8 10.15 10.25 10.25 3.3 Biomass growth rate Figure 1 shows that the biomass growth rate of S. platensis algae in Vinh Hao medium was similar to that in Zarrouk medium. The biomass increased throughout the first 7 d, but on the 8th day it decreased slightly. 0 1 2 3 4 5 6 7 8 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 S1 S2 Zarrouk O D 75 0 τ (d) Figure 1: Biomass growth rate The growth time of S. platensis algae is 7 d, including the lag, log, and stationary phases. On the 8th day, the equilibrium phase is reached. At this point, the algae begin to decline (OD750 is reduced) because the nutrients in the medium are gone and the pH has increased to greater than 10.5. The algae become inhibited, and they grow slowly and begin die. S. platensis algae can be harvested on the 7th day, when their concentration (calculated by Eq(7)) is 1.161 g/L, which is greater than 1 g/L. 3.4 Comparison of the optimised and Zarrouk media The optical densities at OD750 of Vinh Hao and Zarrouk medium are shown in Figure 2. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 S1 S2 Zarrouk O D 75 0 (S i) OD750 (Zarrouk) Figure 2: Optical density (OD750) of Vinh Hao and Zarrouk media 23 The average relative deviation between OD750 (Si) and OD750 (Zar.) ( ) is calculated as Eq(9): = ∑ | | + ∑ | | .100% = 1.49% (9) 4. Conclusions By a linear programming method, the formula for preparing an S. platensis culture medium using Vinh Hao natural mineral water was determined. The cost of preparation according to this formula was less than 252.49 VND/L, approximately 13.05 % less than the cost of Zarrouk medium. The culture growth results are shown in Figure 2. The obtained formula was evaluated by experimentally cultivating S. platensis in the Vinh Hao and Zarrouk media under the same conditions (25–40 °C; pH 8.5–10.2). The biomass growth rate of S. platensis algae in two media were equivalent to each other, reaching 1.0 g/L after 7 d. The average relative deviation of the biomass growth rate (OD750) in these media was 1.49 %, indicating the reliability of the results. This study provides a basis for furthering algae culturing technology using Vinh Hao natural mineral water, which has a low cost in Vietnam. 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