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2018
Investigation on the feasibility of Chlorella vulgaris cultivation in a mixture of pulp and aquaculture
effluents: Treatment of wastewater and lipid extraction
Daneshvar, E
Elsevier BV
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http://dx.doi.org/10.1016/j.biortech.2018.01.101
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Accepted Manuscript
Investigation on the feasibility of Chlorella vulgaris cultivation in a mixture of pulp and aquaculture effluents: treatment of wastewater and lipid extraction Ehsan Daneshvar, Laura Antikainen, Eleni Koutra, Michael Kornaros, Amit Bhatnagar
PII: S0960-8524(18)30115-9
DOI: https://doi.org/10.1016/j.biortech.2018.01.101
Reference: BITE 19458
To appear in: Bioresource Technology Received Date: 10 November 2017 Revised Date: 17 January 2018 Accepted Date: 22 January 2018
Please cite this article as: Daneshvar, E., Antikainen, L., Koutra, E., Kornaros, M., Bhatnagar, A., Investigation on the feasibility of Chlorella vulgaris cultivation in a mixture of pulp and aquaculture effluents: treatment of wastewater and lipid extraction, Bioresource Technology (2018), doi: https://doi.org/10.1016/j.biortech.
2018.01.101
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Page 1 of 28
Investigation on the feasibility of Chlorella vulgaris cultivation in a mixture of pulp and 1
aquaculture effluents: treatment of wastewater and lipid extraction 2
3
Ehsan Daneshvar a,*, Laura Antikainen b, Eleni Koutra c, Michael Kornaros c, Amit Bhatnagar a 4
5
a Department of Environmental and Biological Sciences, University of Eastern Finland, P.O. Box 1627, 6
FI-70211 Kuopio, Finland 7
b Environmental Technology, Savonia University of Applied Sciences, P.O. Box 6 (Microkatu 1 C) FI- 8
70201 Kuopio, Finland 9
c Laboratory of Biochemical Engineering & Environmental Technology (LBEET), Department of 10
Chemical Engineering, University of Patras, 26504 Patras, Greece 11
12 13 14 15 16 17 18 19 20
* Corresponding author 21
E-mail address: ehsan.daneshvar@uef.fi 22
23 24
Page 2 of 28 Nomenclature
DI Deionized water
LW Lake water
AWW Aquaculture wastewater
PWW Pulp wastewater
PLWW Mixture of pulp wastewater and lake water PAWW Mixture of pulp and aquaculture wastewater
PAWW'BBM BBM medium was prepared with PAWW instead of deionized water PAWW'N+P Nitrate and phosphate were added to PAWW
25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
Page 3 of 28 Abstract
43
In this study, feasibility of Chlorella vulgaris cultivation in pulp wastewater (PWW) diluted with lake water 44
(LW) and aquaculture wastewater (AWW) was investigated. The best ratios of PWW and AWW (PAWW) viz., 45
80% PWW:20% AWW and 60% PWW:40% AWW were selected as microalgal culture medium. Algal growth 46
was investigated with and without addition of macro and micronutrients to the cultivation medium. The highest 47
dry algal weight was observed as 1.31 g/L in 60% PWW:40% AWW without adding micronutrients. Nutrients 48
and organic compounds removal efficiencies by microalga were studied in PAWW. Protein, carbohydrate and 49
lipid percentage of harvested microalga from wastewater and Bold's Basal Medium (BBM) solution were 50
analyzed. Fatty acids analysis revealed that C16 and C18 are the major fatty acids in C. vulgaris cultivated in 51
BBM and PAWW. The results of this study revealed that C. vulgaris is a potential candidate for PAWW 52
treatment and lipid and carbohydrate accumulation.
53 54
Keywords: Pulp wastewater (PWW); Aquaculture wastewater (AWW); Chlorella vulgaris; Biochemical 55
components; Lipid profile.
56 57 58 59 60 61 62 63 64 65 66 67
Page 4 of 28 1. Introduction
68
Water pollution, mainly due to anthropogenic activities, has become a serious environmental issue in 69
recent decades. Wastewaters from industrial and agricultural sources contain detrimental contaminants 70
viz., metals, dyes, phenols, detergents, antibiotics, disinfectants, pesticides and nutrients. These 71
contaminants have harmful impact on the health of human beings and ecotoxicological effects on 72
aquatic organisms (Schwarzenbach et al., 2006). Thus, it is necessary to treat wastewater before they 73
enter downstream bodies of water.
74
Besides water quality issue, energy supply is another major problem that humanity faces in the 21st 75
century. Fossil fuels are considered as the main source of primary energy throughout the world 76
(Likozar et al., 2016). However, due to air pollution and decrease in reserves of fossil oil, replacing an 77
alternative source of cheaper, renewable and environmentally friendly energy is necessary (Šoštarič et 78
al., 2012).
79
Microalgae can address both these issues, to a significant extent, in an eco-friendly and inexpensive 80
way. Microalgae are found in freshwater, saline water, brackish water and even in wastewater. Most of 81
them grow photoautotrophically in the presence of sun light, CO2 and nutrients (Kuo et al., 2015).
82
These organisms have their own remarkable advantages, such as fast growth rate, high productivity, 83
non-requirement for arable land and accumulation of valuable biomolecules (Maurya et al., 2016).
84
Nitrogen and phosphorus compounds and the other macro and micronutrients, as the sources of 85
environmental pollution, are considered the main enrichers for cultivation of microalgae (Ruiz et al., 86
2014). Wastewater treatment using microalgae does not require the use of any harmful and expensive 87
chemicals and the produced microalgal mass can be used further to produce bioenergy.
88
The produced biodiesel from microalgae is composed by transesterification of microalgal lipid (Likozar 89
and Levec, 2014). Saturated fatty acids such as palmitic (C16:0) and stearic (C18:0) acids of harvested 90
microalgae from wastewater increase cetane number, energy yield and biodiesel quality (Canakci and 91
Page 5 of 28
Sanli, 2008). The produced biodiesel from algae as clean, renewable and carbon-natural fuel can be 92
considered as an alternative candidate to fossil fuels. Some other useful products like biopolymers, 93
pigments, fertilizers, and biofuel can also be derived from algal biomass, produced in wastewater 94
(Batista et al., 2013).
95
Attention to selecting the appropriate wastewater(s) and microalga(e) is necessary to conduct a 96
successful wastewater treatment plan using microalgae and consequently producing biofuel from algal 97
biomass (Salama et al., 2017). Different types of wastewaters have different concentrations of nitrogen, 98
phosphorous and carbon components (Liu et al., 2016). Sometimes microalgae cannot grow well in a 99
wastewater due to low or high concentration of some nutrients or toxicity of them. To overcome this 100
problem, manipulation of wastewaters such as diluting or mixing them is needed to accomplish 101
effective cultivation and wastewater treatment.
102
The wastewater production by pulp industries is the third largest in terms of amount and has harmful 103
effects on the human health and environment (Ashrafi et al., 2015). However, the concentration of 104
carbon is high in pulp wastewater (PWW) but it has low concentration of nitrate and phosphate 105
(Gentili, 2014). In this study, aquaculture wastewater (AWW) was added to PWW and the feasibility of 106
Chlorella vulgaris cultivation in the mixture of PWW and AWW (PAWW) was investigated.
107
Microalgal growth in PAWW with and without adding different nutrients (individually and together) 108
were studied. The concentrations of nutrients, chemical oxygen demand (COD) and total organic 109
carbon (TOC) in wastewater on the first and final days were measured. Finally, the biochemical 110
compositions and fatty acid methyl esters (FAMEs) quantity of cultivated alga in the mixed wastewater 111
were analyzed. The findings of this study will be helpful for optimization of microalgae cultivation in 112
industrial wastewaters without adding macro and micronutrients to the algal medium.
113 114 115
Page 6 of 28 2. Materials and methods
116
2.1. Mediums and chemicals 117
Deionized water (DI), natural lake water (LW), aquaculture wastewater (AWW), pulp wastewater 118
(PWW) mixture of PAW+LW (PLWW) and PWW+AWW (PAWW) were used to prepare media 119
(Table 1). LW and PWW were collected from local lake in Kuopio and paper making company (from 120
recycled fiber plant before wastewater treatment) in Kuopio, Finland, respectively. AWW was prepared 121
from closed recirculating aquaculture system in the Department of Environmental and Biological 122
Sciences, University of Eastern Finland. The following analytical grade chemicals were used to prepare 123
stocks of Bold's Basal Medium (BBM): NaNO3 (25 g/L), MgSO4.7H2O (7.5 g/L), NaCl (2.5 g/L), 124
K2PO4 (7.5 g/L), KH2PO4 (17.5 g/L), CaCl2.2H2O (2.5 g/L), ZnSO4.7H2O (8.82 g/L), MnCl2.4H2O (3 125
g/L), MoO3 (0.71 g/L), CuSO4.5H2O (1.57 g/L), Co(NO3)2.6H2O (0.49 g/L), H3BO3 (11.42 g/L), 126
EDTA (50 g/L), KOH (31 g/L), FeSO4.7H2O (4.98 g/L), H2SO4 (Conc.) (1.0 mL).
127
2.2. Microalgal strain and cultivation conditions 128
The universal distributed freshwater microalga, C. vulgaris CCAP 211/11B was obtained from the 129
Culture Collection of Algae and Protozoa (CCAP, Scotland, UK). The harvested microalgal pellet after 130
centrifuging at 3000 rpm for 5 min was inoculated in 250 mL bottles including 200 mL medium. Batch 131
cultures were incubated at 25 ± 2 °C under continuous illumination of 85 µmol photon m-2 s-1 provided 132
by white fluorescent lamps for one week. The continuous aeration with 0.04% CO2 was injected to the 133
medium. Microalga was cultivated in BBM and wastewater media without adjusting initial media pH 134
(7.1 and 5.9-6.3, respectively) 135
A calibration curve of OD680 vs. dry algal mass (g/L) was established to convert optical density values 136
to dry mass (g/L) as follows (Ji et al., 2016):
137
Dry algal mass (mg/L): a × OD680 + b (1) 138
where, OD680 is algal density at 680 nm and a and b are the constants of the equation.
139
Page 7 of 28 2.3. Experimental design
140
2.3.1. Effect of PWW concentration on microalgal growth 141
In the first step of experiment, LW, AWW, PWW and mixtures of PLWW and PAWW were used 142
instead of deionized water to prepare the algal medium. For this purpose, different dilution percentages 143
of PWW viz., 0, 20, 40, 60, 80 and 100% were prepared by mixing with LW and AWW. The same 144
concentrations of BBM stocks (section 2.1.) were added to PLWW and PAWW as algal medium 145
(Table 1). Dry algal mass was measured to monitor the best medium for C. vulgaris.
146
2.3.2. Cultivation of microalga with and without adding nutrients 147
Experimental units with the highest algal growth viz., 60%PWW:40%AWW and 148
80%PWW:20%AWW were selected to evaluate the feasibility of C. vulgaris cultivation in PAWW 149
without adding nutrients. Fifteen experimental units (as shown in Fig. 2 and Table 1) were set up with 150
and without adding macro and micronutrients to the medium. The control culture medium was prepared 151
by adding standard concentrations of BBM stocks to deionized water.
152
2.4. Wastewater analysis and removal efficiency of pollutants 153
Wastewater treatment by microalga was conducted in PAWW'BBM, PAWW'N+P and PAWW. The 154
concentrations of nitrate as nitrogen (NO3--N), ammoniacal nitrogen (NH3-N), total phosphorus (PO43-- 155
TNT), chemical oxygen demand (COD), total nitrogen (TN) and total organic carbon (TOC) were 156
analyzed on the first and final day of the experiments. Samples for analysis were taken, centrifuged for 157
10 min at 7000 rpm and filtered using 0.45 µm membrane filters. Cadmium reduction, Nessler, 158
ascorbic acid and dichromate methods were applied to measure the concentrations of NO3-
-N, NH3-N, 159
PO43-TNT and COD, respectively using HACH analysis kits and spectrophotometer (DR 2800 and DR 160
2010). TOC/TN Analyzer (multi N/C 2100S) were used to analysis the concentrations of TN and TOC.
161
The removal efficiencies of pollutants were calculated as follow:
162
R%= Ci-CF/Ci × 100 (2) 163
Page 8 of 28
where, Ci and CF are the concentrations of target pollutant on first and final day of experiment, 164
respectively.
165
2.5. Biochemical components of microalgal biomass 166
After one-week of cultivation time, algal biomass was collected from three media viz., BBM, 167
PAWW'BBM and PAWW'N+P media and biochemical components (total protein, carbohydrates and 168
lipids) of samples were analyzed. Total protein was extracted and analyzed using a modified method 169
reported by Rausch (Rausch, 1981). Ten mg of dried microalgal powder were re-suspended in 10 mL 170
0.5 M NaOH. The mixture was vortexed for 1 min and subsequently sonicated for 30 min. Then the 171
solution was incubated in an oven at 100 °C. After 2 h, microalgal solution was centrifuged at 6000 172
rpm for 5 min. Total protein concentration of supernatant was measured according to Bradford method 173
(Bradford, 1976). Four mL of Bradford reagent was added to 1 mL of supernatant and the absorbance 174
of solution was read at 595 nm after 5 min.
175
The total carbohydrate concentration was determined by the phenol-sulfuric acid technique as reported 176
by Salama et al. (Salama et al., 2014). Briefly, 10 mg of dried microalga powder was added to 10 mL 177
deionized water and vortexed for 1 min. The suspension was incubated in a water bath at 90-100 °C for 178
30 min followed by 30 min sonication. Five mL concentrated sulfuric acid and 1 mL phenol (5%) were 179
added to 1 mL of sample. The mixture was kept in a water bath at 90-100 °C for 5 min. The 180
concentration of carbohydrate in the supernatant was read at 490 nm and compared with glucose 181
standard curve.
182
To extract algal lipids, 5 mL methanol and 2.5 mL chloroform were added to 100 mg dried microalgal 183
powder. After 1 min vortex, the suspension was sonicated for 30 min. Then microalgal biomass at the 184
bottom of the tube was separated by centrifuging and the supernatant was collected to another tube.
185
The extraction procedure was repeated but with the half amount of solvents. The collected supernatant 186
from the previous step was added to the test tube including microalga. Four mL 1% NaCl and 4 mL 187
Page 9 of 28
chloroform were added and the test tube was shaken for 5 min at 80 rpm. After centrifuging at 7000 188
rpm for 5 min, the chloroform phase including lipid (the dark green layer) was carefully collected.
189
Finally, chloroform was evaporated and the weight of lipid was calculated gravimetrically.
190
2.6. Fatty acid (FA) composition 191
For determination of lipid content, microalgal biomass was freeze-dried and fatty acid content was 192
expressed at a dry weight (DW) basis. The analysis was based on a modified one-step in-situ 193
transesterification method (IST), according to Levine et al. (Levine et al., 2011). Fatty acid methyl 194
esters (FAMEs) were subsequently analyzed on a gas chromatograph (Agilent Technologies 7890A) 195
equipped with a flame ionization detector (FID), according to the following method: Oven temperature 196
was increased from 40°C (held for 0.5 min) to 195°C at a rate of 25°C/min, from 195°C to 205°C at a 197
rate of 3°C/min and from 205°C to 230°C (held for 1 min) at a rate of 8°C/min. Helium was used as the 198
carrier gas with an average velocity of 30.34 cm/sec. Temperature of injector and detector was set at 199
250 °C. For the analysis, a capillary column (DB–WAX, 10 m × 0.1 mm × 0.1 μm), as well as a 200
calibration standard (FAMQ-005, AccuStandard) and an internal standard (IS) (C17:0, Sigma) at a final 201
concentration of 50 mg/L, were used. Measurements were carried out in duplicate and mean ±SD 202
values are presented in the results.
203 204 205 206 207 208 209 210 211
Page 10 of 28 3. Results and discussion
212
3.1. Optimization of microalgal growth in PLWW and PAWW 213
In this study, wastewater from local pulp and paper making factory was used and investigated for the 214
feasibility of microalgal growth at different concentration of PWW. For this purpose, PWW was 215
diluted with LW and AWW. Fig. 1 shows the dry algal mass (g/L) at different concentrations of PWW 216
(0, 20, 40, 60, 80 and 100%). In case of PLWW, microalgal dry mass increased from 0.51 to 1.21 g/L 217
as PWW concentration increased from 0 to 40%. Then it decreased at higher concentration of PWW 218
(60 to 100%). Productivity of microalgal dry mass at PAWW is illustrated in Fig. 1. As it can be seen 219
from Fig. 1, microalga grew well in different concentrations of PWW. The maximum dry algal mass 220
observed was 1.1 g/L, at 80% PWW concentration. A literature review reveals that based on the 221
characteristics of wastewater, dilution factor can increase or decrease microalgal growth. Sepúlveda et 222
al. (2015) used 0-80% centrate for the production of the marine microalga Nannochloropsis gaditana 223
(Sepúlveda et al., 2015). In their study, the maximum biomass productivity (g/L/day) was observed as 224
0.4 g/L/day, in the range of 30-50% of centrate. They explained that microalgal growth decreased in 225
centrate concertation lower than 20% and higher than 80%, due to insufficient concentration of 226
nutrients and toxicity of ammonium, respectively. In the current study, microalgal growth in PLWW 227
and PAWW increased as PWW concentration increased. PWW is a complex medium including high 228
contents of BOD, COD, fatty acids, organic and inorganic and nitrogen compounds (Ashrafi et al., 229
2015). Higher biomass production at higher concentrations of PWW can be related to high 230
concentrations of organic substrate (Hwang et al., 2014). This finding is in agreement with another 231
study in which biomass production was higher at higher concentration of acetate and butyrate-rich 232
wastewater effluent (Hwang et al., 2014).
233
3.2. The availability of macro and micronutrients 234
Page 11 of 28
According to the different ratios of PWW:AWW, two ratios with the highest algal dry mass viz.
235
60%:40% and 80%:20% were selected as microalgal culture medium. To confirm that macro and 236
micronutrients of these mixtures can support algal growth, different experimental units were prepared 237
with and without adding nutrients (Table 1). As derives from Fig. 2, microalgal dry weight, in the 238
experimental units without adding nitrate and phosphate (1, 4, 10 and 13), was significantly lower than 239
the others. Decreasing the concentration of PWW from 80 to 60% and increasing the concentration of 240
AWW from 20 to 40% increased microalgal growth in the experimental units without adding nitrate 241
and phosphate. Although, changing the ratios of wastewaters improved microalgal growth, algal dry 242
weight in the aforementioned experimental units was significantly lower than in case of experimental 243
unit 15 having nitrate and phosphate. It can be concluded that microalgal growth is limited in PAWW 244
due to low concentrations of nitrate and phosphate. Microalgae need phosphorous and nitrogen 245
compounds (mainly nitrate and phosphate) for their growth and intracellular metabolism. Nitrogen 246
compounds are fundamental in the structure of protein and nucleic acids. Phosphorus is also an 247
essential nutrient as it has the key role in formation of adenosine triphosphate (ATP) as energy carrier 248
in algal cells (El-Kassas, 2013). Similar to these results, Gao et al. (2017) reported that nutrients 249
concentration in aquaculture wastewater is insufficient for cultivation of C. vulgaris (Gao et al., 2016).
250
Analysis of PWW (Table 2) revealed that concentrations of nitrate and phosphate are not sufficient for 251
cultivation of microalgae and without adding them, biomass productivity is low.
252
Magnesium (Mg), Sodium (Na) and Calcium (Ca) are the other important macronutrients that 253
microalgae need to grow healthy. The results of C. vulgaris cultivation with and without adding Mg, 254
Na and Ca are presented in Fig. 2. Microalgal dry weight (g/L) in 60% PWW+40% AWW medium 255
without adding Mg, Na and Ca (experimental units 2, 3 and 5) were 0.93, 0.99 and 0.98 g/L, 256
respectively. In the experimental unit 15 including all of BBM stocks, and in experimental unit 11 257
without adding Mg, Na and Ca, microalgal dry weight was found to be 1.00 and 1.11 g/L, respectively.
258
Page 12 of 28
Microalga grew well in the medium without adding the above nutrients (Mg, Na and Ca) and even 259
better growth was noticed in the experimental unit having all of BBM stocks. These results clearly 260
show that the concentrations of Mg, Na and Ca in PAWW are enough for the cultivation of C. vulgaris.
261
In another study Ji et al. (2015) investigated the growth of Scenedesmus obliquus in diluted BBM with 262
food wastewater (FW), sodium chloride and sea water (Ji et al., 2015). They concluded that the reason 263
of the highest dry weight of alga (0.41 g/L) in FW can be related to the presence of Ca, Mg, Mn and Fe 264
in the wastewater.
265
Microalgal growth and lipid content depends not only on essential macronutrients (nitrate and 266
phosphate) and major ions such as Mg and Ca, but also on micronutrients such as iron, manganese, 267
cobalt, molybdenum, copper and zinc (Sunda et al., 2005). Here, C. vulgaris grew in the experimental 268
units without adding trace elements, boric acid, Ethylenediaminetetraacetic acid (EDTA) and Fe (6, 7, 269
8 and 9, respectively) as well as in the supplemented medium with all of them (experimental unit 15).
270
The maximum dry biomass of alga was observed as 1.11 g/L (80% PWW+20% AWW) and 1.31 g/L 271
(60% PWW+40% AWW) in the medium without adding micronutrients. This value for the 272
experimental units with the same ratios of PWW:AWW containing all of BBM stocks was 0.93 and 273
1.00 g/L, respectively. Supplementation of micronutrients, as well as EDTA, are necessary in the algal 274
medium to improve CO2 fixation, photosynthesis efficiency and biomass production (Singh et al., 275
2016). Carvalho et al. (2006) showed that biomass concentration of microalga significantly decreased 276
in the absence of micronutrients viz., Mn, Fe, Co and Zn (Carvalho et al., 2006). Results of this 277
experiment show that the mixture of PWW and AWW used in this study has enough amounts of 278
essential micronutrients to support microalgal cultivation.
279
3.3. PAWW wastewater treatment using C. vulgaris 280
Optimal growth of microalgae relies on the concentration of nitrogen, phosphorus and carbon 281
compounds as essential nutrients (Nayak et al., 2016). The concentration of nitrogen, phosphorus and 282
Page 13 of 28
carbon compounds in PAWW'BBM (BBM medium prepared with PAWW instead of deionized water), 283
PAWW'N+P (PAWW containing nitrate and phosphate) and PAWW (without adding nutrients) are 284
presented in Table 2. The concentration of COD and TOC in PWW were quite higher than in AWW.
285
However, the concentration of TN and TP in PWW were lower than in AWW. Therefore, their 286
appropriate mixture balanced the concentration of nutrients, increased microalgal growth and enhanced 287
wastewater treatment efficiency. Fig. 3 depicts the removal efficiency of different nutrients and organic 288
compounds in PAWW by C. vulgaris. Nutrients removal efficiency in the presence and absence of 289
external sources of nutrients (BBM stocks) was assessed. The highest nitrate removal efficiency was 290
69.39 ± 6 in the PAWW'BBM medium. Nitrate removal efficiency was not observed in PAWW 291
without adding BBM nutrients and nitrate concentration increased. The reason might be due to 292
nitrification which takes place in the presence of oxygen, reduced nitrogen and nitrifying bacteria 293
(Jiang et al., 2016). The results corroborated with the study of Jiang et al. where nitrate concentration 294
increased in the high-ammonia complex wastewater after five days cultivation of Monoraphidium spp.
295
(Jiang et al., 2016).
296
NH3-N removal efficiencies in PAWW'BBM, PAWW'N+P and PAWW were found to be 50.00 ± 2%, 297
23.44 ± 3% and 15.42 ± 3%, respectively. The equilibrium of NH4+/NH3 (pKa = 9.25) in aqueous 298
solution is H2O + NH3 ⇌ OH− + NH4+ and is governed by pH and temperature (Ji et al., 2014a). NH4+ 299
andNH3 are the dominant forms of ammonia nitrogen at pH lower than 8.75 and higher than 9.75, 300
respectively (Molins-Legua et al., 2006). As the final pH in this study was 9.43, thus, NH3-N removal 301
could be largely attributed to the presence of C. vulgaris in the medium.
302
The overall TN and TP removal efficiencies were ranged from 55.49 to 94.41% in all tested media.
303
Similar removal efficiencies of TN and TP were observed in the PAWW'BBM and PAWW'N+P 304
media, showing that microalga can grow very well in nitrate and phosphate enriched wastewater and 305
uptake nitrogen and phosphorus from PAWW without adding external source of other nutrients. In 306
Page 14 of 28
general, in different media either BBM or PAWW, removal efficiency of TP was higher than removal 307
efficiency of TN. This observation is in agreement with the results of Ji et al. (Ji et al., 2016). Authors 308
mentioned that high removal efficiency of TP can be related to intracellular uptake of H2PO4 or HPO4−, 309
precipitation of phosphate as calcium phosphate at basic pH and adsorption to cell surface. As can be 310
seen from Fig. 3, TN removal efficiency was higher than NO3-
-N removal efficiency that reveals 311
microalga consumes not only inorganic nitrogen but also organic sources of nitrogen. Nutrients 312
removal efficiency in PAWW was significantly lower than in case of PAWW'BBM and PAWW'N+P.
313
This is strongly related with the microalgal growth as it was 0.21, 1.15 and 0.83 g/L, respectively in the 314
mentioned media.
315
COD indicates the overall concentration of dissolved and suspended organic matter of the wastewater.
316
Higher removal efficiency of COD and lower concentration of organic matter before effluent discharge 317
are important in wastewater treatment plants (Markou, 2015). Removal efficiency of COD and TOC 318
from PAWW by C. vulgaris was investigated in this work. COD and TOC removal efficiency by 319
microalga in the three different media tested followed the order PAWW'BBM> PAWW'N+P> PAWW.
320
The highest values of COD and TOC removal efficiencies were observed as 94.41 and 79.38%, 321
respectively in PAWW'BBM. Normally, microalgae through photoautotrophic metabolism utilize light 322
and CO2 to supply their energy and carbon sources. Some microalgal species such as C. vulgaris can 323
uptake organic carbon under photoautotrophic cultivation (Mujtaba et al., 2017). Zhu et al. reported 324
that 67.25, 65.81, 76.46, 79.84, 78.18 and 74.29% of COD was removed from the 400, 800, 1300, 325
1900, 2500 and 3500 mg L−1 COD cultures under photoautotrophic condition (Zhu et al., 2013). The 326
findings of the current experiment reveal that C. vulgaris is able to use organic carbon and strongly 327
reduces the concentrations of COD and TOC in wastewater.
328 329 330
Page 15 of 28
3.4. Biochemical composition of C. vulgaris in different culture media 331
Nutrients composition and cultivation conditions can affect both growth and biochemical composition 332
of microalgae (Ji et al., 2014b). The content of proteins, carbohydrate and lipid of microalga cultivated 333
in BBM, PAWW'BBM and PAWW'N+P are depicted in Fig. 4. The percentage of accumulated 334
carbohydrate in BBM and wastewater media were ranged from 43.98 to 49.96%. As compared to BBM 335
medium, the concentration of protein increased in PAWW'BBM and PAWW'N+P media. The highest 336
protein content was observed in microalga cultivated in PAWW'BBM, but it was not significantly 337
higher than in PAWW'N+P. In agreement to this study, the same range of protein concentration of C.
338
vulgaris (from 44 to 46%) was observed for control and experimental groups by other researchers 339
(Marudhupandi et al., 2014). The value of carbohydrate as one of the major component of microalgae 340
is around 12-17% in C. vulgaris under normal conditions (Spolaore et al., 2006). Here, the percentage 341
of carbohydrate varied from 18.32 to 19.09% for different media. In addition, accumulated 342
carbohydrates in microalga cultivated in PAWW can be converted to fermentable sugars and employed 343
as an appropriate bio-resource for production of bioethanol (Pancha et al., 2014). The lipid content of 344
C. vulgaris was varied from 7.95 to 12.10%. The highest lipid percentage was found in BBM medium, 345
12.10%. It was 1.52 and 1.33-fold higher than in PAWW'BBM and PAWW'N+P. As derives from Fig.
346
4, lipid concentration was higher in BBM medium while protein concentration was higher in 347
PAWW'BBM and PAWW'N+P media. These findings might be related to higher concentration of TN 348
in PAWW'BBM and PAWW'N+P in comparison with BBM medium. The higher concentration of 349
nitrogen in algae medium will induce the concentration of protein and reduce the concentration of 350
lipids (Martınez et al., 2000; Hsieh and Wu, 2009). The value of biochemical composition of C.
351
vulgaris in PAWW (PAWW'BBM and PAWW'N+P) was more than 75%. Similar results have been 352
reported by other researchers where average concentration of biochemical composition of C. vulgaris, 353
cultivated in diluted monosodium glutamate wastewater, was found to be 77.28% (Ji et al., 2014b).
354
Page 16 of 28 355
3.5. Fatty acid profile of C. vulgaris in different culture media 356
Lipids were extracted from harvested algal biomass of BBM, PAWW'BBM and PAWW'N+P media.
357
The major fatty acids of extracted lipid were identified by a GC system. The nine identical FAMEs 358
with different relative content were found in the investigated media (Fig. 5). The chain length of these 359
FAMEs was found to range between C14 and C20 that is considered as the appropriate FAMEs for 360
biodiesel production (Zheng et al., 2012). Similar to these results, Zheng et al. reported FAMEs chain 361
length of C. vulgaris in the range of C14 and C20 (Zheng et al., 2012). The total percentage of C16 362
(hexadecanoic acid) and C18 (octadecanoic acid) FAMEs were found to be 96.3, 96.40 and 95.98% in 363
the algal biomass from the aforementioned media. Mathimani et al. reported that C16 and C18 are the 364
major FAMEs present in C. vulgaris (Mathimani and Nair, 2016). The sum of saturated and 365
monounsaturated fatty acids (e.g. 16:0, 16:1 and 18:1) percentage as the preferred fatty acids for 366
producing biodiesel, was higher than 50% in the current study. Saturated fatty acids like C16:0 with 367
high cetane number are more oxidative-stable while monounsaturated fatty acids reduce the freezing 368
point and improve the low temperature properties of biodiesel (Hu et al., 2008). Stressful conditions 369
such as nutrient deprivation, lower growth temperature and higher light intensity can change the fatty 370
acids composition of microalgae (Cho et al., 2016). Insignificant variations in the fatty acid profiles of 371
C. vulgaris, both in BBM and PAWW, revealed that PAWW can be used as microalgal medium and 372
preserve diversity of microalgae fatty acids.
373 374 375 376 377 378
Page 17 of 28 379
4. Conclusions 380
Algal growth was investigated with and without the addition of macro and micronutrients to PAWW 381
(mixture of PWW and AWW). The highest dry algal weight was observed as 1.31 g/L in 60%
382
PWW:40% AWW ratio without adding micronutrients. Removal efficiency of nutrients and organic 383
compounds was observed in PAWW'N+P as TN (76.56%), TP (92.72%), COD (75.48%) and TOC 384
(70.67%). Proteins, carbohydrates and lipids concentration were high in algal biomass harvested from 385
optimized medium of PAWW'N+P. Microalgal growth was low in PWW due to low concentration of 386
nitrate and phosphate. Mixing PWW with AWW improved algal growth.
387 388
Acknowledgements 389
Authors wish to thank Harri Kokko from University of Eastern Finland and Maarit Janhunen from 390
Savonia University of Applied Sciences for their help in collecting aquaculture and pulp wastewaters.
391
Authors (ED and AB) gratefully acknowledge financial support from Nordic Centre of Excellence for 392
Sustainable and Resilient Aquatic Production (SUREAQUA), funded by NordForsk (Grant number:
393
82342), within the Nordic bioeconomy programme. Authors also wish to thank all the anonymous 394
reviewers whose comments/suggestions have significantly improved the quality of this manuscript.
395 396 397
398 399 400 401
Page 18 of 28 402
References 403
1. Ashrafi, O., Yerushalmi, L., Haghighat, F., 2015. Wastewater treatment in the pulp-and-paper industry: A 404
review of treatment processes and the associated greenhouse gas emission, J. Environ. Manage. 158, 146- 405
157.
406
2. Batista, A.P., Gouveia, L., Bandarra, N.M., Franco, J.M., Raymundo, A., 2013. Comparison of microalgal 407
biomass profiles as novel functional ingredient for food products, Algal Research. 2, 164-173.
408
3. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein 409
utilizing the principle of protein-dye binding, Anal. Biochem. 72, 248-254.
410
4. Canakci, M., Sanli, H., 2008. Biodiesel production from various feedstocks and their effects on the fuel 411
properties, J. Ind. Microbiol. Biotechnol. 35, 431-441.
412
5. Carvalho, A.P., Pontes, I., Gaspar, H., Malcata, F.X., 2006. Metabolic relationships between macro-and 413
micronutrients, and the eicosapentaenoic acid and docosahexaenoic acid contents of Pavlova lutheri, 414
Enzyme Microb. Technol. 38, 358-366.
415
6. Cho, K., Lee, C., Ko, K., Lee, Y., Kim, K., Kim, M., Chung, Y., Kim, D., Yeo, I., Oda, T., 2016. Use of phenol- 416
induced oxidative stress acclimation to stimulate cell growth and biodiesel production by the oceanic 417
microalga Dunaliella salina, Algal Research. 17, 61-66.
418
7. El-Kassas, H.Y., 2013. Growth and fatty acid profile of the marine microalga Picochlorum sp. grown under 419
nutrient stress conditions, The Egyptian Journal of Aquatic Research. 39, 233-239.
420
8. Gao, F., Li, C., Yang, Z., Zeng, G., Feng, L., Liu, J., Liu, M., Cai, H., 2016. Continuous microalgae cultivation in 421
aquaculture wastewater by a membrane photobioreactor for biomass production and nutrients removal, 422
Ecol. Eng. 92, 55-61.
423
9. Gentili, F.G., 2014. Microalgal biomass and lipid production in mixed municipal, dairy, pulp and paper 424
wastewater together with added flue gases, Bioresour. Technol. 169, 27-32.
425
10.Hsieh, C., Wu, W., 2009. Cultivation of microalgae for oil production with a cultivation strategy of urea 426
limitation, Bioresour. Technol. 100, 3921-3926.
427
11.Hu, Q., Sommerfeld, M., Jarvis, E., Ghirardi, M., Posewitz, M., Seibert, M., Darzins, A., 2008. Microalgal 428
triacylglycerols as feedstocks for biofuel production: perspectives and advances, The plant journal. 54, 621- 429
639.
430
12. Hwang, J., Kabra, A.N., Kim, J.R., Jeon, B., 2014. Photoheterotrophic microalgal hydrogen production using 431
acetate-and butyrate-rich wastewater effluent, Energy. 78, 887-894.
432
13. Ji, M., Kabra, A.N., Salama, E., Roh, H., Kim, J.R., Lee, D.S., Jeon, B., 2014a. Effect of mine wastewater on 433
nutrient removal and lipid production by a green microalga Micratinium reisseri from concentrated 434
municipal wastewater, Bioresour. Technol. 157, 84-90.
435
14.Ji, M., Yun, H., Hwang, B.S., Kabra, A.N., Jeon, B., Choi, J., 2016. Mixotrophic cultivation of Nephroselmis sp.
436
using industrial wastewater for enhanced microalgal biomass production, Ecol. Eng. 95, 527-533.
437
15. Ji, M., Yun, H., Park, S., Lee, H., Park, Y., Bae, S., Ham, J., Choi, J., 2015. Effect of food wastewater on biomass 438
production by a green microalga Scenedesmus obliquus for bioenergy generation, Bioresour. Technol. 179, 439
624-628.
440
16.Ji, Y., Hu, W., Li, X., Ma, G., Song, M., Pei, H., 2014b. Mixotrophic growth and biochemical analysis of 441
Chlorella vulgaris cultivated with diluted monosodium glutamate wastewater, Bioresour. Technol. 152, 471- 442
476.
443
17. Jiang, L., Pei, H., Hu, W., Hou, Q., Han, F., Nie, C., 2016. Biomass production and nutrient assimilation by a 444
novel microalga, Monoraphidium spp. SDEC-17, cultivated in a high-ammonia wastewater, Energy 445
Conversion and Management. 123, 423-430.
446
Page 19 of 28
18. Kuo, C., Chen, T., Lin, T., Kao, C., Lai, J., Chang, J., Lin, C., 2015. Cultivation of Chlorella sp. GD using piggery 447
wastewater for biomass and lipid production, Bioresour. Technol. 194, 326-333.
448
19.Levine, R.B., Costanza-Robinson, M.S., Spatafora, G.A., 2011. Neochloris oleoabundans grown on 449
anaerobically digested dairy manure for concomitant nutrient removal and biodiesel feedstock production, 450
Biomass Bioenergy. 35, 40-49.
451
20. Likozar, B., Levec, J., 2014. Effect of process conditions on equilibrium, reaction kinetics and mass transfer 452
for triglyceride transesterification to biodiesel: experimental and modeling based on fatty acid composition, 453
Fuel Process Technol. 122, 30-41.
454
21.Likozar, B., Pohar, A., Levec, J., 2016. Transesterification of oil to biodiesel in a continuous tubular reactor 455
with static mixers: Modelling reaction kinetics, mass transfer, scale-up and optimization considering fatty 456
acid composition, Fuel Process Technol. 142, 326-336.
457
22. Liu, C., Subashchandrabose, S.R., Megharaj, M., Hu, Z., Xiao, B., 2016. Diplosphaera sp. MM1–A microalga 458
with phycoremediation and biomethane potential, Bioresour. Technol. 218, 1170-1177.
459
23. Markou, G., 2015. Fed-batch cultivation of Arthrospira and Chlorella in ammonia-rich wastewater:
460
optimization of nutrient removal and biomass production, Bioresour. Technol. 193, 35-41.
461
24.Martınez, M., Sánchez, S., Jimenez, J., El Yousfi, F., Munoz, L., 2000. Nitrogen and phosphorus removal from 462
urban wastewater by the microalga Scenedesmus obliquus, Bioresour. Technol. 73, 263-272.
463
25.Marudhupandi, T., Gunasundari, V., Kumar, T.T.A., Tissera, K.R., 2014. Influence of citrate on Chlorella 464
vulgaris for biodiesel production, Biocatalysis and Agricultural Biotechnology. 3, 386-389.
465
26. Mathimani, T., Nair, B.B., 2016. Evaluation of microalga for biodiesel using lipid and fatty acid as a marker–A 466
central composite design approach, Journal of the Energy Institute. 89, 436-446.
467
27.Maurya, R., Paliwal, C., Ghosh, T., Pancha, I., Chokshi, K., Mitra, M., Ghosh, A., Mishra, S., 2016. Applications 468
of de-oiled microalgal biomass towards development of sustainable biorefinery, Bioresour. Technol. 214, 469
787-796.
470
28.Molins-Legua, C., Meseguer-Lloret, S., Moliner-Martinez, Y., Campíns-Falcó, P., 2006. A guide for selecting 471
the most appropriate method for ammonium determination in water analysis, TrAC Trends in Analytical 472
Chemistry. 25, 282-290.
473
29. Mujtaba, G., Rizwan, M., Lee, K., 2017. Removal of nutrients and COD from wastewater using symbiotic co- 474
culture of bacterium Pseudomonas putida and immobilized microalga Chlorella vulgaris, Journal of Industrial 475
and Engineering Chemistry. 49, 145-151.
476
30.Nayak, M., Karemore, A., Sen, R., 2016. Performance evaluation of microalgae for concomitant wastewater 477
bioremediation, CO 2 biofixation and lipid biosynthesis for biodiesel application, Algal Research. 16, 216- 478
223.
479
31. Pancha, I., Chokshi, K., George, B., Ghosh, T., Paliwal, C., Maurya, R., Mishra, S., 2014. Nitrogen stress 480
triggered biochemical and morphological changes in the microalgae Scenedesmus sp. CCNM 1077, 481
Bioresour. Technol. 156, 146-154.
482
32.Rausch, T., 1981. The estimation of micro-algal protein content and its meaning to the evaluation of algal 483
biomass I. Comparison of methods for extracting protein, Hydrobiologia. 78, 237-251.
484
33.Ruiz, J., Arbib, Z., Alvarez-Díaz, P., Garrido-Pérez, C., Barragán, J., Perales, J., 2014. Influence of light 485
presence and biomass concentration on nutrient kinetic removal from urban wastewater by Scenedesmus 486
obliquus, J. Biotechnol. 178, 32-37.
487
34. Salama, E., Kabra, A.N., Ji, M., Kim, J.R., Min, B., Jeon, B., 2014. Enhancement of microalgae growth and fatty 488
acid content under the influence of phytohormones, Bioresour. Technol. 172, 97-103.
489
35.Salama, E., Kurade, M.B., Abou-Shanab, R.A., El-Dalatony, M.M., Yang, I., Min, B., Jeon, B., 2017. Recent 490
progress in microalgal biomass production coupled with wastewater treatment for biofuel generation, 491
Renewable and Sustainable Energy Reviews. 79, 1189-1211.
492
36. Schwarzenbach, R.P., Escher, B.I., Fenner, K., Hofstetter, T.B., Johnson, C.A., von Gunten, U., Wehrli, B., 493
2006. The challenge of micropollutants in aquatic systems, Science. 313, 1072-1077.
494
Page 20 of 28
37. Sepúlveda, C., Acién, F., Gómez, C., Jiménez-Ruíz, N., Riquelme, C., Molina-Grima, E., 2015. Utilization of 495
centrate for the production of the marine microalgae Nannochloropsis gaditana, Algal Research. 9, 107-116.
496
38.Singh, P., Guldhe, A., Kumari, S., Rawat, I., Bux, F., 2016. Combined metals and EDTA control: an integrated 497
and scalable lipid enhancement strategy to alleviate biomass constraints in microalgae under nitrogen 498
limited conditions, Energy Conversion and Management. 114, 100-109.
499
39. Šoštarič, M., Klinar, D., Bricelj, M., Golob, J., Berovič, M., Likozar, B., 2012. Growth, lipid extraction and 500
thermal degradation of the microalga Chlorella vulgaris, New biotechnology. 29, 325-331.
501
40. Spolaore, P., Joannis-Cassan, C., Duran, E., Isambert, A., 2006. Commercial applications of microalgae, 502
Journal of bioscience and bioengineering. 101, 87-96.
503
41.Sunda, W.G., Price, N.M., Morel, F.M., 2005. Trace metal ion buffers and their use in culture studies, Algal 504
culturing techniques. 4, 35-63.
505
42. Zheng, H., Gao, Z., Yin, F., Ji, X., Huang, H., 2012. Effect of CO 2 supply conditions on lipid production of 506
Chlorella vulgaris from enzymatic hydrolysates of lipid-extracted microalgal biomass residues, Bioresour.
507
Technol. 126, 24-30.
508
43. Zhu, L., Wang, Z., Shu, Q., Takala, J., Hiltunen, E., Feng, P., Yuan, Z., 2013. Nutrient removal and biodiesel 509
production by integration of freshwater algae cultivation with piggery wastewater treatment, Water Res.
510
47, 4294-4302.
511 512 513
Page 21 of 28 514
515
Fig. 1. Biomass productivity of microalga in different concentrations of PWW after seven days of 516
cultivation.
517 518 519 520 521 522 523 524 525 526
0:100 20:80 40:60 60:40 80:20 100:0 BBM 0,0
0,2 0,4 0,6 0,8 1,0 1,2
1,4
PLWWPAWW
A lg a l dry w ei g ht (g /L)
PWW dilution ratios (%, PWW:LW/AWW)
1.4 1.2 1.0
0.8 0.6 0.4 0.2
0.0
0:100 20:80 40:60 60:40 80:20 100:0 BBM 0,0
0,2 0,4 0,6 0,8 1,0 1,2
1,4
PLWWPAWW
0:100 20:80 40:60 60:40 80:20 100:0 BBM 0,0
0,2 0,4 0,6 0,8 1,0 1,2
1,4
PLWWPAWW
0:100 20:80 40:60 60:40 80:20 100:0 BBM 0,0
0,2 0,4 0,6 0,8 1,0 1,2 1,4
Control (BBM) PAWW
Control 0:100 20:80 40:60 60:40 80:20 100:0 BBM
0,0 0,2 0,4 0,6 0,8 1,0 1,2
1,4
PLWWPAWW
0:100 20:80 40:60 60:40 80:20 100:0 BBM 0,0
0,2 0,4 0,6 0,8 1,0 1,2
1,4
PLWWPAWW
0:100 20:80 40:60 60:40 80:20 100:0 BBM 0,0
0,2 0,4 0,6 0,8 1,0 1,2
1,4
PLWWPAWW
Page 22 of 28 527
528
Fig. 2.Biomass productivity of microalga in PAWW with and without adding nutrients after seven 529
days of cultivation.
530 531 532 533 534
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 0,0
0,2 0,4 0,6 0,8 1,0 1,2
1,4 B
D
A lg a l dry w ei g ht (g /L)
1.4
1.2 1.0 0.8 0.6 0.4 0.2
0.0
Media (experimental units)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 0,0
0,2 0,4 0,6 0,8 1,0 1,2
1,4 B
D
A lg a l dry w ei g ht (g /L)
1.4 1.2 1.0
0.8 0.6 0.4 0.2 0.0
Media (experimental units)
0 5 10 15 20 25 30 35
0,0 0,2 0,4 0,6 0,8 1,0 1,2
1,4 20% AWW+80% PWW 40% AWW+60% PWW
0 5 10 15 20 25 30 35
0,0 0,2 0,4 0,6 0,8 1,0 1,2
1,4 20% AWW+80% PWW 40% AWW+60% PWW
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4
20% AWW+80% PWW 40% AWW+60% PWW
Control
0 5 10 15 20 25 30 35
0,0 0,2 0,4 0,6 0,8 1,0 1,2
1,4 20% AWW+80% PWW 40% AWW+60% PWW 20% AWW:80% PWW
0 5 10 15 20 25 30 35
0,0 0,2 0,4 0,6 0,8 1,0 1,2
1,4 20% AWW+80% PWW 40% AWW+60% PWW 40% AWW:60% PWW
0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4
20% AWW+80% PWW 40% AWW+60% PWW Control (BBM)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 0,0
0,2 0,4 0,6 0,8 1,0 1,2
1,4 B
D
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 0,0
0,2 0,4 0,6 0,8 1,0 1,2
1,4
B
D
0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4
20% AWW+80% PWW 40% AWW+60% PWW
Control
Page 23 of 28 535
536
Fig. 3. Nutrients and organic matters removal efficiencies by cultivation of microalga in PAWW after 537
seven days of cultivation.
538 539
1 2 3 4 5 6
0 20 40 60 80 100
PAWW'BBM PAWW'N+P PAWW
NO
3-N NH
3-N TP COD TN TOC
P o llu ta n ts re m o v a l ef ficiency (% )
Pollutants
1 2 3 4 5 6
0 20 40 60 80 100
PAWW'BBM PAWW'N+P PAWW
1 2 3 4 5 6
0 20 40 60 80 100
PAWW'BBM PAWW'N+P PAWW
1 2 3 4 5 6
0 20 40 60 80 100
PAWW'BBM
PAWW'N+P
PAWW
Page 24 of 28 540
Fig. 4. Biochemical composition of cultivated microalga in BBM and PAWW after seven days of 541
cultivation.
542
BBM PAWW'BBM PAWW'N+P
0 10 20 30 40 50 60 70 80
B io ch em ica l co m p o sit io n ( % )
Media
BBM PAWW'BBM PAWW'N+P
0 10 20 30 40 50 60 70 80
90 Lipid
Carbohydrate Protein
BBM PAWW'BBM PAWW'N+P
0 10 20 30 40 50 60 70 80
90 Lipid
Carbohydrate Protein
BBM PAWW'BBM PAWW'N+P
0 10 20 30 40 50 60 70 80
90 Lipid
Carbohydrate Protein
43.98 2 49.96 3 47.49 1
12.10 1
7.95 1 9.07 1
18.32 1
18.52 2 19.09 1
Page 25 of 28 543
544
Fig. 5. Fatty acid methyl esters (FAMEs) of cultivated microalga in BBM and PAWW after seven days 545
of cultivation.
546 547 548 549 550 551 552
Fatty acid profiles (% of total FAME)
0 20 40 60 80 100
25.87 0.6 25.64 2 22.22 0.1
28.26 0.0 21.19 1 27.48 0.0
18.67 1 20.23 0.4 23.99 0.4
20.14 2 23.86 0.3 16.63 0.2
0 20 40 60 80 100
C18:3n3 C18:3n6 C18:2 C18:1 C18:0 C17:1 C16:1 C16:0 C14:0
BBM PAWW'BBM PAWW Culture mediums
