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Cultivation of Chlorella sp. GD using piggery wastewater for biomass and lipid production
Accepted Manuscript Cultivation of Chlorella sp. GD using piggery wastewater for biomass and lipid production Chiu-Mei Kuo, Tsai-Yu Chen, Tsung-Hsien Lin, Chien-Ya Kao, Jinn-Tsyy Lai, Jo-Shu Chang, Chih-Sheng Lin PII: DOI: Reference:
S0960-8524(15)00979-7 http://dx.doi.org/10.1016/j.biortech.2015.07.026 BITE 15259
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Bioresource Technology
Received Date: Revised Date: Accepted Date:
13 May 2015 6 July 2015 7 July 2015
Please cite this article as: Kuo, C-M., Chen, T-Y., Lin, T-H., Kao, C-Y., Lai, J-T., Chang, J-S., Lin, C-S., Cultivation of Chlorella sp. GD using piggery wastewater for biomass and lipid production, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.07.026
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Cultivation of Chlorella sp. GD using piggery wastewater for biomass and lipid production Chiu-Mei Kuoa, Tsai-Yu Chena, Tsung-Hsien Lina, Chien-Ya Kao a,b, Jinn-Tsyy Laic, Jo-Shu Changd, e, Chih-Sheng Lina,* a
Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan
b
Aquatic Technology Laboratories, Agricultural Technology Research Institute, Hsinchu, Taiwan
c
Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan
d
Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan
e
Research Center for Energy Technology and Strategy, National Cheng Kung University, Tainan, Taiwan
* Author for correspondence: Chih-Sheng Lin, Ph.D. Department of Biological Science and Technology, National Chiao Tung University, No.75 Bo-Ai Street, Hsinchu 30068, Taiwan Tel: 886-3-5131338 E-mail: [email protected]
1 2
Abstract The development of a culture system for Chlorella sp. GD to efficiently produce
3
biomass and oil for biodiesel production was investigated. Chlorella sp. GD was
4
cultivated with 0, 25, 50, 75 and 100% piggery wastewater (diluted by medium) at 300
5
µmol m-2 s-1, a 2% CO2 aeration rate of 0.2 vvm and 26 ± 1°C; after a 10-day culture in
6
batch cultures, the maximum specific growth rate and biomass productivity of the
7
microalga obtained in 100% piggery wastewater were 0.839 d-1 and 0.681 g L-1 d -1,
8
respectively. The highest lipid content and lipid productivity were 29.3% and 0.155 g
9
L-1 d-1 at 25% wastewater, respectively. In semi-continuous cultures, the biomass and
10
lipid productivities with 25-75% wastewater ratios were greater than 0.852 and 0.128 g
11
L-1 d-1, respectively. These results show that Chlorella sp. GD grows efficiently in
12
piggery wastewater, and that a stable growth performance was achieved for long-term
13
microalgal cultivation in a semi-continuous culture.
14
Keywords: microalgae, wastewater, semi-continuous culture, biomass, lipid
15 16 17 18
1. Introduction As the demand for global energy continues to increase, fossil fuel usage will also
19
continue to increase. Over the long term, fossil fuels are not a sustainable energy
20
resource, and the burning of fossil fuels increases greenhouse gas (GHG) emissions.
21
Therefore, alternative energy sources are being explored to replace fossil fuels. In recent
22
years, microalgae have been considered as a third generation biofuel feedstock because
23
they not only utilize carbon dioxide (CO2) as a carbon source during autotrophic growth
24
to decrease GHGs, but their resulting biomass also contains abundant energy-rich
25
components that can be converted to various biofuels, such as ethanol, butanol, methane 1
26
and biodiesel (Ho et al., 2013). In addition, microalgal biomass is a potential feedstock
27
for biorefineries that can be converted into high value products containing
28
polyunsaturated fatty acids (e.g., DHA and EPA), chlorophylls, carotenoids, phycobilins
29
and others (Yen et al., 2013).
30
Biodiesel is the second largest category of global biofuel, accounting for 6.9 billion
31
gallons globally in 2013, which was 22.6% of the total biofuel production (REN21,
32
2014). Biodiesel is produced via the transesterification process by reacting fats,
33
including vegetable oil, animal fats or waste cooking oils, with an alcohol, such as
34
methanol (Klofutar et al., 2010). Homogeneous alkali-catalyzed transesterification is the
35
most commonly used method, and leads to high conversion levels of triglycerides to
36
their corresponding methyl esters with short reaction times. On the industrial scale,
37
alkalis, such as NaOH and KOH are commonly used as catalysts (Likozar and Levec,
38
2014; Šoštarič et al., 2012). However, with the increasing demand for these oilseed
39
crops there is now an issue regarding food-fuel competition; therefore, an alternative
40
feedstock needs to be developed. Biodiesel derived from microalgae has been
41
considered a promising approach because microalgae have much higher biomass
42
productivity in comparison to terrestrial plants and can be cultivated on non-arable land
43
without competing with other crops (Chisti, 2007; Mata et al., 2010). The main inputs
44
required in addition to microalgae are sunlight, water, CO2 and nutrients, e.g., carbon,
45
nitrogen (N) and phosphorus (P) sources. However, the main drawback for economical
46
biodiesel production from microalgae is the high cost of microalgal cultivation.
47
Approximately 80% of the total medium costs went to microalgae cultivation because
48
of the huge consumption of nutrients and water (Li et al., 2007). When Chlorella
49
zofingiensis was cultivated in diluted piggery wastewater with 1,900 mg L-1 COD
50
culture, the highest biomass productivity was 296 mg L-1 day-1, the total nitrogen (TN) 2
51
and total phosphorus (TP) removal was 83% and almost 100%, respectively (Zhu et al.,
52
2013a). The biomass production of Chlorella sp. growing in the centrate, a highly
53
concentrated municipal wastewater, was 0.212 g L-1 day-1, the TN removal was
54
approximately 90% (Li et al., 2011). Under photoautotrophic cultivation with brewery
55
wastewater for Chlorella vulgaris, the biomass production was 0.149 g L-1 day-1, both
56
TN and TP removal were more than 80% (Farooq et al., 2013). However, microalgal
57
growth is sometimes restricted by the presence of bacteria and protozoa in wastewater.
58
On the laboratory scale, wastewater is often pretreated by autoclave sterilization to
59
reduce the effect of bacteria and protozoa on microalgal growth. However, autoclave
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sterilization pretreatment would increase the cost and difficulty of large-scale
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microalgal cultivation. To guarantee stable microalgal growth using wastewater against
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possible microbial contamination, it is necessary to develop control techniques for
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biotic pollution (Chiu et al., 2015).
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Although many studies have utilized wastewater for biodiesel production in
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microalgal cultivation, few studies have established a continuous stable-growth
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microalgal cultivation system combined with wastewater replacement. Semi-continuous
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microalgal cultivation is an effective strategy to maintain the growth of the exponential
68
phase and enhance the light penetration by culture broth replacement (Chiu et al., 2008).
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The aim of this study is to provide an efficient and stable culturing system for
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microalgal biomass production from biofuel feedstock by using swine wastewater from
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livestock farms. Lipids extracted from Chlorella sp. GD can be transesterified into fatty
72
acid methyl ester (FAME) to produce biodiesel. Moreover, the non-lipid fraction of
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Chlorella sp. GD biomass, which mainly consists of protein (approximately 60% of
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microalgal dry weight), can also be processed to methane and feeds applied in
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aquaculture, poultry and swine. The integrated microalgal culture system with livestock 3
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wastewater may also reduce the cost of biodiesel production and provide an
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environmental benefit of microalgal bioremediation.
78 79 80 81
2. Methods 2.1 Microalgal cultures, medium and chemicals
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The freshwater microalgae Chlorella sp. GD was screened for its potential
83
ability for growth and biomass production according to our previous report (Chiu et
84
al., 2009). The Chlorella sp. GD cells were grown on a modified freshwater medium
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containing 1.25 g L-1 KNO3, 1.25 g L-1 KH2PO4, 1 g L-1 MgSO4．7H2O, 0.5 g L-1
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EDTA．2Na, 83.5 mg L-1 CaCl2．2H2O, 0.1142 g L-1 H3BO3, 49.8 mg L-1 FeSO4．
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7H2O, 88.2 mg L-1 ZnSO4．7H2O, 14.4 mg L-1 MnCl2．4H2O, 10 mg L-1 CuSO4, 7.1
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mg L-1 Na2MoO4 and 4 mg L-1 CoCl2．6H2O. The initial pH of the initial medium
89
was adjusted to 6 with NaOH.
90 91
2.2 Source of wastewater
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The experiments were conducted using piggery wastewater and industrial and
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municipal wastewater from a swine farm (Miaoli, Taiwan), Hsinchu industrial park
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(Hukou, Taiwan) and National Chiao Tung University (Hsinchu, Taiwan),
95
respectively. The wastewater was treated by a three-step waste treatment system
96
consisting of solid-liquid separation, anaerobic treatment and aerobic treatment. The
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samples were periodically collected from the treatment system between April 2013
98
and August 2013. The wastewater consists of 490 ± 60 mg L-1 ammonia–N
99
(NH4+–N), 10 ± 5 mg L-1 nitrite–N (NO2- –N), 2 ± 1 mg L-1 nitrate–N (NO3- –N), 550
100
± 70 mg L-1 total nitrogen (TN), 20 ± 6 mg L-1 total phosphorus (TP), 80 ± 20 mg L-1 4
101
suspended solids (SS) and 430 ± 60 mg L-1 chemical oxygen demand (COD). The pH
102
of the wastewater was 8.5 ± 0.5.
103 104 105
2.3 Pretreatment of wastewater To avoid other interference in the wastewater, as a pretreatment, the wastewater
106
was autoclaved for 30 min at 121°C. Then, the liquid was centrifuged (3,000×g for
107
10 min) to separate the large non-soluble particles from the liquid. The supernatant
108
was stored in a cold room at 4°C and used for further experiments.
109 110 111
2.4 Experimental system of indoor photobioreactors The microalgae cells were cultured in photobioreactors with a working volume
112
of 1 L (Chiu et al., 2008). The photobioreactors were placed in an incubator at 26 ±
113
1°C with a surface light intensity of approximately 300 µmol m-2 s-1 provided by
114
continuous cool-white fluorescent lights. The photobioreactor was a cylindrical glass
115
column with a diameter and length of 6 and 80 cm, respectively. Gas was provided as
116
2% CO2 mixed with ambient air. The microalgal cultures were aerated continuously
117
with gas that was provided via bubbling from the bottom of the photobioreactor with
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an aeration rate of 200 mL min-1 (i.e., 0.2 vvm, volume of gas per volume of broth
119
per min).
120 121
2.5 Preparation of inoculum
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A stock culture of Chlorella sp. GD (approximately 0.3 g L-1) was incubated in
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a column containing 1 L of working volume of the freshwater microalgal medium at
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26 ± 1°C and 300 µmol m-2 s-1. After 3 to 5 days of culture, the biomass
125
concentration was greater than 1.5 g L-1, and the biomass was diluted depending on 5
126
the desired biomass concentration for further experiments.
127 128 129
2.6 Experimental design of batch cultivation The photobioreactor was inoculated with 200 mL of pre-cultured Chlorella sp.
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GD broth and was filled with 800 mL of wastewater at different dilution ratios of
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medium to achieve approximately 0.3 g L-1 of the initial inoculum. Chlorella sp. GD
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was cultured with 0, 25, 50, 75 and 100% piggery wastewater for 10 days. The
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microalgal culture with pure medium (ratio of adding wastewater was 0%) was the
134
control group to compare the difference of growth between the microalgal culture
135
grown with medium and wastewater. Microalgal cultivation was illuminated at 300
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µmol m-2 s-1, a 2% CO2 aeration rate of 0.2 vvm and 26 ± 1°C. The microalgal cells
137
in each culture were sampled at 24 h intervals to determine the optical density, lipid
138
content and fatty acid composition.
139 140
2.7 Experimental design of semi-continuous cultivation
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The semi-continuous culture was carried out in two stages. First, the initial
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biomass concentration of batch cultures was 0.3 g L-1. The biomass concentration of
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Chlorella sp. GD at the stationary phase reached approximately 4 g L-1, which
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occurred after 5 days of incubation (cycle 1 of semi-continuous culture).
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Subsequently, half of the volume of the culture broth was replaced with 25, 50, 75
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and 100% fresh wastewater every 3 days for a period of 12 days (i.e., cycles 2-5 of
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semi-continuous culture). The 25, 50 and 75% piggery wastewater cultures were a
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mix of wastewater and medium. The cultures were illuminated at 300 µmol m-2 s-1, a
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2% CO2 aeration rate of 0.2 vvm and 26 ± 1°C. Every 24 h, each culture was
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sampled to determine the optical density, lipid content and fatty acid composition. 6
151 152 153
2.8 Determination of microalgal cell biomass and growth rate The biomass concentration (dry weight per liter) of the microalgal cultures was
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determined according to a previously reported method (Chiu et al., 2009). A
155
calibration equation considering the optical density and the dry weight of microalgal
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cells was established, as follows:
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Biomass concentration (g L-1) = 0.3101× × A682nm – 0.0065, R² = 0.9981
158
Hence, the biomass concentration could be precisely calculated (R2 = 0.9981; p
159
< 0.001) using the measured optical density (A682) in an Ultrospec 3300 pro
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UV/Visible spectrophotometer (Amersham Biosciences, Cambridge, UK). Each
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sample was diluted to give an absorbance in the range of 0.1 to 1.0 if the optical
162
density was greater than 1.0.
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The optical density was used to evaluate the biomass concentration (g L-1) of
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Chlorella sp. GD in each experiment. The biomass productivity (g L-1 d-1) was
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measured according to the following equation:
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Biomass productivity (g L-1 d-1) = (Wf – Wi) / ∆t
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where Wf and Wi are the final and initial biomass concentrations, respectively,
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and ∆t is the cultivation time in days.
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The specific growth rate (µ, d-1) was calculated as follows:
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µ (d-1) = (lnX2 – lnX1) / (t2 – t1)
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where X2 and X1 are the biomass concentration (g L-1) on t2 and t1 (days),
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respectively. When X2 was equal to 2X1 in the exponential phase of cultivation, the
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period of time required was the doubling time (td, h), which was calculated as
174
follows:
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td (h) = 24 × (ln2) / µ 7
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2.9 Lipid extraction Lipid extraction was performed according to the modification of a method that
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was previously reported (Kao et al., 2014). The microalgae cells were centrifuged,
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washed twice with deionized water and lyophilized to obtain the dry biomass. The
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dried sample (200 mg) was mixed with a chloroform/methanol solution (2/1, v/v) and
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was sonicated for 1 h. The mixture with the chloroform/methanol solution was
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precipitated and chloroform and a 0.9% NaCl solution was added to give a 2:1:1 ratio
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of chloroform, methanol and water. The mixture was centrifuged, and the chloroform
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phase was recovered. Finally, the lipids were weighed after chloroform was removed
186
under a vacuum by a rotary evaporator.
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2.10 Microalgal lipid transesterification and fatty acid profile assay The method used for the transesterification of the microalgal lipids and the fatty
190
acid profile assay was based on previously reported procedures (Chiu et al., 2011).
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The extracted oil samples were placed in a glass test tube and mixed with 2.0 mL of
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chloroform, 1.7 mL of methanol and 0.3 mL of sulfuric acid. The samples were
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sonicated for 60 min. After the reaction completed, the tubes were removed from the
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water bath and were allowed to cool to room temperature. Then, 2 mL of distilled
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water was added to the tube and thoroughly mixed using a vortex. The samples were
196
allowed to separate, forming a biphasic solution. The organic layer containing fatty
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acid methyl ester (FAME) was collected and transferred to a pre-weighed glass vial.
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The solvent was then evaporated using N2 and was heated at 70°C for 40 min.
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Finally the mass of FAME was determined via weighing.
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The fatty acid composition was determined using a FOCUS Gas Chromatograph 8
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(Thermo Fisher Scientific, Waltham, MA, USA) according to the method described
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by Kao et al. (2014).
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2.11 Nutrient analysis in wastewater
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A volume of 50 mL of microalgal broth was collected every day from each
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photobioreactor for nutrient removal analysis starting from inoculation. The samples
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were first centrifuged at 3,000×g for 10 min. Then, the supernatant was analyzed for
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COD, NH4+–N, NO2- –N, NO3- –N, TN and TP following the Hach DR/890
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Colorimeter Procedures Manual (Hach, 2009).
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3. Results and discussion 3.1 Growth profiles of Chlorella sp. GD cultivated with different ratios of wastewater in batch cultivation There are three main sources of wastewater that contain a variety of ingredients,
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i.e., municipal, agricultural and industrial wastewaters. When Chlorella sp. GD was
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cultivated in different sources of wastewater (piggery, industrial and municipal
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wastewater) without dilution and medium in batch cultivation, the maximum biomass
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was obtained in piggery wastewater (Fig. 1).
220
In the present study, the effects of microalgal growth with different ratios of
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piggery wastewater were studied. The microalgae Chlorella sp. GD were cultured
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with 0 (i.e., cultured in medium), 25, 50, 75 and 100% wastewater diluted with
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culture medium for 10 days in batch cultivation. Fig. 2A shows all of the growth
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potentials of Chlorella sp. GD cultured with wastewater were significantly higher
225
than those obtained when the microalga were cultured in medium alone. When 9
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Chlorella sp. GD was cultured with 0, 25, 50, 75 and 100% wastewater in the
227
exponential phase of the batch culture, the specific growth rates (µ) were 0.467,
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0.733, 0.766, 0.797 and 0.839 d-1, respectively, and the doubling times (td) were 35.6,
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22.7, 21.7, 20.9 and 19.8 h, respectively. Higher ratios of wastewater correlated with
230
shorter doubling times and higher maximum biomass concentrations. It was reported
231
that the maximum specific growth rate of Chlorella vulgaris that was cultivated with
232
100% secondary municipal wastewater was 0.52 d -1 (Ebrahimian et al., 2014), which
233
is consistent with our results. When Chlorella protothecoides UTEX 411, C. vulgaris
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UTEX 265 and C. sorokiniana UTEX 1230 were cultivated in heterotrophic growth
235
with a glucose supplement (10 g L-1), the doubling time of the microalgal cultures
236
was 0.2-0.6 times shorter than in the photoautotrophic growth (Rosenberg et al.,
237
2014).
238
Fig. 2B shows the biomass productivity profiles of Chlorella sp. GD cultivated
239
with different ratios of wastewater. The biomass productivity was higher in the
240
cultures with wastewater than that in the culture medium alone. It is proposed that
241
livestock wastewater has many nutrient compounds for microalgal growth, such as
242
NH4 +-N, N and P (Chiu et al., 2015). The highest biomass productivity of Chlorella
243
sp. GD in the batch culture from this study was 0.681 g L-1 d-1, which was much
244
higher than that of Chlorella pyrenoidosa (90 mg L-1 d -1) and Chodatella sp. (115 mg
245
L-1 d-1) cultured in mixotrophic growth with piggery wastewater, as previously
246
reported (Li et al., 2014a; Wang et al., 2012). With higher ratios of wastewater,
247
higher biomass productivities and shorter doubling times of Chlorella sp. GD
248
cultivation were achieved. These results indicate that Chlorella sp. GD is well
249
adapted to grow in piggery wastewater for mixotrophic cultivation without additional
250
expensive carbon sources. The cost-effectiveness of microalgal biodiesel production 10
251
could be improved by cultivation with piggery wastewater.
252 253
3.2 Lipid content and productivity of Chlorella sp. GD cultivated with different
254
ratios of wastewater in batch cultivation
255
To investigate the effect of microalgal lipid accumulation and productivity with
256
different ratios of wastewater, Chlorella sp. GD was cultured with 0, 25, 50, 75 and
257
100% wastewater. After 10 days in culture, Chlorella sp. GD was harvested to
258
measure the lipid content and to calculate the lipid productivity (Fig. 3). When
259
Chlorella sp. GD was cultured with wastewater, the lipid content was increased 1.5-
260
to 2-fold compared with that in culture medium alone. Among the conditions,
261
maximum biomass productivity was obtained in the 25% wastewater of microalgal
262
cultivation. A similar result was found that the maximum lipid content (23%) of
263
Chlorella pyrenoidosa, which was cultivated in water-diluted piggery wastewater
264
(Wang et al., 2012). The lipid productivity of Chlorella sp. GD cultured with
265
wastewater was approximately 2-fold higher compared with samples cultured in
266
medium. Moreover, it also showed that the biomass productivity increased along
267
with the wastewater ratio added to the cultures. These results indicate that
268
wastewater usage for Chlorella sp. GD cultivation could enhance microalgal lipid
269
accumulation. The neutral lipids, composed of triacylglycerol (TAG), diacylglycerol
270
(DAG) and monoacylglycerol (MAG), accounted for 78-84% of the total lipids. The
271
polar lipids, composed of phospholipid and glycolipid, represented 16-22% of the
272
total lipids. The results indicate that the lipid produced from Chlorella sp. GD
273
cultured with the piggery wastewater is applicable as the precursor for biodiesel.
274 275
In the microalgal cultivation duration, starch is first synthesized to store energy and then lipids accumulate to overcome possible environment stress in long-term 11
276
cultivation (Siaut et al., 2011). Lipid induction in microalgae occurs under stress
277
conditions, such as nutrient stress (N and/or P starvation), light intensity, pH and
278
temperature (Li et al., 2014b; Ho et al., 2012; Sharma et al., 2012). The nutrient
279
content (such as N and P) is of particular importance as both a key macronutrient and
280
a trigger for lipid accumulation in microalgal cells.
281
In our study, the concentrations of N and P in wastewater were lower than in the
282
culture medium. This indicates that the N and P limitations of wastewater could
283
contribute to the accumulation of lipid content from microalgal cultivation. Moreover,
284
some studies indicated that microalgae were cultured in heterotrophic or mixotrophic
285
growth and that the biomass and lipid production was effectively improve compared
286
to that from autotrophic growth (Wan et al., 2012; Yeh and Chang, 2012).
287 288 289 290
3.3 Fatty acid compositions of Chlorella sp. GD cultivated with different ratios of wastewater in batch cultivation The fatty acid compositions of Chlorella sp. GD cultured with 0, 25, 50, 75 and
291
100% wastewater in batch cultures for 10 days are shown in Fig. 4. Fatty acids
292
C16:0, C18:0, C18:1 and C18:2, which are beneficial for producing biodiesel,
293
abound in Chlorella sp., including the strains kept in our laboratory (Kao et al., 2012).
294
As shown in Fig. 4, the microalgal fatty acids of C16:0, C18:0, C18:1 and C18:2
295
accounted for over 70% of the total fatty acids in the Chlorella sp. GD cultures.
296
Compared with the culture medium, there was a relative decreased of the C16:0 and
297
increase of the C18:1 contents of the 25% diluted wastewater and an increase of the
298
C18:2 of the cultures with 50-100% diluted wastewater. This result indicates that the
299
variation in the fatty acid profile of Chlorella sp. GD was probably associated with
300
certain components in wastewater, which are most likely certain minerals or 12
301
stress-accumulated conditions (e.g., growth-inhibiting ingredients, wastewater pH,
302
toxic organic compounds, etc.). There were many reports performed that discuss the
303
effects of culture broth derived from wastewater on the microalgal lipid content and
304
fatty acids profiles. With a reduction of the COD of the medium by dilution, the fatty
305
acids C18:1 and C16:0 of Chlorella sp. were increased and C16:2 was decreased
306
(Wang et al., 2010). C16:0, C18:2 and C18:3 were the abundant fatty acids of
307
Chlorella pyrenoidosa when grown on diluted piggery wastewater and Bristol’s
308
solution, and the total decrease of C16:2 and C18:1 was equal to an increase of
309
C16:0 (Wang et al., 2012). When Chlorella sp. was cultivated in treated and
310
untreated concentrated municipal wastewater, the relative content of monoenoic fatty
311
acids (C16:1 and C18:1) increased, polyenoic fatty acids (C16:2, C18:2 and C18:3)
312
decreased and saturated fatty acids were not different (Li et al., 2011). The results
313
mentioned above suggest that the variation in the fatty acid profiles of microalgal
314
species grown on different wastewater-containing medium seems to be
315
strain-dependent. In addition, the kinematic viscosity (4.6 mm2 s-1), flash point
316
(140 oC) and cetane number (61) of the biodiesel generated from Chlorella sp. GD
317
microalgae oil are fit with the ASTM D6751 or EN 14214 biodiesel standard. From
318
the fractions of lipids, composition profiles of fatty acids and properties of the
319
generated biodiesel, the lipid from Chlorella sp. GD in piggery wastewater
320
cultivation was suitable for biodiesel production.
321 322 323 324 325
3.4 Growth profiles of Chlorella sp. GD cultivated with different ratios of wastewater in semi-continuous cultivation The growth profiles of Chlorella sp. GD using different ratios of wastewater diluted with medium in semi-continuous cultures are shown in Fig. 5. In cycles 2-5, 13
326
the maximum biomass concentrations of Chlorella sp. GD with 25, 50 and 75%
327
wastewater reached 4 g L-1 or more, which were similar to the results from cycle 1.
328
However, the maximum biomass concentration of Chlorella sp. GD with 100%
329
wastewater decreased gradually after every replacement. This decrease was most
330
likely because some of the wastewater components could be accumulated with every
331
replacement; these components may inhibit microalgal growth, but the components
332
still need to be further investigated. The growth of Chlorella sp. GD was decreased
333
because the pH of the initial culture broth was gradually increased after every 100%
334
wastewater replacement. The average maximum biomass concentration in the culture
335
with 25, 50, 75 and 100% wastewater were 4.73, 4.81, 4.86 and 4.14 g L-1,
336
respectively. The average of biomass productivity in the culture with 25, 50, 75 and
337
100% wastewater was 0.852, 0.870, 0.859 and 0.701 g L-1 d-1, respectively. The
338
biomass productivity of Chlorella sp. GD cultured with diluted wastewater was
339
significantly higher than that with 100% wastewater. We emphasize that the biomass
340
productivity of Chlorella sp. GD in the semi-continuous cultures was approximately
341
2-fold higher than that in the batch cultures (Fig. 2 vs. Fig. 5). It may be that
342
replacing fresh diluted wastewater could maintain Chlorella sp. GD growth in the
343
exponential phase. Another reason could be that when the high biomass
344
concentration of microalgal cultivation was half replaced by fresh nutrients, the
345
culture broth replacement in semi-continuous cultivation decreased the self-shading
346
phenomenon, turbidity and light penetration of the microalgal cultures, resulting in
347
an increase in the photosynthesis efficiency (Li et al., 2011; Wang et al., 2012; Zhu et
348
al., 2013b). Chlorella zofingiensis was incubated with the replacement half fresh
349
piggery wastewater in semi-continuous cultures every 1.5 days after 6 days of batch
350
cultivation to day 15. The stable biomass concentration was maintained at 14
351
approximately 2 g L-1, and the net biomass productivity was 3-fold higher than that
352
in the batch culture (Zhu et al., 2013b). Similar results were shown in our study that
353
the semi-continuous replacement culture strategy could be a practical approach to
354
establish a stable microalgal cultivation process.
355 356 357
3.5 Lipid content and productivity of Chlorella sp. GD cultivated with different ratios of wastewater in semi-continuous cultivation
358
The lipid content and lipid productivity of Chlorella sp. GD, which was
359
harvested on day 17 in semi-continuous cultivation, are illustrated in Table 1. The
360
lipid contents of Chlorella sp. GD cultured with 75 and 100% wastewater were
361
higher than those cultured with 25 and 50% wastewater. This shows that the
362
microalgae that were cultivated with high ratios of wastewater enhanced the lipid
363
contents. Compared with the batch cultures, the averages of the microalgal lipid
364
contents in semi-continuous cultures were lower. The probable reason for this is that
365
the growth rate of Chlorella sp. GD in semi-continuous cultures was faster than that
366
in batch cultures; therefore, the uptake of nutrients by microalgae was used to
367
synthesize essential cell structures, such as proteins and carbohydrates, rather than
368
microalgal lipids. Although the lipid content decreased, increasing biomass
369
productivity was obtained in the semi-continuous Chlorella sp. GD cultures.
370
Therefore, higher lipid productivity could be gained in semi-continuous cultures
371
using diluted piggery wastewater. Higher biomass and lipid productivity were
372
similarly obtained when Chlorella sp. NJ-18 was cultivated outdoors with medium
373
replacement in semi-continuous cultures (Zhou et al., 2013). Because the lipid
374
content of Desmodesmus sp. F2 was decreased along with the medium replacement
375
ratios in semi-continuous cultures, the overall biomass productivity was significantly 15
376
increased. Because of the higher overall lipid productivity and the longer stable
377
operation time, the costs of the semi-continuous cultivation strategy for biodiesel
378
production could be cheaper (Ho et al., 2014).
379
It is frequently noted that many challenges are cost-associated, and cannot be
380
overcome without technical breakthroughs and innovative system integration for the
381
production of microalgae biomass. Using wastewater as a source and combining
382
wastewater treatment with the production of microalgal based bioproducts can
383
overcome several of the identified challenges. In our opinion, the cost reduction of
384
using wastewater for microalgae cultivation may include the use of fresh water and
385
chemicals for culture nutrition. However, there are still several important technical
386
challenges that need to be overcome before the large-scale production of microalgal
387
derived biofuels can become commercially viable, such as wastewater pretreatment.
388 389 390 391
3.6 Fatty acid compositions of Chlorella sp. GD cultivated with different ratios of wastewater in semi-continuous cultivation In addition to increasing the total lipid production of Chlorella sp. GD, it is also
392
important to explore fatty acid compositions for biofuel production. The fatty acid
393
compositions of Chlorella sp. GD cultivated with 25, 50, 75 and 100% wastewater in
394
semi-continuous cultivation at the end of the semi-continuous cultivations (i.e., on
395
day 17) are presented in Table 1. The relative contents of C16:0 (22.8-31.4%) and
396
C18:2 (22.1-31.5%) were higher than other fatty acids in microalgal lipids. These
397
results were similar to those obtained in the batch cultures. The suitable fatty acid
398
compositions for biofuel production in semi-continuous cultivation experienced no
399
obvious changes with the changing diluted wastewater ratio. C16:0 (15.2-19.1%) and
400
C18:2 (14.4-22.4%) were the main fatty acids of Chlorella sp. that were cultivated in 16
401
the highly concentrated municipal wastewater (Li et al., 2011). Because Chlorella
402
vulgaris cultivated with wastewater contained high levels of ammonia, C18:2
403
(28.2-37.0%) was the most abundant and C16:0 (26.2-36.3%) was the second most
404
abundant fatty acid (He et al., 2013). The results suggest that in semi-continuous
405
cultivation with wastewater, Chlorella sp. GD oil is an appropriate feedstock for
406
biodiesel production.
407 408 409 410
4. Conclusions This study shows that the maximum specific growth rate of Chlorella sp. GD is
411
obtained in piggery wastewater without dilution in batch cultivation. In a
412
semi-continuous cultivation, biomass productivity with 25-75% piggery wastewater was
413
significantly higher than that of batch cultivation. With higher biomass productivity, the
414
lipid productivity is increased with the benefit of enhancing the total lipid production.
415
The maximum lipid productivity was 0.176 g L-1 day-1 with 75% piggery wastewater
416
replacement. This study demonstrated a potential approach of stable growth
417
performance for long-term microalgal cultivation in semi-continuous operation
418
cultivation system with piggery wastewater.
419 420 421 422
Acknowledgements The work was financially supported by grants MOST 103-3113-E-006-006 from
423
the Ministry of Science and Technology. This work was also supported in part by the
424
Aim for the Top University Program of the National Chiao Tung University and
425
Ministry of Education, Taiwan. 17
426
Reference
427
1.
294−306.
428 429
Chisti, Y., 2007. Biodiesel from microalgae. Biotechnology Advances. 25,
2.
Chiu, S.Y., Kao, C.Y., Chen, C.H., Kuan, T.C., Ong, S.C., Lin, C.S., 2008.
430
Reduction of CO2 by a high-density culture of Chlorella sp. in a semicontinuous
431
photobioreactor. Bioresour. Technol. 99, 3389−3396.
432
3.
Chiu, S.Y., Kao, C.Y., Tsai, M.T., Ong, S.C., Chen, C.H., Lin, C.S., 2009. Lipid
433
accumulation and CO2 utilization of Nannochloropsis oculata in response to CO2
434
aeration. Bioresour. Technol. 100, 833−838.
435
4.
Chiu, S.Y., Kao, C.Y., Huang, T.T., Lin, C.J., Ong, S.C., Chen, C.D., Chang, S.H.,
436
Lin, C.S., 2011. Microalgal biomass production and on-site bioremediation of
437
carbon dioxide, nitrogen oxide and sulfur dioxide from flue gas using Chlorella sp.
438
cultures. Bioresour. Technol. 102, 9135−9142.
439
5.
Chiu, S.Y., Kao, C.Y., Chen, T.Y., Chang, Y.B., Kuo, C.M., Lin, C.S., 2015.
440
Cultivation of microalgal Chlorella for biomass and lipid production using
441
wastewater as nutrient resource. Bioresour. Technol. 184, 179−189.
442
6.
Ebrahimian, A., Kariminia, H.R., Vosoughi, M., 2014. Lipid production in
443
mixotrophic cultivation of Chlorella vulgaris in a mixture of primary and
444
secondary municipal wastewater. Renewable Energy. 71, 502−508.
445
7.
Farooq, W., Lee, Y.C., Ryu, B.G., Kim, B.H., Kim, H.S., Choi, Y.E., Yang, J.W.,
446
2013. Two-stage cultivation of two Chlorella sp. strains by simultaneous treatment
447
of brewery wastewater and maximizing lipid productivity. Bioresour.Technol. 132,
448
230–238.
449 450
8.
Hach Company, 2009. Dr/890 Colorimeter Procedures Manual, ninth ed. Hach Company, Loveland, CO, USA. 18
451
9.
He, P.J., Mao, B., Shen, C.M., Shao, L.M., Lee, D.J., Chang, J.S., 2013.
452
Cultivation of Chlorella vulgaris on wastewater containing high levels of ammonia
453
for biodiesel production. Bioresour. Technol. 129, 177−181.
454
10. Ho, S.H., Chen, C.Y., Chang, J.S., 2012. Effect of light intensity and nitrogen
455
starvation on CO2 fixation and lipid/carbohydrate production of an indigenous
456
microalga Scenedesmus obliquus CNW-N. Bioresour. Technol. 113, 244−252.
457
11. Ho, S.H., Li, P.J., Liu, C.C., Chang, J.S., 2013. Bioprocess development on
458
microalgae-based CO2 fixation and bioethanol production using Scenedesmus
459
obliquus CNW-N. Bioresour. Technol. 145, 142−149.
460
12. Ho, S.H., Chen, C.N., Lai, Y.Y., Lu, W.B., Chang, J.S., 2014. Exploring the high
461
lipid production potential of a thermotolerant microalga using statistical
462
optimization and semi-continuous cultivation. Bioresour. Technol. 163, 128−135.
463
13. Kao, C.Y., Chiu, S.Y., Huang, T.T., Dai, L., Hsu, L.K., Lin, C.S., 2012. Ability of a
464
mutant strain of the microalga Chlorella sp. to capture carbon dioxide for biogas
465
upgrading. Appl. Energy 93, 176−183.
466
14. Kao, C.Y., Chen, T.Y., Chang, Y.B., Chiu, T.W., Lin, H.Y., Chen, C.D., Chang, J.S.,
467
Lin, C.S., 2014. Utilization of carbon dioxide in industrial flue gases for the
468
cultivation of microalga Chlorella sp. Bioresour. Technol. 166, 485−493.
469
15. Klofutar, B., Golob, J., Likozar, B., Klofutar, C., Žagar, E., Poljanšek, I., 2010. The
470
transesterification of rapeseed and waste sunflower oils: Mass-transfer and kinetics
471
in a laboratory batch reactor and in an industrial-scale reactor/separator setup.
472
Bioresource Technology 101, 3333-3344.
473
16. Li, X., Xu, H., Wu, Q., 2007. Large-scale biodiesel production from microalga
474
Chlorella protothecoides through heterotrophic cultivation in bioreactors.
475
Biotechnol. Bioeng. 98, 764−771. 19
476
17. Li, Y., Chen, Y.F., Chen, P., Min, M., Zhou, W., Martinez, B., Zhu, J., Ruan, R.,
477
2011. Characterization of a microalga Chlorella sp. well adapted to highly
478
concentrated municipal wastewater for nutrient removal and biodiesel production.
479
Bioresour. Technol. 102, 5138−5144.
480
18. Li, Y.R., Tsai, W.T., Hsu, Y.C., Xie, M.Z., Chen, J.J., 2014a. Comparison of
481
autotrophic and mixotrophic cultivation of green microalgal for biodiesel
482
production. Energy Procedia. 52, 371−376.
483
19. Li, Y., Han, F., Xu, H., Mu, J., Chen, D., Feng, B., Zeng, H., 2014b. Potential lipid
484
accumulation and growth characteristic of the green alga Chlorella with
485
combination cultivation mode of nitrogen (N) and phosphorus (P). Bioresour.
486
Technol. 174, 24−32.
487
20. Likozar, B., Levec, J., 2014. Transesterification of canola, palm, peanut, soybean
488
and sunflower oil with methanol, ethanol, isopropanol, butanol and tert-butanol to
489
biodiesel: Modelling of chemical equilibrium, reaction kinetics and mass transfer
490
based on fatty acid composition. Applied Energy. 123, 108−120.
491
21. Mata, T.M., Martins, A.A., Caetano, N.S., 2010. Microalgae for biodiesel
492
production and other applications: a review. Renew. Sustain. Energy Rev. 14,
493
217−232.
494
22. REN21, 2014. Renewables 2014 Global Status Report. pp. 34–38.
495
23. Rosenberg, J.N., Kobayashi, N., Barnes, A., Noel, E.A., Betenbaugh, M.J., Oyler,
496
G.A., 2014. Comparative analyses of three Chlorella species in response to light
497
and sugar reveal distinctive lipid accumulation patterns in the microalga C.
498
sorokiniana. PLoS One 9: e92460.
499 500
24. Sharma, K.K., Schuhmann, H., Schenk, P.M., 2012. High lipid induction in microalgae for biodiesel production. Energies 5, 1532−1553. 20
501
25. Siaut, M., Cuiné S., Cagnon, C., Fessler, B., Nguyen, M., Carrier, P., Beyly, A.,
502
Beisson, F., Triantaphylidès, C., Li-Beisson, Y., Peltier, G., 2011. Oil accumulation
503
in the model green alga Chlamydomonas reinhardtii : characterization, variability
504
between common laboratory strains and relationship with starch reserves. BMC
505
Biotechnology 11, 1−15.
506
26. Šoštarič, M., Klinar, D., Bricelj, M., Golob, J., Berovič, M., Likozar, B., 2012.
507
Growth, lipid extraction and thermal degradation of the microalga Chlorella
508
vulgaris. New Biotechnology 29, 325–331.
509
27. Wan, M.X., Wang, R.M., Xia, J.L., Rosenberg, J.N., Nie, Z.Y., Kobayashi, N.,
510
Oyler, G.A., Betenbaugh, M.J., 2012. Physiological evaluation of a new Chlorella
511
sorokiniana isolate for its biomass production and lipid accumulation in
512
photoautotrophic and heterotrophic cultures. Biotechnol. Bioeng. 109, 1958−1964.
513
28. Wang, H., Xiong, H., Hui, Z., Zeng, X., 2012. Mixotrophic cultivation of Chlorella
514
pyrenoidosa with diluted primary piggery wastewater to produce lipids. Bioresour.
515
Technol. 104, 215−220.
516
29. Wang, L., Li, Y., Chen, P., Min, M., Chen, Y., Zhu, J., Ruan, R.R., 2010. Anaerobic
517
digested dairy manure as a nutrient supplement for cultivation of oil-rich green
518
microalgae Chlorella sp. Bioresour. Technol. 101, 2623−2628.
519
30. Yeh, K.L., Chang, J.S., 2012. Effects of cultivation conditions and media
520
composition on cell growth and lipid productivity of indigenous microalga
521
Chlorella vulgaris. Bioresour. Technol. 105, 120−127.
522
31. Yen, H.W., Hu, I.C., Chen, C.Y., Ho, S.H., Lee, D.J., Chang, J.S., 2013.
523
Microalgae-based biorefinery – from biofuels to natural products. Bioresour.
524
Technol. 135, 166−174.
525
32. Zhou, X., Ge, H., Xia, L., Zhang, D., Hu, C., 2013. Evaluation of oil-producing 21
526 527
algae as potential biodiesel feedstock. Bioresour. Technol. 134, 24−29. 33. Zhu, L., Wang, Z., Shu, Q., Takala, J., Hiltunen, E., Feng, P., Yuan, Z., 2013a.
528
Nutrient removal and biodiesel production by integration of freshwater algae
529
cultivation with piggery wastewater treatment. Water Res. 47, 4294−4302.
530
34. Zhu, L., Wang, Z., Takala, J., Hiltunen, E., Qin, L., Xu, Z., Qin, X., Yuan, Z.,
531
2013b. Scale- up potential of cultivating Chlorella zofingiensis in piggery
532
wastewater for biodiesel production. Bioresour. Technol. 137, 318−325.
22
533
Figures captions
534
Fig. 1. Growth profiles of Chlorella sp. GD cultured with different sources of
535
wastewater (piggery, industrial and municipal wastewater) without dilution and medium
536
in batch cultures. Piggery, industrial and municipal wastewaters were from a swine farm
537
(Miaoli, Taiwan), Hsinchu industrial park (Hukou, Taiwan) and National Chiao Tung
538
University (Hsinchu, Taiwan), respectively. The initial biomass concentration was
539
approximately 0.3 g L-1. The cultures were illuminated at 300 µmol m-2 s-1 and with a
540
2% CO2 aeration rate of 0.2 vvm at 26 ± 1°C. The batch culture was operated for 10
541
days, and the microalgal cells were sampled every 24 h for growth determinations.
542 543
Fig. 2. Growth (A) and biomass productivity (B) profiles of Chlorella sp. GD cultured
544
with different ratios of piggery wastewater (0, 25, 50, 75 and 100%) diluted by culture
545
medium in batch cultures. The initial biomass concentration was approximately 0.3 g
546
L-1. The cultures were illuminated at 300 µmol m-2 s-1 and with a 2% CO2 aeration rate
547
of 0.2 vvm at 26 ± 1°C. The batch culture was operated for 10 days, and the microalgal
548
cells were sampled every 24 h for growth determinations. The biomass productivity was
549
calculated from biomass concentration.
550 551
Fig. 3. Lipid content, lipid productivity and biomass productivity of Chlorella sp. GD
552
using different ratios (0, 25, 50, 75 and 100%) of piggery wastewater diluted with
553
culture medium in a 10-day batch culture. The initial biomass concentration was
554
approximately 0.3 g L-1. The cultures were illuminated at 300 µmol m-2 s-1 and with a
555
2% CO2 aeration rate of 0.2 vvm at 26 ± 1°C.
23
556
Fig. 4. The main fatty acid profiles of Chlorella sp. GD cultured with different ratios of
557
piggery wastewater (0, 25, 50, 75 and 100%) diluted by culture medium in 10-day batch
558
cultures.
559 560
Fig. 5. Growth profiles of Chlorella sp. GD using the 100% (A), 75% (B), 50% (C) and
561
25% (D) piggery wastewater diluted with culture medium in semi-continuous cultures.
562
The initial biomass concentration was approximately 0.3 g L-1. The cultures were
563
illuminated at 300 µmol m-2 s-1 and with a 2% CO2 aeration rate of 0.2 vvm at 26 ± 1°C.
564
The microalgal cultures were started as a batch culture for 5 days (cycle 1 of
565
semi-continuous culture). Half of the culture broth was replaced with the same amount
566
of fresh wastewater every 3 days for a period of 12 days (i.e., cycles 2-5 of
567
semi-continuous culture). The microalgal cells were sampled every day for growth
568
determinations.
569
24
570
Table 1. Lipid content, lipid productivity and main fatty acid compositions of Chlorella
571
sp. GD using the different ratios of wastewater diluted with culture medium in
572
semi-continuous cultures. Wastewater ratio
Lipid content (%) a Lipid productivity (g L-1 day-1) b
25%
50%
75%
100%
15 ± 0
15 ± 1
21 ± 1
21 ± 2
0.123 ± 0.001 0.130 ± 0.010 0.173 ± 0.001 0.140 ± 0.028
Fatty acid composition Saturated fatty acids
Monoenoic fatty acids
Polyenoic fatty acids
Others 573
a
C16:0
30 ± 8
31 ± 5
23 ± 3
28 ± 5
C18:0
7±1
3±0
5±1
4±0
subtotal
36 ± 9
35 ± 5
28 ± 4
32 ± 5
C16:1
2±0
2±0
3±0
2±0
C18:1
12 ± 0
10 ± 1
6±0
13 ± 1
subtotal
15 ± 1
12 ± 1
9±0
16 ± 2
C16:2
6±0
11 ± 1
16 ± 2
10 ± 2
C18:2
22 ± 3
28 ± 4
32 ± 6
29 ± 7
C18:3
16 ± 1
13 ± 0
14 ± 0
13 ± 1
subtotal
44 ± 4
52 ± 6
62 ± 9
52 ± 10
5±2
1±1
2±0
1±0
Lipid content (%) = (lipid dry weight / biomass dry weight) × 100%. Each data indicates the mean ± SD from three experiments.
574 575
Relative content (%)
b
Lipid productivity (g L-1 day-1) = (biomass productivity × lipid content) / 100.
576 577
25
Figure 1
-1
Biomass concentration (g L )
10
1
Piggery wastewater Industrial wastewater Municipal wastewater Medium
0.1
0
1
2
3
4 5 6 7 8 Days of cultivation
9
10
Figure 2
A. -1
Biomass concentration (g L )
10
1 Medium 25% WW 50% WW 75% WW 100% WW 0.1
0
1
2
3
B.
4
5
6
7
Days of cultivation
9
10
Medium 25% WW 50% WW 75% WW 100% WW
-1
-1
Biomass productivity (g L d )
1.6
8
1.2
0.8
0.4
0.0
0
1
2
3
4
5
6
7
Days of cultivation
8
9
10
Figure 3
0.8
40 Biomass productivity Lipid productivity Lipid content
30
0.6 0.5
20
0.4 0.2
10
0.1
0.0
0
25
50
75
Wastewater ratio (%)
100
0
Lipid content (%)
-1
-1
Productivity (g L d )
0.7
Fatty acid compositions
40
Other
C18:3
C18:2
C18:1
C18:0
C16:2
C16:1
C16:0
Relative content of fatty acid (%)
Figure 4
50
Medium 25% WW 50% WW 75% WW 100% WW
30
20
10
0
Figure 5
A. 100% WW 6 4 2
Biomass concentration (g L-1)
0
0
2
4
6
8
10
12
14
16
18
14
16
18
14
16
18
14
16
18
B. 75% WW and 25% medium 6 4 2 0
0
2
4
6
8
10
12
C. 50% WW and 50% medium 6 4 2 0
0
2
4
6
8
10
12
D. 25% WW and 75% medium 6 4 2 0
0
2
4
6
8
10
12
Days of cultivation
578
Highlights
579
Piggery wastewater was able to be directly used for Chlorella sp. GD cultivation.
580
The maximum specific growth rate of Chlorella sp. GD with 100% piggery wastewater was 0.839 d-1.
581 582
semi-continuous culture.
583 584 585
The biomass and lipid productivity of Chlorella sp. GD was enhanced in a
Stable growth performance for long-term microalgal cultivation in semi-continuous operations is promising.
586 587
26
Graphical abstract
S0960-8524(15)00979-7 http://dx.doi.org/10.1016/j.biortech.2015.07.026 BITE 15259
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
13 May 2015 6 July 2015 7 July 2015
Please cite this article as: Kuo, C-M., Chen, T-Y., Lin, T-H., Kao, C-Y., Lai, J-T., Chang, J-S., Lin, C-S., Cultivation of Chlorella sp. GD using piggery wastewater for biomass and lipid production, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.07.026
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Cultivation of Chlorella sp. GD using piggery wastewater for biomass and lipid production Chiu-Mei Kuoa, Tsai-Yu Chena, Tsung-Hsien Lina, Chien-Ya Kao a,b, Jinn-Tsyy Laic, Jo-Shu Changd, e, Chih-Sheng Lina,* a
Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan
b
Aquatic Technology Laboratories, Agricultural Technology Research Institute, Hsinchu, Taiwan
c
Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan
d
Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan
e
Research Center for Energy Technology and Strategy, National Cheng Kung University, Tainan, Taiwan
* Author for correspondence: Chih-Sheng Lin, Ph.D. Department of Biological Science and Technology, National Chiao Tung University, No.75 Bo-Ai Street, Hsinchu 30068, Taiwan Tel: 886-3-5131338 E-mail: [email protected]
1 2
Abstract The development of a culture system for Chlorella sp. GD to efficiently produce
3
biomass and oil for biodiesel production was investigated. Chlorella sp. GD was
4
cultivated with 0, 25, 50, 75 and 100% piggery wastewater (diluted by medium) at 300
5
µmol m-2 s-1, a 2% CO2 aeration rate of 0.2 vvm and 26 ± 1°C; after a 10-day culture in
6
batch cultures, the maximum specific growth rate and biomass productivity of the
7
microalga obtained in 100% piggery wastewater were 0.839 d-1 and 0.681 g L-1 d -1,
8
respectively. The highest lipid content and lipid productivity were 29.3% and 0.155 g
9
L-1 d-1 at 25% wastewater, respectively. In semi-continuous cultures, the biomass and
10
lipid productivities with 25-75% wastewater ratios were greater than 0.852 and 0.128 g
11
L-1 d-1, respectively. These results show that Chlorella sp. GD grows efficiently in
12
piggery wastewater, and that a stable growth performance was achieved for long-term
13
microalgal cultivation in a semi-continuous culture.
14
Keywords: microalgae, wastewater, semi-continuous culture, biomass, lipid
15 16 17 18
1. Introduction As the demand for global energy continues to increase, fossil fuel usage will also
19
continue to increase. Over the long term, fossil fuels are not a sustainable energy
20
resource, and the burning of fossil fuels increases greenhouse gas (GHG) emissions.
21
Therefore, alternative energy sources are being explored to replace fossil fuels. In recent
22
years, microalgae have been considered as a third generation biofuel feedstock because
23
they not only utilize carbon dioxide (CO2) as a carbon source during autotrophic growth
24
to decrease GHGs, but their resulting biomass also contains abundant energy-rich
25
components that can be converted to various biofuels, such as ethanol, butanol, methane 1
26
and biodiesel (Ho et al., 2013). In addition, microalgal biomass is a potential feedstock
27
for biorefineries that can be converted into high value products containing
28
polyunsaturated fatty acids (e.g., DHA and EPA), chlorophylls, carotenoids, phycobilins
29
and others (Yen et al., 2013).
30
Biodiesel is the second largest category of global biofuel, accounting for 6.9 billion
31
gallons globally in 2013, which was 22.6% of the total biofuel production (REN21,
32
2014). Biodiesel is produced via the transesterification process by reacting fats,
33
including vegetable oil, animal fats or waste cooking oils, with an alcohol, such as
34
methanol (Klofutar et al., 2010). Homogeneous alkali-catalyzed transesterification is the
35
most commonly used method, and leads to high conversion levels of triglycerides to
36
their corresponding methyl esters with short reaction times. On the industrial scale,
37
alkalis, such as NaOH and KOH are commonly used as catalysts (Likozar and Levec,
38
2014; Šoštarič et al., 2012). However, with the increasing demand for these oilseed
39
crops there is now an issue regarding food-fuel competition; therefore, an alternative
40
feedstock needs to be developed. Biodiesel derived from microalgae has been
41
considered a promising approach because microalgae have much higher biomass
42
productivity in comparison to terrestrial plants and can be cultivated on non-arable land
43
without competing with other crops (Chisti, 2007; Mata et al., 2010). The main inputs
44
required in addition to microalgae are sunlight, water, CO2 and nutrients, e.g., carbon,
45
nitrogen (N) and phosphorus (P) sources. However, the main drawback for economical
46
biodiesel production from microalgae is the high cost of microalgal cultivation.
47
Approximately 80% of the total medium costs went to microalgae cultivation because
48
of the huge consumption of nutrients and water (Li et al., 2007). When Chlorella
49
zofingiensis was cultivated in diluted piggery wastewater with 1,900 mg L-1 COD
50
culture, the highest biomass productivity was 296 mg L-1 day-1, the total nitrogen (TN) 2
51
and total phosphorus (TP) removal was 83% and almost 100%, respectively (Zhu et al.,
52
2013a). The biomass production of Chlorella sp. growing in the centrate, a highly
53
concentrated municipal wastewater, was 0.212 g L-1 day-1, the TN removal was
54
approximately 90% (Li et al., 2011). Under photoautotrophic cultivation with brewery
55
wastewater for Chlorella vulgaris, the biomass production was 0.149 g L-1 day-1, both
56
TN and TP removal were more than 80% (Farooq et al., 2013). However, microalgal
57
growth is sometimes restricted by the presence of bacteria and protozoa in wastewater.
58
On the laboratory scale, wastewater is often pretreated by autoclave sterilization to
59
reduce the effect of bacteria and protozoa on microalgal growth. However, autoclave
60
sterilization pretreatment would increase the cost and difficulty of large-scale
61
microalgal cultivation. To guarantee stable microalgal growth using wastewater against
62
possible microbial contamination, it is necessary to develop control techniques for
63
biotic pollution (Chiu et al., 2015).
64
Although many studies have utilized wastewater for biodiesel production in
65
microalgal cultivation, few studies have established a continuous stable-growth
66
microalgal cultivation system combined with wastewater replacement. Semi-continuous
67
microalgal cultivation is an effective strategy to maintain the growth of the exponential
68
phase and enhance the light penetration by culture broth replacement (Chiu et al., 2008).
69
The aim of this study is to provide an efficient and stable culturing system for
70
microalgal biomass production from biofuel feedstock by using swine wastewater from
71
livestock farms. Lipids extracted from Chlorella sp. GD can be transesterified into fatty
72
acid methyl ester (FAME) to produce biodiesel. Moreover, the non-lipid fraction of
73
Chlorella sp. GD biomass, which mainly consists of protein (approximately 60% of
74
microalgal dry weight), can also be processed to methane and feeds applied in
75
aquaculture, poultry and swine. The integrated microalgal culture system with livestock 3
76
wastewater may also reduce the cost of biodiesel production and provide an
77
environmental benefit of microalgal bioremediation.
78 79 80 81
2. Methods 2.1 Microalgal cultures, medium and chemicals
82
The freshwater microalgae Chlorella sp. GD was screened for its potential
83
ability for growth and biomass production according to our previous report (Chiu et
84
al., 2009). The Chlorella sp. GD cells were grown on a modified freshwater medium
85
containing 1.25 g L-1 KNO3, 1.25 g L-1 KH2PO4, 1 g L-1 MgSO4．7H2O, 0.5 g L-1
86
EDTA．2Na, 83.5 mg L-1 CaCl2．2H2O, 0.1142 g L-1 H3BO3, 49.8 mg L-1 FeSO4．
87
7H2O, 88.2 mg L-1 ZnSO4．7H2O, 14.4 mg L-1 MnCl2．4H2O, 10 mg L-1 CuSO4, 7.1
88
mg L-1 Na2MoO4 and 4 mg L-1 CoCl2．6H2O. The initial pH of the initial medium
89
was adjusted to 6 with NaOH.
90 91
2.2 Source of wastewater
92
The experiments were conducted using piggery wastewater and industrial and
93
municipal wastewater from a swine farm (Miaoli, Taiwan), Hsinchu industrial park
94
(Hukou, Taiwan) and National Chiao Tung University (Hsinchu, Taiwan),
95
respectively. The wastewater was treated by a three-step waste treatment system
96
consisting of solid-liquid separation, anaerobic treatment and aerobic treatment. The
97
samples were periodically collected from the treatment system between April 2013
98
and August 2013. The wastewater consists of 490 ± 60 mg L-1 ammonia–N
99
(NH4+–N), 10 ± 5 mg L-1 nitrite–N (NO2- –N), 2 ± 1 mg L-1 nitrate–N (NO3- –N), 550
100
± 70 mg L-1 total nitrogen (TN), 20 ± 6 mg L-1 total phosphorus (TP), 80 ± 20 mg L-1 4
101
suspended solids (SS) and 430 ± 60 mg L-1 chemical oxygen demand (COD). The pH
102
of the wastewater was 8.5 ± 0.5.
103 104 105
2.3 Pretreatment of wastewater To avoid other interference in the wastewater, as a pretreatment, the wastewater
106
was autoclaved for 30 min at 121°C. Then, the liquid was centrifuged (3,000×g for
107
10 min) to separate the large non-soluble particles from the liquid. The supernatant
108
was stored in a cold room at 4°C and used for further experiments.
109 110 111
2.4 Experimental system of indoor photobioreactors The microalgae cells were cultured in photobioreactors with a working volume
112
of 1 L (Chiu et al., 2008). The photobioreactors were placed in an incubator at 26 ±
113
1°C with a surface light intensity of approximately 300 µmol m-2 s-1 provided by
114
continuous cool-white fluorescent lights. The photobioreactor was a cylindrical glass
115
column with a diameter and length of 6 and 80 cm, respectively. Gas was provided as
116
2% CO2 mixed with ambient air. The microalgal cultures were aerated continuously
117
with gas that was provided via bubbling from the bottom of the photobioreactor with
118
an aeration rate of 200 mL min-1 (i.e., 0.2 vvm, volume of gas per volume of broth
119
per min).
120 121
2.5 Preparation of inoculum
122
A stock culture of Chlorella sp. GD (approximately 0.3 g L-1) was incubated in
123
a column containing 1 L of working volume of the freshwater microalgal medium at
124
26 ± 1°C and 300 µmol m-2 s-1. After 3 to 5 days of culture, the biomass
125
concentration was greater than 1.5 g L-1, and the biomass was diluted depending on 5
126
the desired biomass concentration for further experiments.
127 128 129
2.6 Experimental design of batch cultivation The photobioreactor was inoculated with 200 mL of pre-cultured Chlorella sp.
130
GD broth and was filled with 800 mL of wastewater at different dilution ratios of
131
medium to achieve approximately 0.3 g L-1 of the initial inoculum. Chlorella sp. GD
132
was cultured with 0, 25, 50, 75 and 100% piggery wastewater for 10 days. The
133
microalgal culture with pure medium (ratio of adding wastewater was 0%) was the
134
control group to compare the difference of growth between the microalgal culture
135
grown with medium and wastewater. Microalgal cultivation was illuminated at 300
136
µmol m-2 s-1, a 2% CO2 aeration rate of 0.2 vvm and 26 ± 1°C. The microalgal cells
137
in each culture were sampled at 24 h intervals to determine the optical density, lipid
138
content and fatty acid composition.
139 140
2.7 Experimental design of semi-continuous cultivation
141
The semi-continuous culture was carried out in two stages. First, the initial
142
biomass concentration of batch cultures was 0.3 g L-1. The biomass concentration of
143
Chlorella sp. GD at the stationary phase reached approximately 4 g L-1, which
144
occurred after 5 days of incubation (cycle 1 of semi-continuous culture).
145
Subsequently, half of the volume of the culture broth was replaced with 25, 50, 75
146
and 100% fresh wastewater every 3 days for a period of 12 days (i.e., cycles 2-5 of
147
semi-continuous culture). The 25, 50 and 75% piggery wastewater cultures were a
148
mix of wastewater and medium. The cultures were illuminated at 300 µmol m-2 s-1, a
149
2% CO2 aeration rate of 0.2 vvm and 26 ± 1°C. Every 24 h, each culture was
150
sampled to determine the optical density, lipid content and fatty acid composition. 6
151 152 153
2.8 Determination of microalgal cell biomass and growth rate The biomass concentration (dry weight per liter) of the microalgal cultures was
154
determined according to a previously reported method (Chiu et al., 2009). A
155
calibration equation considering the optical density and the dry weight of microalgal
156
cells was established, as follows:
157
Biomass concentration (g L-1) = 0.3101× × A682nm – 0.0065, R² = 0.9981
158
Hence, the biomass concentration could be precisely calculated (R2 = 0.9981; p
159
< 0.001) using the measured optical density (A682) in an Ultrospec 3300 pro
160
UV/Visible spectrophotometer (Amersham Biosciences, Cambridge, UK). Each
161
sample was diluted to give an absorbance in the range of 0.1 to 1.0 if the optical
162
density was greater than 1.0.
163
The optical density was used to evaluate the biomass concentration (g L-1) of
164
Chlorella sp. GD in each experiment. The biomass productivity (g L-1 d-1) was
165
measured according to the following equation:
166
Biomass productivity (g L-1 d-1) = (Wf – Wi) / ∆t
167
where Wf and Wi are the final and initial biomass concentrations, respectively,
168
and ∆t is the cultivation time in days.
169
The specific growth rate (µ, d-1) was calculated as follows:
170
µ (d-1) = (lnX2 – lnX1) / (t2 – t1)
171
where X2 and X1 are the biomass concentration (g L-1) on t2 and t1 (days),
172
respectively. When X2 was equal to 2X1 in the exponential phase of cultivation, the
173
period of time required was the doubling time (td, h), which was calculated as
174
follows:
175
td (h) = 24 × (ln2) / µ 7
176 177 178
2.9 Lipid extraction Lipid extraction was performed according to the modification of a method that
179
was previously reported (Kao et al., 2014). The microalgae cells were centrifuged,
180
washed twice with deionized water and lyophilized to obtain the dry biomass. The
181
dried sample (200 mg) was mixed with a chloroform/methanol solution (2/1, v/v) and
182
was sonicated for 1 h. The mixture with the chloroform/methanol solution was
183
precipitated and chloroform and a 0.9% NaCl solution was added to give a 2:1:1 ratio
184
of chloroform, methanol and water. The mixture was centrifuged, and the chloroform
185
phase was recovered. Finally, the lipids were weighed after chloroform was removed
186
under a vacuum by a rotary evaporator.
187 188 189
2.10 Microalgal lipid transesterification and fatty acid profile assay The method used for the transesterification of the microalgal lipids and the fatty
190
acid profile assay was based on previously reported procedures (Chiu et al., 2011).
191
The extracted oil samples were placed in a glass test tube and mixed with 2.0 mL of
192
chloroform, 1.7 mL of methanol and 0.3 mL of sulfuric acid. The samples were
193
sonicated for 60 min. After the reaction completed, the tubes were removed from the
194
water bath and were allowed to cool to room temperature. Then, 2 mL of distilled
195
water was added to the tube and thoroughly mixed using a vortex. The samples were
196
allowed to separate, forming a biphasic solution. The organic layer containing fatty
197
acid methyl ester (FAME) was collected and transferred to a pre-weighed glass vial.
198
The solvent was then evaporated using N2 and was heated at 70°C for 40 min.
199
Finally the mass of FAME was determined via weighing.
200
The fatty acid composition was determined using a FOCUS Gas Chromatograph 8
201
(Thermo Fisher Scientific, Waltham, MA, USA) according to the method described
202
by Kao et al. (2014).
203 204
2.11 Nutrient analysis in wastewater
205
A volume of 50 mL of microalgal broth was collected every day from each
206
photobioreactor for nutrient removal analysis starting from inoculation. The samples
207
were first centrifuged at 3,000×g for 10 min. Then, the supernatant was analyzed for
208
COD, NH4+–N, NO2- –N, NO3- –N, TN and TP following the Hach DR/890
209
Colorimeter Procedures Manual (Hach, 2009).
210 211 212 213 214 215
3. Results and discussion 3.1 Growth profiles of Chlorella sp. GD cultivated with different ratios of wastewater in batch cultivation There are three main sources of wastewater that contain a variety of ingredients,
216
i.e., municipal, agricultural and industrial wastewaters. When Chlorella sp. GD was
217
cultivated in different sources of wastewater (piggery, industrial and municipal
218
wastewater) without dilution and medium in batch cultivation, the maximum biomass
219
was obtained in piggery wastewater (Fig. 1).
220
In the present study, the effects of microalgal growth with different ratios of
221
piggery wastewater were studied. The microalgae Chlorella sp. GD were cultured
222
with 0 (i.e., cultured in medium), 25, 50, 75 and 100% wastewater diluted with
223
culture medium for 10 days in batch cultivation. Fig. 2A shows all of the growth
224
potentials of Chlorella sp. GD cultured with wastewater were significantly higher
225
than those obtained when the microalga were cultured in medium alone. When 9
226
Chlorella sp. GD was cultured with 0, 25, 50, 75 and 100% wastewater in the
227
exponential phase of the batch culture, the specific growth rates (µ) were 0.467,
228
0.733, 0.766, 0.797 and 0.839 d-1, respectively, and the doubling times (td) were 35.6,
229
22.7, 21.7, 20.9 and 19.8 h, respectively. Higher ratios of wastewater correlated with
230
shorter doubling times and higher maximum biomass concentrations. It was reported
231
that the maximum specific growth rate of Chlorella vulgaris that was cultivated with
232
100% secondary municipal wastewater was 0.52 d -1 (Ebrahimian et al., 2014), which
233
is consistent with our results. When Chlorella protothecoides UTEX 411, C. vulgaris
234
UTEX 265 and C. sorokiniana UTEX 1230 were cultivated in heterotrophic growth
235
with a glucose supplement (10 g L-1), the doubling time of the microalgal cultures
236
was 0.2-0.6 times shorter than in the photoautotrophic growth (Rosenberg et al.,
237
2014).
238
Fig. 2B shows the biomass productivity profiles of Chlorella sp. GD cultivated
239
with different ratios of wastewater. The biomass productivity was higher in the
240
cultures with wastewater than that in the culture medium alone. It is proposed that
241
livestock wastewater has many nutrient compounds for microalgal growth, such as
242
NH4 +-N, N and P (Chiu et al., 2015). The highest biomass productivity of Chlorella
243
sp. GD in the batch culture from this study was 0.681 g L-1 d-1, which was much
244
higher than that of Chlorella pyrenoidosa (90 mg L-1 d -1) and Chodatella sp. (115 mg
245
L-1 d-1) cultured in mixotrophic growth with piggery wastewater, as previously
246
reported (Li et al., 2014a; Wang et al., 2012). With higher ratios of wastewater,
247
higher biomass productivities and shorter doubling times of Chlorella sp. GD
248
cultivation were achieved. These results indicate that Chlorella sp. GD is well
249
adapted to grow in piggery wastewater for mixotrophic cultivation without additional
250
expensive carbon sources. The cost-effectiveness of microalgal biodiesel production 10
251
could be improved by cultivation with piggery wastewater.
252 253
3.2 Lipid content and productivity of Chlorella sp. GD cultivated with different
254
ratios of wastewater in batch cultivation
255
To investigate the effect of microalgal lipid accumulation and productivity with
256
different ratios of wastewater, Chlorella sp. GD was cultured with 0, 25, 50, 75 and
257
100% wastewater. After 10 days in culture, Chlorella sp. GD was harvested to
258
measure the lipid content and to calculate the lipid productivity (Fig. 3). When
259
Chlorella sp. GD was cultured with wastewater, the lipid content was increased 1.5-
260
to 2-fold compared with that in culture medium alone. Among the conditions,
261
maximum biomass productivity was obtained in the 25% wastewater of microalgal
262
cultivation. A similar result was found that the maximum lipid content (23%) of
263
Chlorella pyrenoidosa, which was cultivated in water-diluted piggery wastewater
264
(Wang et al., 2012). The lipid productivity of Chlorella sp. GD cultured with
265
wastewater was approximately 2-fold higher compared with samples cultured in
266
medium. Moreover, it also showed that the biomass productivity increased along
267
with the wastewater ratio added to the cultures. These results indicate that
268
wastewater usage for Chlorella sp. GD cultivation could enhance microalgal lipid
269
accumulation. The neutral lipids, composed of triacylglycerol (TAG), diacylglycerol
270
(DAG) and monoacylglycerol (MAG), accounted for 78-84% of the total lipids. The
271
polar lipids, composed of phospholipid and glycolipid, represented 16-22% of the
272
total lipids. The results indicate that the lipid produced from Chlorella sp. GD
273
cultured with the piggery wastewater is applicable as the precursor for biodiesel.
274 275
In the microalgal cultivation duration, starch is first synthesized to store energy and then lipids accumulate to overcome possible environment stress in long-term 11
276
cultivation (Siaut et al., 2011). Lipid induction in microalgae occurs under stress
277
conditions, such as nutrient stress (N and/or P starvation), light intensity, pH and
278
temperature (Li et al., 2014b; Ho et al., 2012; Sharma et al., 2012). The nutrient
279
content (such as N and P) is of particular importance as both a key macronutrient and
280
a trigger for lipid accumulation in microalgal cells.
281
In our study, the concentrations of N and P in wastewater were lower than in the
282
culture medium. This indicates that the N and P limitations of wastewater could
283
contribute to the accumulation of lipid content from microalgal cultivation. Moreover,
284
some studies indicated that microalgae were cultured in heterotrophic or mixotrophic
285
growth and that the biomass and lipid production was effectively improve compared
286
to that from autotrophic growth (Wan et al., 2012; Yeh and Chang, 2012).
287 288 289 290
3.3 Fatty acid compositions of Chlorella sp. GD cultivated with different ratios of wastewater in batch cultivation The fatty acid compositions of Chlorella sp. GD cultured with 0, 25, 50, 75 and
291
100% wastewater in batch cultures for 10 days are shown in Fig. 4. Fatty acids
292
C16:0, C18:0, C18:1 and C18:2, which are beneficial for producing biodiesel,
293
abound in Chlorella sp., including the strains kept in our laboratory (Kao et al., 2012).
294
As shown in Fig. 4, the microalgal fatty acids of C16:0, C18:0, C18:1 and C18:2
295
accounted for over 70% of the total fatty acids in the Chlorella sp. GD cultures.
296
Compared with the culture medium, there was a relative decreased of the C16:0 and
297
increase of the C18:1 contents of the 25% diluted wastewater and an increase of the
298
C18:2 of the cultures with 50-100% diluted wastewater. This result indicates that the
299
variation in the fatty acid profile of Chlorella sp. GD was probably associated with
300
certain components in wastewater, which are most likely certain minerals or 12
301
stress-accumulated conditions (e.g., growth-inhibiting ingredients, wastewater pH,
302
toxic organic compounds, etc.). There were many reports performed that discuss the
303
effects of culture broth derived from wastewater on the microalgal lipid content and
304
fatty acids profiles. With a reduction of the COD of the medium by dilution, the fatty
305
acids C18:1 and C16:0 of Chlorella sp. were increased and C16:2 was decreased
306
(Wang et al., 2010). C16:0, C18:2 and C18:3 were the abundant fatty acids of
307
Chlorella pyrenoidosa when grown on diluted piggery wastewater and Bristol’s
308
solution, and the total decrease of C16:2 and C18:1 was equal to an increase of
309
C16:0 (Wang et al., 2012). When Chlorella sp. was cultivated in treated and
310
untreated concentrated municipal wastewater, the relative content of monoenoic fatty
311
acids (C16:1 and C18:1) increased, polyenoic fatty acids (C16:2, C18:2 and C18:3)
312
decreased and saturated fatty acids were not different (Li et al., 2011). The results
313
mentioned above suggest that the variation in the fatty acid profiles of microalgal
314
species grown on different wastewater-containing medium seems to be
315
strain-dependent. In addition, the kinematic viscosity (4.6 mm2 s-1), flash point
316
(140 oC) and cetane number (61) of the biodiesel generated from Chlorella sp. GD
317
microalgae oil are fit with the ASTM D6751 or EN 14214 biodiesel standard. From
318
the fractions of lipids, composition profiles of fatty acids and properties of the
319
generated biodiesel, the lipid from Chlorella sp. GD in piggery wastewater
320
cultivation was suitable for biodiesel production.
321 322 323 324 325
3.4 Growth profiles of Chlorella sp. GD cultivated with different ratios of wastewater in semi-continuous cultivation The growth profiles of Chlorella sp. GD using different ratios of wastewater diluted with medium in semi-continuous cultures are shown in Fig. 5. In cycles 2-5, 13
326
the maximum biomass concentrations of Chlorella sp. GD with 25, 50 and 75%
327
wastewater reached 4 g L-1 or more, which were similar to the results from cycle 1.
328
However, the maximum biomass concentration of Chlorella sp. GD with 100%
329
wastewater decreased gradually after every replacement. This decrease was most
330
likely because some of the wastewater components could be accumulated with every
331
replacement; these components may inhibit microalgal growth, but the components
332
still need to be further investigated. The growth of Chlorella sp. GD was decreased
333
because the pH of the initial culture broth was gradually increased after every 100%
334
wastewater replacement. The average maximum biomass concentration in the culture
335
with 25, 50, 75 and 100% wastewater were 4.73, 4.81, 4.86 and 4.14 g L-1,
336
respectively. The average of biomass productivity in the culture with 25, 50, 75 and
337
100% wastewater was 0.852, 0.870, 0.859 and 0.701 g L-1 d-1, respectively. The
338
biomass productivity of Chlorella sp. GD cultured with diluted wastewater was
339
significantly higher than that with 100% wastewater. We emphasize that the biomass
340
productivity of Chlorella sp. GD in the semi-continuous cultures was approximately
341
2-fold higher than that in the batch cultures (Fig. 2 vs. Fig. 5). It may be that
342
replacing fresh diluted wastewater could maintain Chlorella sp. GD growth in the
343
exponential phase. Another reason could be that when the high biomass
344
concentration of microalgal cultivation was half replaced by fresh nutrients, the
345
culture broth replacement in semi-continuous cultivation decreased the self-shading
346
phenomenon, turbidity and light penetration of the microalgal cultures, resulting in
347
an increase in the photosynthesis efficiency (Li et al., 2011; Wang et al., 2012; Zhu et
348
al., 2013b). Chlorella zofingiensis was incubated with the replacement half fresh
349
piggery wastewater in semi-continuous cultures every 1.5 days after 6 days of batch
350
cultivation to day 15. The stable biomass concentration was maintained at 14
351
approximately 2 g L-1, and the net biomass productivity was 3-fold higher than that
352
in the batch culture (Zhu et al., 2013b). Similar results were shown in our study that
353
the semi-continuous replacement culture strategy could be a practical approach to
354
establish a stable microalgal cultivation process.
355 356 357
3.5 Lipid content and productivity of Chlorella sp. GD cultivated with different ratios of wastewater in semi-continuous cultivation
358
The lipid content and lipid productivity of Chlorella sp. GD, which was
359
harvested on day 17 in semi-continuous cultivation, are illustrated in Table 1. The
360
lipid contents of Chlorella sp. GD cultured with 75 and 100% wastewater were
361
higher than those cultured with 25 and 50% wastewater. This shows that the
362
microalgae that were cultivated with high ratios of wastewater enhanced the lipid
363
contents. Compared with the batch cultures, the averages of the microalgal lipid
364
contents in semi-continuous cultures were lower. The probable reason for this is that
365
the growth rate of Chlorella sp. GD in semi-continuous cultures was faster than that
366
in batch cultures; therefore, the uptake of nutrients by microalgae was used to
367
synthesize essential cell structures, such as proteins and carbohydrates, rather than
368
microalgal lipids. Although the lipid content decreased, increasing biomass
369
productivity was obtained in the semi-continuous Chlorella sp. GD cultures.
370
Therefore, higher lipid productivity could be gained in semi-continuous cultures
371
using diluted piggery wastewater. Higher biomass and lipid productivity were
372
similarly obtained when Chlorella sp. NJ-18 was cultivated outdoors with medium
373
replacement in semi-continuous cultures (Zhou et al., 2013). Because the lipid
374
content of Desmodesmus sp. F2 was decreased along with the medium replacement
375
ratios in semi-continuous cultures, the overall biomass productivity was significantly 15
376
increased. Because of the higher overall lipid productivity and the longer stable
377
operation time, the costs of the semi-continuous cultivation strategy for biodiesel
378
production could be cheaper (Ho et al., 2014).
379
It is frequently noted that many challenges are cost-associated, and cannot be
380
overcome without technical breakthroughs and innovative system integration for the
381
production of microalgae biomass. Using wastewater as a source and combining
382
wastewater treatment with the production of microalgal based bioproducts can
383
overcome several of the identified challenges. In our opinion, the cost reduction of
384
using wastewater for microalgae cultivation may include the use of fresh water and
385
chemicals for culture nutrition. However, there are still several important technical
386
challenges that need to be overcome before the large-scale production of microalgal
387
derived biofuels can become commercially viable, such as wastewater pretreatment.
388 389 390 391
3.6 Fatty acid compositions of Chlorella sp. GD cultivated with different ratios of wastewater in semi-continuous cultivation In addition to increasing the total lipid production of Chlorella sp. GD, it is also
392
important to explore fatty acid compositions for biofuel production. The fatty acid
393
compositions of Chlorella sp. GD cultivated with 25, 50, 75 and 100% wastewater in
394
semi-continuous cultivation at the end of the semi-continuous cultivations (i.e., on
395
day 17) are presented in Table 1. The relative contents of C16:0 (22.8-31.4%) and
396
C18:2 (22.1-31.5%) were higher than other fatty acids in microalgal lipids. These
397
results were similar to those obtained in the batch cultures. The suitable fatty acid
398
compositions for biofuel production in semi-continuous cultivation experienced no
399
obvious changes with the changing diluted wastewater ratio. C16:0 (15.2-19.1%) and
400
C18:2 (14.4-22.4%) were the main fatty acids of Chlorella sp. that were cultivated in 16
401
the highly concentrated municipal wastewater (Li et al., 2011). Because Chlorella
402
vulgaris cultivated with wastewater contained high levels of ammonia, C18:2
403
(28.2-37.0%) was the most abundant and C16:0 (26.2-36.3%) was the second most
404
abundant fatty acid (He et al., 2013). The results suggest that in semi-continuous
405
cultivation with wastewater, Chlorella sp. GD oil is an appropriate feedstock for
406
biodiesel production.
407 408 409 410
4. Conclusions This study shows that the maximum specific growth rate of Chlorella sp. GD is
411
obtained in piggery wastewater without dilution in batch cultivation. In a
412
semi-continuous cultivation, biomass productivity with 25-75% piggery wastewater was
413
significantly higher than that of batch cultivation. With higher biomass productivity, the
414
lipid productivity is increased with the benefit of enhancing the total lipid production.
415
The maximum lipid productivity was 0.176 g L-1 day-1 with 75% piggery wastewater
416
replacement. This study demonstrated a potential approach of stable growth
417
performance for long-term microalgal cultivation in semi-continuous operation
418
cultivation system with piggery wastewater.
419 420 421 422
Acknowledgements The work was financially supported by grants MOST 103-3113-E-006-006 from
423
the Ministry of Science and Technology. This work was also supported in part by the
424
Aim for the Top University Program of the National Chiao Tung University and
425
Ministry of Education, Taiwan. 17
426
Reference
427
1.
294−306.
428 429
Chisti, Y., 2007. Biodiesel from microalgae. Biotechnology Advances. 25,
2.
Chiu, S.Y., Kao, C.Y., Chen, C.H., Kuan, T.C., Ong, S.C., Lin, C.S., 2008.
430
Reduction of CO2 by a high-density culture of Chlorella sp. in a semicontinuous
431
photobioreactor. Bioresour. Technol. 99, 3389−3396.
432
3.
Chiu, S.Y., Kao, C.Y., Tsai, M.T., Ong, S.C., Chen, C.H., Lin, C.S., 2009. Lipid
433
accumulation and CO2 utilization of Nannochloropsis oculata in response to CO2
434
aeration. Bioresour. Technol. 100, 833−838.
435
4.
Chiu, S.Y., Kao, C.Y., Huang, T.T., Lin, C.J., Ong, S.C., Chen, C.D., Chang, S.H.,
436
Lin, C.S., 2011. Microalgal biomass production and on-site bioremediation of
437
carbon dioxide, nitrogen oxide and sulfur dioxide from flue gas using Chlorella sp.
438
cultures. Bioresour. Technol. 102, 9135−9142.
439
5.
Chiu, S.Y., Kao, C.Y., Chen, T.Y., Chang, Y.B., Kuo, C.M., Lin, C.S., 2015.
440
Cultivation of microalgal Chlorella for biomass and lipid production using
441
wastewater as nutrient resource. Bioresour. Technol. 184, 179−189.
442
6.
Ebrahimian, A., Kariminia, H.R., Vosoughi, M., 2014. Lipid production in
443
mixotrophic cultivation of Chlorella vulgaris in a mixture of primary and
444
secondary municipal wastewater. Renewable Energy. 71, 502−508.
445
7.
Farooq, W., Lee, Y.C., Ryu, B.G., Kim, B.H., Kim, H.S., Choi, Y.E., Yang, J.W.,
446
2013. Two-stage cultivation of two Chlorella sp. strains by simultaneous treatment
447
of brewery wastewater and maximizing lipid productivity. Bioresour.Technol. 132,
448
230–238.
449 450
8.
Hach Company, 2009. Dr/890 Colorimeter Procedures Manual, ninth ed. Hach Company, Loveland, CO, USA. 18
451
9.
He, P.J., Mao, B., Shen, C.M., Shao, L.M., Lee, D.J., Chang, J.S., 2013.
452
Cultivation of Chlorella vulgaris on wastewater containing high levels of ammonia
453
for biodiesel production. Bioresour. Technol. 129, 177−181.
454
10. Ho, S.H., Chen, C.Y., Chang, J.S., 2012. Effect of light intensity and nitrogen
455
starvation on CO2 fixation and lipid/carbohydrate production of an indigenous
456
microalga Scenedesmus obliquus CNW-N. Bioresour. Technol. 113, 244−252.
457
11. Ho, S.H., Li, P.J., Liu, C.C., Chang, J.S., 2013. Bioprocess development on
458
microalgae-based CO2 fixation and bioethanol production using Scenedesmus
459
obliquus CNW-N. Bioresour. Technol. 145, 142−149.
460
12. Ho, S.H., Chen, C.N., Lai, Y.Y., Lu, W.B., Chang, J.S., 2014. Exploring the high
461
lipid production potential of a thermotolerant microalga using statistical
462
optimization and semi-continuous cultivation. Bioresour. Technol. 163, 128−135.
463
13. Kao, C.Y., Chiu, S.Y., Huang, T.T., Dai, L., Hsu, L.K., Lin, C.S., 2012. Ability of a
464
mutant strain of the microalga Chlorella sp. to capture carbon dioxide for biogas
465
upgrading. Appl. Energy 93, 176−183.
466
14. Kao, C.Y., Chen, T.Y., Chang, Y.B., Chiu, T.W., Lin, H.Y., Chen, C.D., Chang, J.S.,
467
Lin, C.S., 2014. Utilization of carbon dioxide in industrial flue gases for the
468
cultivation of microalga Chlorella sp. Bioresour. Technol. 166, 485−493.
469
15. Klofutar, B., Golob, J., Likozar, B., Klofutar, C., Žagar, E., Poljanšek, I., 2010. The
470
transesterification of rapeseed and waste sunflower oils: Mass-transfer and kinetics
471
in a laboratory batch reactor and in an industrial-scale reactor/separator setup.
472
Bioresource Technology 101, 3333-3344.
473
16. Li, X., Xu, H., Wu, Q., 2007. Large-scale biodiesel production from microalga
474
Chlorella protothecoides through heterotrophic cultivation in bioreactors.
475
Biotechnol. Bioeng. 98, 764−771. 19
476
17. Li, Y., Chen, Y.F., Chen, P., Min, M., Zhou, W., Martinez, B., Zhu, J., Ruan, R.,
477
2011. Characterization of a microalga Chlorella sp. well adapted to highly
478
concentrated municipal wastewater for nutrient removal and biodiesel production.
479
Bioresour. Technol. 102, 5138−5144.
480
18. Li, Y.R., Tsai, W.T., Hsu, Y.C., Xie, M.Z., Chen, J.J., 2014a. Comparison of
481
autotrophic and mixotrophic cultivation of green microalgal for biodiesel
482
production. Energy Procedia. 52, 371−376.
483
19. Li, Y., Han, F., Xu, H., Mu, J., Chen, D., Feng, B., Zeng, H., 2014b. Potential lipid
484
accumulation and growth characteristic of the green alga Chlorella with
485
combination cultivation mode of nitrogen (N) and phosphorus (P). Bioresour.
486
Technol. 174, 24−32.
487
20. Likozar, B., Levec, J., 2014. Transesterification of canola, palm, peanut, soybean
488
and sunflower oil with methanol, ethanol, isopropanol, butanol and tert-butanol to
489
biodiesel: Modelling of chemical equilibrium, reaction kinetics and mass transfer
490
based on fatty acid composition. Applied Energy. 123, 108−120.
491
21. Mata, T.M., Martins, A.A., Caetano, N.S., 2010. Microalgae for biodiesel
492
production and other applications: a review. Renew. Sustain. Energy Rev. 14,
493
217−232.
494
22. REN21, 2014. Renewables 2014 Global Status Report. pp. 34–38.
495
23. Rosenberg, J.N., Kobayashi, N., Barnes, A., Noel, E.A., Betenbaugh, M.J., Oyler,
496
G.A., 2014. Comparative analyses of three Chlorella species in response to light
497
and sugar reveal distinctive lipid accumulation patterns in the microalga C.
498
sorokiniana. PLoS One 9: e92460.
499 500
24. Sharma, K.K., Schuhmann, H., Schenk, P.M., 2012. High lipid induction in microalgae for biodiesel production. Energies 5, 1532−1553. 20
501
25. Siaut, M., Cuiné S., Cagnon, C., Fessler, B., Nguyen, M., Carrier, P., Beyly, A.,
502
Beisson, F., Triantaphylidès, C., Li-Beisson, Y., Peltier, G., 2011. Oil accumulation
503
in the model green alga Chlamydomonas reinhardtii : characterization, variability
504
between common laboratory strains and relationship with starch reserves. BMC
505
Biotechnology 11, 1−15.
506
26. Šoštarič, M., Klinar, D., Bricelj, M., Golob, J., Berovič, M., Likozar, B., 2012.
507
Growth, lipid extraction and thermal degradation of the microalga Chlorella
508
vulgaris. New Biotechnology 29, 325–331.
509
27. Wan, M.X., Wang, R.M., Xia, J.L., Rosenberg, J.N., Nie, Z.Y., Kobayashi, N.,
510
Oyler, G.A., Betenbaugh, M.J., 2012. Physiological evaluation of a new Chlorella
511
sorokiniana isolate for its biomass production and lipid accumulation in
512
photoautotrophic and heterotrophic cultures. Biotechnol. Bioeng. 109, 1958−1964.
513
28. Wang, H., Xiong, H., Hui, Z., Zeng, X., 2012. Mixotrophic cultivation of Chlorella
514
pyrenoidosa with diluted primary piggery wastewater to produce lipids. Bioresour.
515
Technol. 104, 215−220.
516
29. Wang, L., Li, Y., Chen, P., Min, M., Chen, Y., Zhu, J., Ruan, R.R., 2010. Anaerobic
517
digested dairy manure as a nutrient supplement for cultivation of oil-rich green
518
microalgae Chlorella sp. Bioresour. Technol. 101, 2623−2628.
519
30. Yeh, K.L., Chang, J.S., 2012. Effects of cultivation conditions and media
520
composition on cell growth and lipid productivity of indigenous microalga
521
Chlorella vulgaris. Bioresour. Technol. 105, 120−127.
522
31. Yen, H.W., Hu, I.C., Chen, C.Y., Ho, S.H., Lee, D.J., Chang, J.S., 2013.
523
Microalgae-based biorefinery – from biofuels to natural products. Bioresour.
524
Technol. 135, 166−174.
525
32. Zhou, X., Ge, H., Xia, L., Zhang, D., Hu, C., 2013. Evaluation of oil-producing 21
526 527
algae as potential biodiesel feedstock. Bioresour. Technol. 134, 24−29. 33. Zhu, L., Wang, Z., Shu, Q., Takala, J., Hiltunen, E., Feng, P., Yuan, Z., 2013a.
528
Nutrient removal and biodiesel production by integration of freshwater algae
529
cultivation with piggery wastewater treatment. Water Res. 47, 4294−4302.
530
34. Zhu, L., Wang, Z., Takala, J., Hiltunen, E., Qin, L., Xu, Z., Qin, X., Yuan, Z.,
531
2013b. Scale- up potential of cultivating Chlorella zofingiensis in piggery
532
wastewater for biodiesel production. Bioresour. Technol. 137, 318−325.
22
533
Figures captions
534
Fig. 1. Growth profiles of Chlorella sp. GD cultured with different sources of
535
wastewater (piggery, industrial and municipal wastewater) without dilution and medium
536
in batch cultures. Piggery, industrial and municipal wastewaters were from a swine farm
537
(Miaoli, Taiwan), Hsinchu industrial park (Hukou, Taiwan) and National Chiao Tung
538
University (Hsinchu, Taiwan), respectively. The initial biomass concentration was
539
approximately 0.3 g L-1. The cultures were illuminated at 300 µmol m-2 s-1 and with a
540
2% CO2 aeration rate of 0.2 vvm at 26 ± 1°C. The batch culture was operated for 10
541
days, and the microalgal cells were sampled every 24 h for growth determinations.
542 543
Fig. 2. Growth (A) and biomass productivity (B) profiles of Chlorella sp. GD cultured
544
with different ratios of piggery wastewater (0, 25, 50, 75 and 100%) diluted by culture
545
medium in batch cultures. The initial biomass concentration was approximately 0.3 g
546
L-1. The cultures were illuminated at 300 µmol m-2 s-1 and with a 2% CO2 aeration rate
547
of 0.2 vvm at 26 ± 1°C. The batch culture was operated for 10 days, and the microalgal
548
cells were sampled every 24 h for growth determinations. The biomass productivity was
549
calculated from biomass concentration.
550 551
Fig. 3. Lipid content, lipid productivity and biomass productivity of Chlorella sp. GD
552
using different ratios (0, 25, 50, 75 and 100%) of piggery wastewater diluted with
553
culture medium in a 10-day batch culture. The initial biomass concentration was
554
approximately 0.3 g L-1. The cultures were illuminated at 300 µmol m-2 s-1 and with a
555
2% CO2 aeration rate of 0.2 vvm at 26 ± 1°C.
23
556
Fig. 4. The main fatty acid profiles of Chlorella sp. GD cultured with different ratios of
557
piggery wastewater (0, 25, 50, 75 and 100%) diluted by culture medium in 10-day batch
558
cultures.
559 560
Fig. 5. Growth profiles of Chlorella sp. GD using the 100% (A), 75% (B), 50% (C) and
561
25% (D) piggery wastewater diluted with culture medium in semi-continuous cultures.
562
The initial biomass concentration was approximately 0.3 g L-1. The cultures were
563
illuminated at 300 µmol m-2 s-1 and with a 2% CO2 aeration rate of 0.2 vvm at 26 ± 1°C.
564
The microalgal cultures were started as a batch culture for 5 days (cycle 1 of
565
semi-continuous culture). Half of the culture broth was replaced with the same amount
566
of fresh wastewater every 3 days for a period of 12 days (i.e., cycles 2-5 of
567
semi-continuous culture). The microalgal cells were sampled every day for growth
568
determinations.
569
24
570
Table 1. Lipid content, lipid productivity and main fatty acid compositions of Chlorella
571
sp. GD using the different ratios of wastewater diluted with culture medium in
572
semi-continuous cultures. Wastewater ratio
Lipid content (%) a Lipid productivity (g L-1 day-1) b
25%
50%
75%
100%
15 ± 0
15 ± 1
21 ± 1
21 ± 2
0.123 ± 0.001 0.130 ± 0.010 0.173 ± 0.001 0.140 ± 0.028
Fatty acid composition Saturated fatty acids
Monoenoic fatty acids
Polyenoic fatty acids
Others 573
a
C16:0
30 ± 8
31 ± 5
23 ± 3
28 ± 5
C18:0
7±1
3±0
5±1
4±0
subtotal
36 ± 9
35 ± 5
28 ± 4
32 ± 5
C16:1
2±0
2±0
3±0
2±0
C18:1
12 ± 0
10 ± 1
6±0
13 ± 1
subtotal
15 ± 1
12 ± 1
9±0
16 ± 2
C16:2
6±0
11 ± 1
16 ± 2
10 ± 2
C18:2
22 ± 3
28 ± 4
32 ± 6
29 ± 7
C18:3
16 ± 1
13 ± 0
14 ± 0
13 ± 1
subtotal
44 ± 4
52 ± 6
62 ± 9
52 ± 10
5±2
1±1
2±0
1±0
Lipid content (%) = (lipid dry weight / biomass dry weight) × 100%. Each data indicates the mean ± SD from three experiments.
574 575
Relative content (%)
b
Lipid productivity (g L-1 day-1) = (biomass productivity × lipid content) / 100.
576 577
25
Figure 1
-1
Biomass concentration (g L )
10
1
Piggery wastewater Industrial wastewater Municipal wastewater Medium
0.1
0
1
2
3
4 5 6 7 8 Days of cultivation
9
10
Figure 2
A. -1
Biomass concentration (g L )
10
1 Medium 25% WW 50% WW 75% WW 100% WW 0.1
0
1
2
3
B.
4
5
6
7
Days of cultivation
9
10
Medium 25% WW 50% WW 75% WW 100% WW
-1
-1
Biomass productivity (g L d )
1.6
8
1.2
0.8
0.4
0.0
0
1
2
3
4
5
6
7
Days of cultivation
8
9
10
Figure 3
0.8
40 Biomass productivity Lipid productivity Lipid content
30
0.6 0.5
20
0.4 0.2
10
0.1
0.0
0
25
50
75
Wastewater ratio (%)
100
0
Lipid content (%)
-1
-1
Productivity (g L d )
0.7
Fatty acid compositions
40
Other
C18:3
C18:2
C18:1
C18:0
C16:2
C16:1
C16:0
Relative content of fatty acid (%)
Figure 4
50
Medium 25% WW 50% WW 75% WW 100% WW
30
20
10
0
Figure 5
A. 100% WW 6 4 2
Biomass concentration (g L-1)
0
0
2
4
6
8
10
12
14
16
18
14
16
18
14
16
18
14
16
18
B. 75% WW and 25% medium 6 4 2 0
0
2
4
6
8
10
12
C. 50% WW and 50% medium 6 4 2 0
0
2
4
6
8
10
12
D. 25% WW and 75% medium 6 4 2 0
0
2
4
6
8
10
12
Days of cultivation
578
Highlights
579
Piggery wastewater was able to be directly used for Chlorella sp. GD cultivation.
580
The maximum specific growth rate of Chlorella sp. GD with 100% piggery wastewater was 0.839 d-1.
581 582
semi-continuous culture.
583 584 585
The biomass and lipid productivity of Chlorella sp. GD was enhanced in a
Stable growth performance for long-term microalgal cultivation in semi-continuous operations is promising.
586 587
26
Graphical abstract