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Sustainable biomass production under CO2 conditions and effective wet microalgae lipid extraction for biodiesel production
Journal Pre-proof Sustainable biomass production under CO2 conditions and effective wet microalgae lipid extraction for biodiesel production M. Lakshmikandan, A.G. Murugesan, Shuang Wang, Abd El-Fatah Abomohra, P. Anjelin Jovita, S. Kiruthiga PII:
S0959-6526(19)34268-4
DOI:
https://doi.org/10.1016/j.jclepro.2019.119398
Reference:
JCLP 119398
To appear in:
Journal of Cleaner Production
Received Date: 4 July 2019 Revised Date:
2 November 2019
Accepted Date: 19 November 2019
Please cite this article as: Lakshmikandan M, Murugesan AG, Wang S, Abomohra AE-F, Jovita PA, Kiruthiga S, Sustainable biomass production under CO2 conditions and effective wet microalgae lipid extraction for biodiesel production, Journal of Cleaner Production (2019), doi: https://doi.org/10.1016/ j.jclepro.2019.119398. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Ltd.
Opt i mi z at i onofbi omas sandl i pi dpr oduc t i vi t y
EXTRACTI ON Pr e s s ur e 2kg/ c m2
Pr e he at e d( 60˚ C)gl as spl at e
Th i nl a y e ro f we t mi c r o a l g a l b i o ma s s
0. 04%
4%
Li pi d
8%
Pr e he at e d( 60˚ C)gl as spl at e
Dur i ngmi l dpr e s s ur eand he ats hoc kt r e at me nt
ds o i r pe o t Pho
Bi omas s
Ce l lr upt ur eandl i pi dbodi e sr e l e as e
1
Sustainable biomass production under CO2 conditions and effective wet microalgae
2
lipid extraction for biodiesel production
3
M. Lakshmikandan
4
Anjelin Jovita b, S. Kiruthiga b
5
a
6
b
7
Sundaranar University, Alwarkurichi 627 412, Tamil Nadu, India
8
c
a,b
, A.G. Murugesan b, Shuang Wang a*, Abd El-Fatah Abomohra
a,c*
, P.
School of Energy and Power Engineering, Jiangsu University, Jiangsu 212013, China Sri Paramakalyani Centre of Excellence in Environmental Sciences, Manonmaniam
Botany Department, Faculty of Science, Tanta University, Tanta 31527, Egypt
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
*Correspondence
24
[email protected](Shuang
25
Abomohra)
Wang);
[email protected](Abd
1
El-Fatah
26
Abstract
27
The freshwater microalga Chlorella vulgaris MSU AGM 14 were cultured at different CO2
28
conditions (up to 8%) with 100 µmol photons m-2 s-1 to evaluate biomass and lipid
29
productivity. For effective extraction of intra cellular lipids, a novel method based on mild
30
pressure (1-2.5 kg/cm2) with short period (5-15 min) of heat shock (50-70˚C) were studied
31
for the wet biomass. The transesterified lipids were qualitatively and quantitatively analysed
32
by using Gas Chromatography coupled with Mass Spectrometry (GC-MS). The higher CO2
33
aeration (8%) significantly increased the biomass productivity (23%) when compared with
34
control and CO2 (4%) aeration. Total lipid production (93%) acquired by conventional
35
extraction procedure showed enhanced production simultaneously. The maximum lipid
36
recovery (0.225g g-1 dw) was obtained at a pressure of 2 kg/cm2 and heating for 10 min at
37
60˚C. The transesterified lipids showed that oleic acid (C18:1 - 51.62%) was the main
38
component in both conventional and suggested lipid extraction process. The suggested
39
extraction process showed significant increase in biodiesel yield by 26.7%. The energy
40
outputs of biodiesel by conventional and suggested extraction process were 417.7 and 533.6
41
MJ ton-1, respectively. The overall results indicated that 8% of CO2 induced the biomass and
42
lipid productivity by 94% and 54.8%, respectively, when compared with control. In addition,
43
the suggested mild pressure with heat shock extraction process further enhanced the lipid
44
recovery by 21% which serves as a cost-effective lipid extraction process for microalgae.
45
46
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Keywords: Biodiesel; Microalgae; Chlorella vulgaris; Mild pressure; Heat shock; Lipid
48
extraction
2
49
1. Introduction
50
Millions of years ago dead organic matter were been transformed into black gold by natural
51
events like prolonged period of time and pressurized geothermal process. So far, crude oil is
52
one of the most significant sources of energy on the planet. Since petroleum is a part of the
53
global economy with limited reservoirs (Hamilton, 2009; He et al., 2010); Jones et al. (2004),
54
experts are predicting that the end of petroleum era is near. But footsteps remain in the
55
environment due to toxic pollution leading to climate change (Prasad and Kumari, 1987;
56
Wang et al., 2017). The search for alternative fuel will tread on renewable energy resources
57
peculiarly without damaging the bionomical balance (Elsayed et al., 2018; Yuan et al., 2019).
58
Biodiesel gives some hope for renewable and clean energy with reduced carbon footprint.
59
Predominately, biodiesel is produced from plant oil (canola, sunflower, soybean and rapeseed
60
oil) by alkali-based transesterification process (Meher et al., 2006). But using food crops for
61
biodiesel production has never been the right solution for fuel crisis problems since they
62
interfere with food production for high grade arable land, initiating the food demand leading
63
to hike in the food price (Chisti, 2007; Lin et al., 2011; Silitonga et al., 2013). The true fact is
64
global oblige for food crops and products is awaited to double within 5 decades and in case of
65
transportation fuels it is still more worsened (Hill et al., 2006).
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In such challenging situations, microalgae has attracted greater attentions for various forms of
67
biofuel production like biodiesel (Abinandan et al., 2019; Jain et al., 2019), bioethanol
68
(Sanchez Rizza et al., 2019), biohydrogen (Lakshmikandan and Murugesan, 2016a, b),
69
bioelectricity (Powell et al., 2009) and biomethane (Raheem et al., 2018), since microalgae
70
cultivation has positive environmental impact and doesn’t compete with food supplies.
71
Furthermore, microalgae possess important advantages like higher photosynthetic efficiency
72
leading to 10-50 times more significant CO2 mitigation when compared to terrestrial floras
73
(Anto et al., 2019; Mondal et al., 2017). Prominent quantity of biomass and lipid production 3
74
rate in oil bearing perennial crops like Jatropha, biomass productivity is about 4.1 ton ha-1 y-1
75
whereas microalgae owes to two fold higher capability with less land demand and efficient
76
non-edible oil source (Mathimani and Mallick, 2019; Mathimani et al., 2018). In addition,
77
higher growth rate on harsh environmental conditions and its ability to grow on wastewater
78
with various biorefinery applications are the significant features of microalgae (Abomohra et
79
al., 2017; Nascimento et al., 2014). At normal environmental conditions, microalgae can
80
accumulate appreciable quantity of lipids (structural lipids) but certain specific environmental
81
stress conditions will stimulate the elevated level of lipid accumulation in microalgae (Tu et
82
al., 2015; Widjaja et al., 2009). High CO2 concentration and length of photoperiod were
83
reported to play a significant role in elevated biomass recovery and lipid content of
84
microalgae (Che et al., 2019; Lv et al., 2010; Wahidin et al., 2013). From this phenomenon,
85
CO2 and photoperiod-based microalgal lipid enhancement has been investigated widely on
86
higher productivity and adaptive nature microalgal species.
87
In the current study, eukaryotic green microalga Chlorella vulgaris was investigated as it is
88
very commonly obtained in both fresh and seawater with high photosynthetic efficiency and
89
lipid productivity (Battah et al., 2015). The crucial characters are; they are strongly resistant
90
to any harsh conditions, and are invaders with high biomass productivity within a quick
91
period of time (Ebrahiminezhad et al., 2014; Frumento et al., 2013; Safi et al., 2014). The
92
crucial challenge of lipid extraction is selecting suitable cell disruption method to obtain
93
maximum productivity. Several cell disruption techniques have been employed to recover
94
lipids from the whole cells. Cell disruption techniques are grouped into mechanical and non-
95
mechanical process; mechanical extraction method includes bead milling, sonication, high
96
pressure homogenization, oil expeller, autoclaving, grinding and microwave techniques
97
(Günerken et al., 2015; Mathimani et al., 2017; Postma et al., 2015). The commonly used
98
non-mechanical extraction processes are chemical based Soxhlet, supercritical fluids and 4
99
enzymatic extractions. Peralta-Ruiz et al. (2013) reported that 85% total energy inputs of
100
biodiesel production has been shared by lipid extraction process itself. Conventional lipid
101
extraction process requires microalgal biomass dewatering before lipid extraction process and
102
85% of total energy input were exhausted by drying process (Patil et al., 2012). The crucial
103
part of cell disruption is to increase the disruption rates, minimize damage of targeted product
104
and lower the capital cost (Yap et al., 2015). High pressure based mechanical cell disruption
105
methods are promising techniques for complete disruption of cells (Carullo et al., 2018).
106
However, the high energy consumption, non-targeted intracellular compounds disruption and
107
difficult downstream process make it more complicated to the economic viability. The
108
applications of mild pressure in microalgae has been rarely investigated and are mainly
109
focused on encouraging the growth and lipid accumulation (Praveenkumar et al., 2016).
110
Lorenzen et al. (2017) reported that the significance of pressure (12 MPa) and heat (20˚C)
111
combination increased higher efficiency of lipid extraction from dried biomass. In the present
112
investigation mild pressure with heat shock were used to disrupt the wet cells of C. vulgaris
113
to maximise the lipid recovery and reduce energy consumption in pressure-based cell
114
disruption process.
115
With the above perspective in view, detailed investigations were carried out to optimize cell
116
growth and lipid productivity at higher CO2 concentrations and different photoperiod
117
conditions. The effect of wet cell disruption by novel method based on mild pressure and heat
118
shock treatment were then thoroughly investigated. The variations between conventional
119
solvent extraction and the suggested extraction were analysed by GC-MS. Finally, microalgal
120
cultivation, harvest cost, energy input and output of biodiesel from conventional and
121
suggested extraction process were evaluated.
122
2. Materials and Methods
5
123
2.1. Microalgal strain and inoculum preparation
124
Chlorella vulgaris MSU-AGM 14 (accession number KM189121) was obtained from
125
Department of Environmental Science, Sri Paramakalyani Centre of Excellence in
126
Environmental Sciences, Manonmaniam Sundaranar University, Tirunelveli, India. The
127
microalga was grown in the Algae Culture Broth (Hi-Media, Mumbai) composed of NaNO3,
128
1g; K2HPO4, 0.25g; MgSO47H2O, 0.513g; NH4Cl, 0.05g; CaCl22H2O, 0.02g; FeCl3, 0.003g
129
per liter of deionized water at pH of 7.0±0.2. Cultures were incubated at 27±1˚C with
130
continuous illumination (100 µmol photons m-2 s-1) and 4% CO2 aeration. When the cultures
131
reached the exponential phase, inoculum was introduced into 30 litres cylindrical photo
132
bioreactor for microalgal biomass accumulation. As per experimental setup, different
133
photoperiod [light (L): dark (D) cycles] (24L, 18L+6D and 12L+12D) and different CO2
134
supply (4% and 8%) were applied. Atmospheric air (with 0.04% CO2) was used as control.
135
Microalgal growth was monitored every 12 h by using spectrophotometer (Au-2701,
136
Systronics) at 680nm (OD680). After 12 days of incubation (stationary phase), cultures were
137
harvested by centrifugation (Eppendorf 5804R, Germany) for 10 min at 3000×g to obtain the
138
wet microalgal biomass. The cumulative biomass (g L-1 day-1) and lipid productivity (mg L-1
139
day-1) were calculated by using the following Eq. (1) Biomass / Lipid productivity = (DWt - DWo)/t
140
(1)
141
Where DWt and DWoare the final and initial dry weights of the sample, respectively; while t
142
is the time of harvest.
143
2.2. Cell disruption and lipid extraction
144
Approximately 11.2 g (adequate to 1g of dry weight) of harvested wet microalgal biomass
145
were placed between two preheated (50˚C, 60˚C and 70˚C) annealed glass plates (2 mm
6
146
thickness) at different pressured conditions (0.98, 1.47, 1.96 and 2.45 bar) and different
147
exposure times (5, 10 and 15 min). After mild pressure with heat shock treatment, lipids were
148
extracted by hexane: isopropanol (3:2 v/v) (Bian et al., 2018) and were kept in ice bath for 15
149
min. For control, 1g of dried microalgal sample was used for cell disruption and lipid
150
extraction process following the Bligh and Dyer (1959) method. The solvent extracts were
151
filtered by using Whatman filter paper no.1 and were allowed to evaporate to obtain the total
152
lipids which were stored at -20˚C in 1 mL chloroform for further analysis.
153
2.3.Transesterification of sample
154
In 10 mL screw caped glass tube, 25 mg of lipid sample was weighed and mixed with 1.5 mL
155
of 0.5 N methanolic NaOH (Nascimento et al., 2012). The solution was mixed gently, and
156
were sealed after nitrogen gas flushing, and were incubated in oven at 100˚C for 15 min.
157
After heat treatment, samples were allowed to cool at room temperature, followed by addition
158
of 2 mL of 12% boron trifluoride in methanol. Then the samples were sealed after nitrogen
159
gas flushing. To initiate a transesterification reaction, samples were placed at 100˚C for 45
160
min. After the transesterification, samples were allowed to cool at room temperature.
161
Approximately 2 mL of isooctane were added into each sample and were shaked vigorously
162
for 3 min. After mixing, the samples were immediately treated with 5 mL of saturated sodium
163
chloride solution followed by gentle agitation. After mild centrifugation the isooctane layer
164
was separated and were transferred into a clean glass tube and were stored at -20˚C for
165
further analysis.
166
2.4. Fatty acid analysis
167
The fatty acid composition of the prepared fatty acid methyl esters (FAMEs) were assessed
168
by Gas Chromatography (GC- Clarus 680 – Perkin Elmer) linked with Mass Spectrometry
169
(MS- Clarus SQ8T - Perkin Elmer) equipped with capillary column Elite-5 MS (30 m 7
170
length and 0.25 mm - internal diameter) and a flame ionization detector. Helium was used as
171
a carrier gas at a flow rate of 1.3 mL min-1. The electron ionization mode (70 eV) was
172
selected for mass spectrometry with full scan mode (range: 30–300 Da). The identification of
173
fatty acid components were based on their retention times, abundance and fragmentation
174
patterns comparison with NIST spectral library 2.0 g (2011).
175
2.5. Determination of biodiesel properties
176
Molecular characteristics of FAMEs describes the parameters of biodiesel quality such as
177
saponification value (SV), cetane number (CN), iodine value (IV), long chain saturated factor
178
(LCSF), cold filter plugging point (CFPP), allylic position equivalents (APE), bis- allylic
179
position equivalents (BAPE), kinematic viscosity (υ), density (ρ) and higher heating value
180
(HHV) (Islam et al., 2013; Talebi et al., 2013).
181
The empirical Eq. (2-4) was applied to calculate the SV, IV and CN depending on the FAME
182
profile. SV =
IV =
CN = 46.3 +
560 × ℎ 254 × ℎ
(2) ×
(3)
5458 − (0.225 × (&) (4) %&
183
Where Ni is the percentage of ith FAME, Di is the number of double bonds in ith FAME,
184
MW is the molecular weight.
8
185
The degree of unsaturation (DU) were calculated based on the mass fraction of mono
186
(MUFA) and poly unsaturated fatty acids (PUFA) by using the following Eq. (5). The LCSF,
187
CFPP, APE and BAPE were calculated by applying the following Eqs. (6-9) DU =
+,- + (2 × .+,-) (5)
/0%, = (0.1 × 016: 0) + (0.5 × 018: 0) + (1 × 020: 0 (6) 0,.. = (3.1417 × /0%,) − 16.477 (7) APE =
( 78 × -98 ) (8)
BAPE =
(;78 × -98 ) (9)
188
Where apn and bpn are the allylic and bis-allylic positions, respectively, in specific fatty acid;
189
while Acn is the mass percentage of each fatty acid.
190
FAMEs υ, ρ and HHV were calculated by using the following Eqs. (10-12) and final value of
191
each factors were obtained by summation of all FAMEs fuel properties. ln(υ? ) = −12.503 + 2.496 × ln( @? = 0.8463 +
4.9
AA&? = 46.19 −
?
?)
− 0.178 × (10)
+ 0.0118 × (11)
1794 ?
− 0.21 × (12)
192
Where Mi is the molecular weight; while N is thenumber of double bonds in ith fatty acid.
193
2.6. Statistical analysis
9
194
The results of means were calculated by using three replicates and were described as mean ±
195
standard deviation. The significance of differences (p < 0.05) were analyzed using one-way
196
analysis of variance (ANOVA) complied by least significant difference (LSD) using SPSS
197
(IBM, v.20).
198
3. Results and Discussion
199
3.1. Microalgal growth and lipid accumulation
200
C. vulgaris MSU AGM 14 were cultivated at two different percentages (4% and 8%) of CO2
201
aeration for 16 days and were monitored for cell growth and intracellular lipid accumulations.
202
While providing 8% aeration, cells have grown very well when compared to 4% and control
203
(Fig.1). Both samples reached their stationery phase on day 10. Chan et al. (2010) reported
204
that increasing CO2 aeration elevated the growth of microalgae in certain species such as B.
205
braunii, C. vulgaris and Scenedesmus sp. Similarly de Morais and Costa (2007) confirmed
206
that C. vulgaris can actively grow under high CO2 conditions. The microalgal production and
207
productivity (Fig. 2) also evidenced enhanced production of biomass and lipids at elevated
208
CO2 aeration when compared with control (0.04%). The cultures with 8% CO2 aeration
209
produced 0.79±0.04 g L-1 of biomass and the productivity indicated a maximum significant
210
amount of 0.064±0.003 g L-1 day-1 which represented 23% higher than that of the control.
211
Whereas lipid production recorded 186±11 mg g-1 and productivity reached to 11.89±0.48 mg
212
L-1 day-1, representing 93.96% significant increase over the control (Fig. 2b). In the present
213
study, the effect of photoperiod and CO2 aeration on C. vulgaris MSU AGM 14 biomass and
214
lipid accumulation were also evaluated (Table 1). The maximum biomass (1.44±0.02 g L-1
215
dw) were observed at 24 h illumination with 8% CO2 aeration, with total lipids of 0.18±0.02
216
g g-1. Whereas at 12 h L+12 h D illumination with 8% CO2, maximum lipid content of
217
0.2±0.02 g g-1 were recorded with dry weight of 1.37±0.03 g L-1. These results revealed that
218
microalgae require both light and dark conditions for effective lipid production. In addition, 10
219
increased CO2 aeration proved to be one of the important factors for the elevation of lipid
220
accumulation in microalgae. Previously, Chan et al. (2010) reported that the total lipid
221
content of C. vulgaris were 11.92% of the dry weight, while the present study revealed that at
222
suitable photoperiod with elevated CO2 condition C. vulgaris MSU AGM 14 has the potential
223
to produce 20% of total lipids. The present investigation clearly represents that CO2 condition
224
and photoperiod has significant impact on biomass and lipid accumulation of microalgae.
225
3.2. Mild pressure with heat shock treatment
226
Effective extraction of lipids from microalgal cells depends upon cell disruption technique
227
and selection of extraction process (Halim et al., 2012; Prabakaran and Ravindran, 2011).
228
There are several cell disruption techniques employed to extract intracellular lipids from
229
microalgal cells. Previously, some research works explained significant advantages of mild
230
pressure treatment on lipid accumulation during growth. Praveenkumar et al. (2016) reported
231
that mild pressure treatment (10-15 bar) induces accumulation of neutral lipid in Chlorella
232
sp., and few reports revealed that pressure tolerance ability differs from strain to strain.
233
Seckbach (1971) reported that microalga Cyanidium caldarium can tolerate mild (1- 10 bar)
234
pressures for 19 days at room temperature. While elevating the pressure significantly, growth
235
has been inhibited with change in pigmentation. In addition, several studies revealed that high
236
pressure can inhibit the growth or cause lethal damage to cells by affecting the normal
237
functions of cell organelles (Mañas and Mackey, 2004; Robey et al., 2001). However, there is
238
a gap in literature about the effect of pressure combined with heat shock on lipid extraction.
239
In this study, wet thin layered C. vulgaris MSU AGM 14 was disrupted by mild pressure
240
along with heat shock. The designed experiment showed good response in extracting the lipid
241
content from the selected strain (Fig. 3). Results confirmed that 2 kg/cm2 (1.96 bar) pressure
242
and 60˚C heat treatment with 10 min exposure time proved maximum effective extraction of
243
0.225±0.013 g g-1 total lipid. Overall, experiment demonstrated that exposure time of 11
244
microalgae in selected temperature is more important for effective extraction. When
245
compared with control, the present study showed 12.5% additional recovery of total lipid.
246
3.3.Composition of biodiesel
247
The successful conversion of total lipids into FAME was examined by GC-MS (Table 2).
248
Three most abundant methyl esters were determined in the biodiesel of the selected strain.
249
Among them octadecenoic acid methyl ester (oleic acid) showed the highest relative content
250
in the pressurised sample (51.62%) when compared to other methyl esters. The other two
251
methyl esters such as hexadecenoic acid methyl esters (14.53%) and octadecanoic acid
252
methyl ester (12.08%) possessed considerable relative contents in the biodiesel. Similarly,
253
Gao et al. (2010) reported that biodiesel of Chlorella protothecoides showed higher relative
254
content for these three methyl esters. Mathimani et al. (2015) also reported that palmitic,
255
oleic and linolenic acids are the major methyl esters of Chlorella sp. BDUG 91771biodiesel.
256
The suggested lipid extraction process evidenced no significant differences were reported in
257
the SFAs, MUFAs and PUFAs ratios with the control. The good quality biodiesel possesses
258
certain significant properties like heat of combustion, ignition quality, viscosity, cold filter
259
plugging point, lubricity and importantly oxidative stability which are defined by the
260
structure of its FAME components. The productive amount of oleic acid content represents its
261
enhanced oxidative stability for longer storage (Knothe, 2005). In addition, higher oxidizing
262
tendency of biodiesel was evaluated by the presence of degree of unsaturation on FAMEs
263
(Hoekman et al., 2012). Ramos et al. (2009) also described that higher degree of unsaturation
264
(>137) does not meet the European biodiesel standards. But in the present study, biodiesel of
265
C. vulgaris MSU AGM 14 showed within the limit (86.13 and 86.71) of unsaturation. Allylic
266
positions of biodiesel plays a crucial role in autoxidation/oxidation of unsaturated FAME.
267
The linear relationship of oxidizability on bis- allylic position equivalents and allylic position
268
equivalents has capablity to induce non-uniform oxidation in biodiesel (Iyer, 2017). In this 12
269
study, biodiesel of C. vulgaris MSU AGM 14 showed the presence of lower value allylic
270
position equivalents and bis- allylic position equivalents. These calculated allytic position
271
results have evidenced that FAME composition of biodiesel possess reasonably good
272
oxidation stability. The combustion behaviour of biodiesel has been estimated based on
273
cetane number (Koley et al., 2018). Depending upon fuel cetane number, engines ignition
274
delay time may vary, shorter ignition time has been recorded in higher cetane numbered fuel
275
(Islam et al., 2013). Overall, the suggested lipid extraction process showed slight neglectable
276
changes in all studied parameters with those of the control (Table 3).The resemblance
277
analyses with standard biodiesel EN14214 and ASTM D6751-02 indicated that higher cetane
278
number and moderate quantity on degree of unsaturation. Higher viscosity and density of
279
biodiesel may create many problems like fuel pump failure, carbon deposit, ring sticking and
280
poor atomization. Whereas, density values of biodiesel were within the range of standard
281
while the kinetic viscosity were slightly below the range. As reported by Yang et al. (2014)
282
suitable amount of viscosity and density was observed in higher concentrations of oleic,
283
palmitoleic and palmitic acid containing biodiesel. The determination of FAME derived HHV
284
(39.22 and 39.46MJ kg-1) showed 14% less than the conventional petroleum derived diesel
285
(46MJ kg-1). Thus, further studies are required to improve the biodiesel quality with respect
286
to the HHV of C. vulgaris lipids.
287
3.4. Economic assessment
288
The economic feasibility of the present study was evaluated following Wang et al. (2019)
289
energy calculations. The system was designed to cultivate 30L of algal culture with energy
290
consumption of 77.65 kWh per cycle. Totally 24 cycles were performed to acquire 1 kg of the
291
dry biomass by utilizing 1863.72 kWh kg-1 of input energy and the estimated cost of expenses
292
was 140.27 USD including chemicals used for cultivation purpose (Fig. 4). In addition,
293
microalgae harvest required 438.3 kWh kg-1 of energy with 26.38 USD kg-1 of dry biomass 13
294
recovery. Whereas extraction of lipids recorded that the suggested process required 13.7%
295
more expense to perform when compared to conventional method. However, almost 13% of
296
the effective lipid yield and elevated total FAMEs level carried out the attention to economic
297
significance. In addition, energy outputs of conventional and suggested methods were
298
calculated based on calorific value (HHV) of FAME and were recorded as 417.7 and 533.6
299
MJ ton-1 respectively. Despite higher energy consumption of the suggested extraction
300
method, it showed 27.7% higher energy output than the conventional extraction method. In
301
addition, the possible improvement in cultivation and harvesting process will encounter
302
higher cost and energy consumption in economical point of view. The large-scale cultivation
303
system and low energy consuming harvesting process like flocculation and bio-flocculation
304
allows wide possibility for feasible biodiesel production.
305
Conclusion
306
C. vulgaris MSU AGM 14 lipid production has been promoted by simultaneous increase of
307
biomass by higher CO2 conditions of microalgae. The increased amount of total lipid yield
308
(22.5 %) has been extracted by employing mild pressure and heat shock treatment. FAMEs
309
analysis of C. vulgaris MSU AGM 14 lipid evidenced the presence of high content of
310
monounsaturated and saturated fatty acids. In addition, degree of unsaturation, iodine value,
311
cold filter plugging point, kinematic viscosity and density of biodiesel strengthened the
312
appropriate characteristics of fuel standards. The overall results revealed that C. vulgaris
313
MSU AGM 14 is a potential strain for biodiesel production. The novelty of this study reveals
314
that mild pressure and heat shock cell disruption evidenced 27.7% elevated energy output,
315
which could reduce the total cost of energy consumption and increase the lipid recovery from
316
the selected strain.
317
Acknowledgement
14
318
The authors sincerely acknowledge National Natural Science Foundation of China (No.
319
51676091), China Postdoctoral Science Foundation (2019T120408, 2018M630529),
320
Foundation of State Key Laboratory of Coal Combustion (FSKLCCA1904), the Six talent
321
peaks project in Jiangsu Province (XNY-007, 2018) for the financial support. Authors also
322
thank Jiangsu University, Zhenjiang, China and Sri Paramakalyani Centre of Excellence in
323
Environmental Sciences, Manonmaniam Sundaranar University for providing facilities
324
required to carry out this work.
325
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Figures and tables captions
508
Figure 1: Growth curve of Chlorella vulgaris at different percentages of CO2 with confocal
509
microscopy images stained with Nile red to visualize the intracellular lipids.
510
Figure 2: Microalgal biomass and total lipid production (a) and productivity (b) of C.
511
vulgaris MSU AGM 14 at different percentages of CO2. The same series with the same letter
512
showed insignificant difference (at P<0.05).
22
513
Figure 3: Optimized extraction of total lipids from C. vulgaris MSU AGM 14 by suggested
514
mild pressure and heat treated method. The horizontal dashed line represents the lipid
515
recovery by the conventional extraction method.
516
Figure 4: Cultivation, harvest and extraction process cost and energy output for biodiesel
517
produced from microalgae C. vulgaris MSU AGM 14.
518
Table 1: Selection of suitable photoperiod lengths for optimizing the growth and lipid
519
productivity.
520
Table 2: Composition of biodiesel produced from different lipid extraction process.
521
Table 3: Resemblance between calculated properties of conventional and suggested
522
extraction processed biodiesel with biodiesel standards.
23
TABLES
Table 1. Selection of suitable photoperiod lengths for optimizing the growth and lipid productivity. Illumination time
Biomass CO2
(Hrs at 100 µmol -2 -1
photons m s )
18L+6D
12L+12D
24L
dry
Total lipid
weight
(gg-1 dw)
-1
Biomass
Lipid
productivity (gL-
Productivity (gL-
1
day-1)
1
day-1)
(%)
(gL )
Control
0.66±0.02a
0.12±0.02a
0.052±0.003a
0.006±0.002a
4%
0.69±0.05a
0.15±0.04ab
0.054±0.004ab
0.008±0.003a
8%
0.79±0.04b
0.19±0.02b
0.064±0.003b
0.012±0.002b
Control
0.49±0.05a
0.14±0.03a
0.038±.004a
0.005±.002a
4%
0.93±0.02b
0.17±0.05ab
0.075±.003b
0.013±.004b
8%
1.37±0.03c
0.2±0.02b
0.111±.002c
0.022±.003c
Control
0.38±0.05a
0.13±0.02a
0.029±.002a
0.004±.002a
4%
0.91±0.02b
0.16±0.04ab
0.074±.003b
0.012±.003b
8%
1.44±0.02c
0.18±0.02b
0.117±.003c
0.021±.003c
L- Light; D- Dark, values are mean ± SD, n=3. The same parameter at the same light regime with the same letter showed insignificant difference (at P<0.05).
Table 2. Composition of biodiesel produced from different lipid extraction process. Fatty Acid Methyl Esters
Relative content Control
Mild pressure and heat treated
Myristic acid (C14:0)
-
1.40
Palmitic acid (C16:0)
7.82
6.78
Palmitoleic acid (C16:1n-7)
15.11
14.53
Stearic acid (C18:0)
12.57
12.08
Oleic acid (C18:1n-9)
50.66
51.62
α - Linolenic acid (C18:3n-3)
10.18
10.24
γ - Linolenic acid (C18:3n-6)
-
0.04
Arachidic acid (C20:0)
2.98
3.27
Saturated fatty acids (%)
23.37
23.53
Monounsaturated fatty acids (%)
65.77
66.15ns
Polyunsaturated fatty acids (%)
10.18
10.28ns
Total FAMEs (%)
99.32
99.96ns
Unidentified (%)
0.68
0.04
ns
Showed insignificant difference with the corresponding control (at P<0.05).
Table 3.Resemblance between calculated properties of conventional and suggested extraction processed biodiesel with biodiesel standards. Biodiesel standards Properties
ASTM D6751-02
Degree of unsaturation
Mild pressure Conventional
EN 14214
and heat treated
-
-
86.13
86.71
-
-
201.48
203.04
Iodine value (I2100g )
NA
≤120
88.50
89.06
Cetane number
≥47
≥51
53.48
53.14
-
-
10.05
9.99
NA
≤5/≤-20
15.1
14.91
-
-
71.02
72.18
-
-
26.32
27.10
-
≤12
10.18
10.28
1.9-6.0
3.5-5.0
1.35
1.35
Density (g cm-3)
NA
0.86-0.90
0.86
0.87
Higher heating value (MJ kg-1)
NA
NA
39.22
39.46
-1
Saponification value (mg KOHg ) -1
Long chain saturated factor Cold filter plugging point (ºC) Allylic position equivalents (APE) Bis- allylic position equivalents (BAPE) C18:3 (%) Kinematic viscosity (mm2s-1)
FIGURES
Figure 1. Growth curve of Chlorella vulgaris at different percentages of CO2.
Figure 2. Microalgal biomass and total lipid production (a) and productivity (b) of C. vulgaris MSU AGM 14 at different percentages of CO2. The same series with the same letter showed insignificant difference (at P<0.05).
0.25
5 min 15 min
10 min
-1
Lipid recovery (g g dw)
0.20
0.15
0.10
0.05
o
70C +C N 70C +1 70C +1. 5 70C +2 70C +2. 5
60C +C N 60C +1 60C +1. 5 60C +2 60C +2. 5
50C +C N 50C +1 50C +1. 5 50C +2 50C +2. 5
0.00
2
Temperature ( C) + Pressure (kg/cm ) Figure 3. Optimized extraction of total lipids from C. vulgaris MSU AGM 14 by suggested mild pressure and heat-treated method. The horizontal dashed line represents the lipid recovery by the conventional extraction method.
Figure 4.Cultivation, harvest and extraction process cost and energy output for biodiesel produced from microalgae C. vulgaris MSU AGM 14.
Highlights: •
Elevated CO2 (8%) promoted biomass (23%) and lipid productivity (94%).
•
Mild pressure with heat shock evidenced 12.5% additional recovery of total lipids.
•
Suggested extraction increased 1.96% of PUFA and 0.58% of MUFA recovery.
•
FAMEs of suggested process proved 27.8% elevated energy output.
Declaration of interests ☐The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
No conflict of interest to declare
S0959-6526(19)34268-4
DOI:
https://doi.org/10.1016/j.jclepro.2019.119398
Reference:
JCLP 119398
To appear in:
Journal of Cleaner Production
Received Date: 4 July 2019 Revised Date:
2 November 2019
Accepted Date: 19 November 2019
Please cite this article as: Lakshmikandan M, Murugesan AG, Wang S, Abomohra AE-F, Jovita PA, Kiruthiga S, Sustainable biomass production under CO2 conditions and effective wet microalgae lipid extraction for biodiesel production, Journal of Cleaner Production (2019), doi: https://doi.org/10.1016/ j.jclepro.2019.119398. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Ltd.
Opt i mi z at i onofbi omas sandl i pi dpr oduc t i vi t y
EXTRACTI ON Pr e s s ur e 2kg/ c m2
Pr e he at e d( 60˚ C)gl as spl at e
Th i nl a y e ro f we t mi c r o a l g a l b i o ma s s
0. 04%
4%
Li pi d
8%
Pr e he at e d( 60˚ C)gl as spl at e
Dur i ngmi l dpr e s s ur eand he ats hoc kt r e at me nt
ds o i r pe o t Pho
Bi omas s
Ce l lr upt ur eandl i pi dbodi e sr e l e as e
1
Sustainable biomass production under CO2 conditions and effective wet microalgae
2
lipid extraction for biodiesel production
3
M. Lakshmikandan
4
Anjelin Jovita b, S. Kiruthiga b
5
a
6
b
7
Sundaranar University, Alwarkurichi 627 412, Tamil Nadu, India
8
c
a,b
, A.G. Murugesan b, Shuang Wang a*, Abd El-Fatah Abomohra
a,c*
, P.
School of Energy and Power Engineering, Jiangsu University, Jiangsu 212013, China Sri Paramakalyani Centre of Excellence in Environmental Sciences, Manonmaniam
Botany Department, Faculty of Science, Tanta University, Tanta 31527, Egypt
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
*Correspondence
24
[email protected](Shuang
25
Abomohra)
Wang);
[email protected](Abd
1
El-Fatah
26
Abstract
27
The freshwater microalga Chlorella vulgaris MSU AGM 14 were cultured at different CO2
28
conditions (up to 8%) with 100 µmol photons m-2 s-1 to evaluate biomass and lipid
29
productivity. For effective extraction of intra cellular lipids, a novel method based on mild
30
pressure (1-2.5 kg/cm2) with short period (5-15 min) of heat shock (50-70˚C) were studied
31
for the wet biomass. The transesterified lipids were qualitatively and quantitatively analysed
32
by using Gas Chromatography coupled with Mass Spectrometry (GC-MS). The higher CO2
33
aeration (8%) significantly increased the biomass productivity (23%) when compared with
34
control and CO2 (4%) aeration. Total lipid production (93%) acquired by conventional
35
extraction procedure showed enhanced production simultaneously. The maximum lipid
36
recovery (0.225g g-1 dw) was obtained at a pressure of 2 kg/cm2 and heating for 10 min at
37
60˚C. The transesterified lipids showed that oleic acid (C18:1 - 51.62%) was the main
38
component in both conventional and suggested lipid extraction process. The suggested
39
extraction process showed significant increase in biodiesel yield by 26.7%. The energy
40
outputs of biodiesel by conventional and suggested extraction process were 417.7 and 533.6
41
MJ ton-1, respectively. The overall results indicated that 8% of CO2 induced the biomass and
42
lipid productivity by 94% and 54.8%, respectively, when compared with control. In addition,
43
the suggested mild pressure with heat shock extraction process further enhanced the lipid
44
recovery by 21% which serves as a cost-effective lipid extraction process for microalgae.
45
46
47
Keywords: Biodiesel; Microalgae; Chlorella vulgaris; Mild pressure; Heat shock; Lipid
48
extraction
2
49
1. Introduction
50
Millions of years ago dead organic matter were been transformed into black gold by natural
51
events like prolonged period of time and pressurized geothermal process. So far, crude oil is
52
one of the most significant sources of energy on the planet. Since petroleum is a part of the
53
global economy with limited reservoirs (Hamilton, 2009; He et al., 2010); Jones et al. (2004),
54
experts are predicting that the end of petroleum era is near. But footsteps remain in the
55
environment due to toxic pollution leading to climate change (Prasad and Kumari, 1987;
56
Wang et al., 2017). The search for alternative fuel will tread on renewable energy resources
57
peculiarly without damaging the bionomical balance (Elsayed et al., 2018; Yuan et al., 2019).
58
Biodiesel gives some hope for renewable and clean energy with reduced carbon footprint.
59
Predominately, biodiesel is produced from plant oil (canola, sunflower, soybean and rapeseed
60
oil) by alkali-based transesterification process (Meher et al., 2006). But using food crops for
61
biodiesel production has never been the right solution for fuel crisis problems since they
62
interfere with food production for high grade arable land, initiating the food demand leading
63
to hike in the food price (Chisti, 2007; Lin et al., 2011; Silitonga et al., 2013). The true fact is
64
global oblige for food crops and products is awaited to double within 5 decades and in case of
65
transportation fuels it is still more worsened (Hill et al., 2006).
66
In such challenging situations, microalgae has attracted greater attentions for various forms of
67
biofuel production like biodiesel (Abinandan et al., 2019; Jain et al., 2019), bioethanol
68
(Sanchez Rizza et al., 2019), biohydrogen (Lakshmikandan and Murugesan, 2016a, b),
69
bioelectricity (Powell et al., 2009) and biomethane (Raheem et al., 2018), since microalgae
70
cultivation has positive environmental impact and doesn’t compete with food supplies.
71
Furthermore, microalgae possess important advantages like higher photosynthetic efficiency
72
leading to 10-50 times more significant CO2 mitigation when compared to terrestrial floras
73
(Anto et al., 2019; Mondal et al., 2017). Prominent quantity of biomass and lipid production 3
74
rate in oil bearing perennial crops like Jatropha, biomass productivity is about 4.1 ton ha-1 y-1
75
whereas microalgae owes to two fold higher capability with less land demand and efficient
76
non-edible oil source (Mathimani and Mallick, 2019; Mathimani et al., 2018). In addition,
77
higher growth rate on harsh environmental conditions and its ability to grow on wastewater
78
with various biorefinery applications are the significant features of microalgae (Abomohra et
79
al., 2017; Nascimento et al., 2014). At normal environmental conditions, microalgae can
80
accumulate appreciable quantity of lipids (structural lipids) but certain specific environmental
81
stress conditions will stimulate the elevated level of lipid accumulation in microalgae (Tu et
82
al., 2015; Widjaja et al., 2009). High CO2 concentration and length of photoperiod were
83
reported to play a significant role in elevated biomass recovery and lipid content of
84
microalgae (Che et al., 2019; Lv et al., 2010; Wahidin et al., 2013). From this phenomenon,
85
CO2 and photoperiod-based microalgal lipid enhancement has been investigated widely on
86
higher productivity and adaptive nature microalgal species.
87
In the current study, eukaryotic green microalga Chlorella vulgaris was investigated as it is
88
very commonly obtained in both fresh and seawater with high photosynthetic efficiency and
89
lipid productivity (Battah et al., 2015). The crucial characters are; they are strongly resistant
90
to any harsh conditions, and are invaders with high biomass productivity within a quick
91
period of time (Ebrahiminezhad et al., 2014; Frumento et al., 2013; Safi et al., 2014). The
92
crucial challenge of lipid extraction is selecting suitable cell disruption method to obtain
93
maximum productivity. Several cell disruption techniques have been employed to recover
94
lipids from the whole cells. Cell disruption techniques are grouped into mechanical and non-
95
mechanical process; mechanical extraction method includes bead milling, sonication, high
96
pressure homogenization, oil expeller, autoclaving, grinding and microwave techniques
97
(Günerken et al., 2015; Mathimani et al., 2017; Postma et al., 2015). The commonly used
98
non-mechanical extraction processes are chemical based Soxhlet, supercritical fluids and 4
99
enzymatic extractions. Peralta-Ruiz et al. (2013) reported that 85% total energy inputs of
100
biodiesel production has been shared by lipid extraction process itself. Conventional lipid
101
extraction process requires microalgal biomass dewatering before lipid extraction process and
102
85% of total energy input were exhausted by drying process (Patil et al., 2012). The crucial
103
part of cell disruption is to increase the disruption rates, minimize damage of targeted product
104
and lower the capital cost (Yap et al., 2015). High pressure based mechanical cell disruption
105
methods are promising techniques for complete disruption of cells (Carullo et al., 2018).
106
However, the high energy consumption, non-targeted intracellular compounds disruption and
107
difficult downstream process make it more complicated to the economic viability. The
108
applications of mild pressure in microalgae has been rarely investigated and are mainly
109
focused on encouraging the growth and lipid accumulation (Praveenkumar et al., 2016).
110
Lorenzen et al. (2017) reported that the significance of pressure (12 MPa) and heat (20˚C)
111
combination increased higher efficiency of lipid extraction from dried biomass. In the present
112
investigation mild pressure with heat shock were used to disrupt the wet cells of C. vulgaris
113
to maximise the lipid recovery and reduce energy consumption in pressure-based cell
114
disruption process.
115
With the above perspective in view, detailed investigations were carried out to optimize cell
116
growth and lipid productivity at higher CO2 concentrations and different photoperiod
117
conditions. The effect of wet cell disruption by novel method based on mild pressure and heat
118
shock treatment were then thoroughly investigated. The variations between conventional
119
solvent extraction and the suggested extraction were analysed by GC-MS. Finally, microalgal
120
cultivation, harvest cost, energy input and output of biodiesel from conventional and
121
suggested extraction process were evaluated.
122
2. Materials and Methods
5
123
2.1. Microalgal strain and inoculum preparation
124
Chlorella vulgaris MSU-AGM 14 (accession number KM189121) was obtained from
125
Department of Environmental Science, Sri Paramakalyani Centre of Excellence in
126
Environmental Sciences, Manonmaniam Sundaranar University, Tirunelveli, India. The
127
microalga was grown in the Algae Culture Broth (Hi-Media, Mumbai) composed of NaNO3,
128
1g; K2HPO4, 0.25g; MgSO47H2O, 0.513g; NH4Cl, 0.05g; CaCl22H2O, 0.02g; FeCl3, 0.003g
129
per liter of deionized water at pH of 7.0±0.2. Cultures were incubated at 27±1˚C with
130
continuous illumination (100 µmol photons m-2 s-1) and 4% CO2 aeration. When the cultures
131
reached the exponential phase, inoculum was introduced into 30 litres cylindrical photo
132
bioreactor for microalgal biomass accumulation. As per experimental setup, different
133
photoperiod [light (L): dark (D) cycles] (24L, 18L+6D and 12L+12D) and different CO2
134
supply (4% and 8%) were applied. Atmospheric air (with 0.04% CO2) was used as control.
135
Microalgal growth was monitored every 12 h by using spectrophotometer (Au-2701,
136
Systronics) at 680nm (OD680). After 12 days of incubation (stationary phase), cultures were
137
harvested by centrifugation (Eppendorf 5804R, Germany) for 10 min at 3000×g to obtain the
138
wet microalgal biomass. The cumulative biomass (g L-1 day-1) and lipid productivity (mg L-1
139
day-1) were calculated by using the following Eq. (1) Biomass / Lipid productivity = (DWt - DWo)/t
140
(1)
141
Where DWt and DWoare the final and initial dry weights of the sample, respectively; while t
142
is the time of harvest.
143
2.2. Cell disruption and lipid extraction
144
Approximately 11.2 g (adequate to 1g of dry weight) of harvested wet microalgal biomass
145
were placed between two preheated (50˚C, 60˚C and 70˚C) annealed glass plates (2 mm
6
146
thickness) at different pressured conditions (0.98, 1.47, 1.96 and 2.45 bar) and different
147
exposure times (5, 10 and 15 min). After mild pressure with heat shock treatment, lipids were
148
extracted by hexane: isopropanol (3:2 v/v) (Bian et al., 2018) and were kept in ice bath for 15
149
min. For control, 1g of dried microalgal sample was used for cell disruption and lipid
150
extraction process following the Bligh and Dyer (1959) method. The solvent extracts were
151
filtered by using Whatman filter paper no.1 and were allowed to evaporate to obtain the total
152
lipids which were stored at -20˚C in 1 mL chloroform for further analysis.
153
2.3.Transesterification of sample
154
In 10 mL screw caped glass tube, 25 mg of lipid sample was weighed and mixed with 1.5 mL
155
of 0.5 N methanolic NaOH (Nascimento et al., 2012). The solution was mixed gently, and
156
were sealed after nitrogen gas flushing, and were incubated in oven at 100˚C for 15 min.
157
After heat treatment, samples were allowed to cool at room temperature, followed by addition
158
of 2 mL of 12% boron trifluoride in methanol. Then the samples were sealed after nitrogen
159
gas flushing. To initiate a transesterification reaction, samples were placed at 100˚C for 45
160
min. After the transesterification, samples were allowed to cool at room temperature.
161
Approximately 2 mL of isooctane were added into each sample and were shaked vigorously
162
for 3 min. After mixing, the samples were immediately treated with 5 mL of saturated sodium
163
chloride solution followed by gentle agitation. After mild centrifugation the isooctane layer
164
was separated and were transferred into a clean glass tube and were stored at -20˚C for
165
further analysis.
166
2.4. Fatty acid analysis
167
The fatty acid composition of the prepared fatty acid methyl esters (FAMEs) were assessed
168
by Gas Chromatography (GC- Clarus 680 – Perkin Elmer) linked with Mass Spectrometry
169
(MS- Clarus SQ8T - Perkin Elmer) equipped with capillary column Elite-5 MS (30 m 7
170
length and 0.25 mm - internal diameter) and a flame ionization detector. Helium was used as
171
a carrier gas at a flow rate of 1.3 mL min-1. The electron ionization mode (70 eV) was
172
selected for mass spectrometry with full scan mode (range: 30–300 Da). The identification of
173
fatty acid components were based on their retention times, abundance and fragmentation
174
patterns comparison with NIST spectral library 2.0 g (2011).
175
2.5. Determination of biodiesel properties
176
Molecular characteristics of FAMEs describes the parameters of biodiesel quality such as
177
saponification value (SV), cetane number (CN), iodine value (IV), long chain saturated factor
178
(LCSF), cold filter plugging point (CFPP), allylic position equivalents (APE), bis- allylic
179
position equivalents (BAPE), kinematic viscosity (υ), density (ρ) and higher heating value
180
(HHV) (Islam et al., 2013; Talebi et al., 2013).
181
The empirical Eq. (2-4) was applied to calculate the SV, IV and CN depending on the FAME
182
profile. SV =
IV =
CN = 46.3 +
560 × ℎ 254 × ℎ
(2) ×
(3)
5458 − (0.225 × (&) (4) %&
183
Where Ni is the percentage of ith FAME, Di is the number of double bonds in ith FAME,
184
MW is the molecular weight.
8
185
The degree of unsaturation (DU) were calculated based on the mass fraction of mono
186
(MUFA) and poly unsaturated fatty acids (PUFA) by using the following Eq. (5). The LCSF,
187
CFPP, APE and BAPE were calculated by applying the following Eqs. (6-9) DU =
+,- + (2 × .+,-) (5)
/0%, = (0.1 × 016: 0) + (0.5 × 018: 0) + (1 × 020: 0 (6) 0,.. = (3.1417 × /0%,) − 16.477 (7) APE =
( 78 × -98 ) (8)
BAPE =
(;78 × -98 ) (9)
188
Where apn and bpn are the allylic and bis-allylic positions, respectively, in specific fatty acid;
189
while Acn is the mass percentage of each fatty acid.
190
FAMEs υ, ρ and HHV were calculated by using the following Eqs. (10-12) and final value of
191
each factors were obtained by summation of all FAMEs fuel properties. ln(υ? ) = −12.503 + 2.496 × ln( @? = 0.8463 +
4.9
AA&? = 46.19 −
?
?)
− 0.178 × (10)
+ 0.0118 × (11)
1794 ?
− 0.21 × (12)
192
Where Mi is the molecular weight; while N is thenumber of double bonds in ith fatty acid.
193
2.6. Statistical analysis
9
194
The results of means were calculated by using three replicates and were described as mean ±
195
standard deviation. The significance of differences (p < 0.05) were analyzed using one-way
196
analysis of variance (ANOVA) complied by least significant difference (LSD) using SPSS
197
(IBM, v.20).
198
3. Results and Discussion
199
3.1. Microalgal growth and lipid accumulation
200
C. vulgaris MSU AGM 14 were cultivated at two different percentages (4% and 8%) of CO2
201
aeration for 16 days and were monitored for cell growth and intracellular lipid accumulations.
202
While providing 8% aeration, cells have grown very well when compared to 4% and control
203
(Fig.1). Both samples reached their stationery phase on day 10. Chan et al. (2010) reported
204
that increasing CO2 aeration elevated the growth of microalgae in certain species such as B.
205
braunii, C. vulgaris and Scenedesmus sp. Similarly de Morais and Costa (2007) confirmed
206
that C. vulgaris can actively grow under high CO2 conditions. The microalgal production and
207
productivity (Fig. 2) also evidenced enhanced production of biomass and lipids at elevated
208
CO2 aeration when compared with control (0.04%). The cultures with 8% CO2 aeration
209
produced 0.79±0.04 g L-1 of biomass and the productivity indicated a maximum significant
210
amount of 0.064±0.003 g L-1 day-1 which represented 23% higher than that of the control.
211
Whereas lipid production recorded 186±11 mg g-1 and productivity reached to 11.89±0.48 mg
212
L-1 day-1, representing 93.96% significant increase over the control (Fig. 2b). In the present
213
study, the effect of photoperiod and CO2 aeration on C. vulgaris MSU AGM 14 biomass and
214
lipid accumulation were also evaluated (Table 1). The maximum biomass (1.44±0.02 g L-1
215
dw) were observed at 24 h illumination with 8% CO2 aeration, with total lipids of 0.18±0.02
216
g g-1. Whereas at 12 h L+12 h D illumination with 8% CO2, maximum lipid content of
217
0.2±0.02 g g-1 were recorded with dry weight of 1.37±0.03 g L-1. These results revealed that
218
microalgae require both light and dark conditions for effective lipid production. In addition, 10
219
increased CO2 aeration proved to be one of the important factors for the elevation of lipid
220
accumulation in microalgae. Previously, Chan et al. (2010) reported that the total lipid
221
content of C. vulgaris were 11.92% of the dry weight, while the present study revealed that at
222
suitable photoperiod with elevated CO2 condition C. vulgaris MSU AGM 14 has the potential
223
to produce 20% of total lipids. The present investigation clearly represents that CO2 condition
224
and photoperiod has significant impact on biomass and lipid accumulation of microalgae.
225
3.2. Mild pressure with heat shock treatment
226
Effective extraction of lipids from microalgal cells depends upon cell disruption technique
227
and selection of extraction process (Halim et al., 2012; Prabakaran and Ravindran, 2011).
228
There are several cell disruption techniques employed to extract intracellular lipids from
229
microalgal cells. Previously, some research works explained significant advantages of mild
230
pressure treatment on lipid accumulation during growth. Praveenkumar et al. (2016) reported
231
that mild pressure treatment (10-15 bar) induces accumulation of neutral lipid in Chlorella
232
sp., and few reports revealed that pressure tolerance ability differs from strain to strain.
233
Seckbach (1971) reported that microalga Cyanidium caldarium can tolerate mild (1- 10 bar)
234
pressures for 19 days at room temperature. While elevating the pressure significantly, growth
235
has been inhibited with change in pigmentation. In addition, several studies revealed that high
236
pressure can inhibit the growth or cause lethal damage to cells by affecting the normal
237
functions of cell organelles (Mañas and Mackey, 2004; Robey et al., 2001). However, there is
238
a gap in literature about the effect of pressure combined with heat shock on lipid extraction.
239
In this study, wet thin layered C. vulgaris MSU AGM 14 was disrupted by mild pressure
240
along with heat shock. The designed experiment showed good response in extracting the lipid
241
content from the selected strain (Fig. 3). Results confirmed that 2 kg/cm2 (1.96 bar) pressure
242
and 60˚C heat treatment with 10 min exposure time proved maximum effective extraction of
243
0.225±0.013 g g-1 total lipid. Overall, experiment demonstrated that exposure time of 11
244
microalgae in selected temperature is more important for effective extraction. When
245
compared with control, the present study showed 12.5% additional recovery of total lipid.
246
3.3.Composition of biodiesel
247
The successful conversion of total lipids into FAME was examined by GC-MS (Table 2).
248
Three most abundant methyl esters were determined in the biodiesel of the selected strain.
249
Among them octadecenoic acid methyl ester (oleic acid) showed the highest relative content
250
in the pressurised sample (51.62%) when compared to other methyl esters. The other two
251
methyl esters such as hexadecenoic acid methyl esters (14.53%) and octadecanoic acid
252
methyl ester (12.08%) possessed considerable relative contents in the biodiesel. Similarly,
253
Gao et al. (2010) reported that biodiesel of Chlorella protothecoides showed higher relative
254
content for these three methyl esters. Mathimani et al. (2015) also reported that palmitic,
255
oleic and linolenic acids are the major methyl esters of Chlorella sp. BDUG 91771biodiesel.
256
The suggested lipid extraction process evidenced no significant differences were reported in
257
the SFAs, MUFAs and PUFAs ratios with the control. The good quality biodiesel possesses
258
certain significant properties like heat of combustion, ignition quality, viscosity, cold filter
259
plugging point, lubricity and importantly oxidative stability which are defined by the
260
structure of its FAME components. The productive amount of oleic acid content represents its
261
enhanced oxidative stability for longer storage (Knothe, 2005). In addition, higher oxidizing
262
tendency of biodiesel was evaluated by the presence of degree of unsaturation on FAMEs
263
(Hoekman et al., 2012). Ramos et al. (2009) also described that higher degree of unsaturation
264
(>137) does not meet the European biodiesel standards. But in the present study, biodiesel of
265
C. vulgaris MSU AGM 14 showed within the limit (86.13 and 86.71) of unsaturation. Allylic
266
positions of biodiesel plays a crucial role in autoxidation/oxidation of unsaturated FAME.
267
The linear relationship of oxidizability on bis- allylic position equivalents and allylic position
268
equivalents has capablity to induce non-uniform oxidation in biodiesel (Iyer, 2017). In this 12
269
study, biodiesel of C. vulgaris MSU AGM 14 showed the presence of lower value allylic
270
position equivalents and bis- allylic position equivalents. These calculated allytic position
271
results have evidenced that FAME composition of biodiesel possess reasonably good
272
oxidation stability. The combustion behaviour of biodiesel has been estimated based on
273
cetane number (Koley et al., 2018). Depending upon fuel cetane number, engines ignition
274
delay time may vary, shorter ignition time has been recorded in higher cetane numbered fuel
275
(Islam et al., 2013). Overall, the suggested lipid extraction process showed slight neglectable
276
changes in all studied parameters with those of the control (Table 3).The resemblance
277
analyses with standard biodiesel EN14214 and ASTM D6751-02 indicated that higher cetane
278
number and moderate quantity on degree of unsaturation. Higher viscosity and density of
279
biodiesel may create many problems like fuel pump failure, carbon deposit, ring sticking and
280
poor atomization. Whereas, density values of biodiesel were within the range of standard
281
while the kinetic viscosity were slightly below the range. As reported by Yang et al. (2014)
282
suitable amount of viscosity and density was observed in higher concentrations of oleic,
283
palmitoleic and palmitic acid containing biodiesel. The determination of FAME derived HHV
284
(39.22 and 39.46MJ kg-1) showed 14% less than the conventional petroleum derived diesel
285
(46MJ kg-1). Thus, further studies are required to improve the biodiesel quality with respect
286
to the HHV of C. vulgaris lipids.
287
3.4. Economic assessment
288
The economic feasibility of the present study was evaluated following Wang et al. (2019)
289
energy calculations. The system was designed to cultivate 30L of algal culture with energy
290
consumption of 77.65 kWh per cycle. Totally 24 cycles were performed to acquire 1 kg of the
291
dry biomass by utilizing 1863.72 kWh kg-1 of input energy and the estimated cost of expenses
292
was 140.27 USD including chemicals used for cultivation purpose (Fig. 4). In addition,
293
microalgae harvest required 438.3 kWh kg-1 of energy with 26.38 USD kg-1 of dry biomass 13
294
recovery. Whereas extraction of lipids recorded that the suggested process required 13.7%
295
more expense to perform when compared to conventional method. However, almost 13% of
296
the effective lipid yield and elevated total FAMEs level carried out the attention to economic
297
significance. In addition, energy outputs of conventional and suggested methods were
298
calculated based on calorific value (HHV) of FAME and were recorded as 417.7 and 533.6
299
MJ ton-1 respectively. Despite higher energy consumption of the suggested extraction
300
method, it showed 27.7% higher energy output than the conventional extraction method. In
301
addition, the possible improvement in cultivation and harvesting process will encounter
302
higher cost and energy consumption in economical point of view. The large-scale cultivation
303
system and low energy consuming harvesting process like flocculation and bio-flocculation
304
allows wide possibility for feasible biodiesel production.
305
Conclusion
306
C. vulgaris MSU AGM 14 lipid production has been promoted by simultaneous increase of
307
biomass by higher CO2 conditions of microalgae. The increased amount of total lipid yield
308
(22.5 %) has been extracted by employing mild pressure and heat shock treatment. FAMEs
309
analysis of C. vulgaris MSU AGM 14 lipid evidenced the presence of high content of
310
monounsaturated and saturated fatty acids. In addition, degree of unsaturation, iodine value,
311
cold filter plugging point, kinematic viscosity and density of biodiesel strengthened the
312
appropriate characteristics of fuel standards. The overall results revealed that C. vulgaris
313
MSU AGM 14 is a potential strain for biodiesel production. The novelty of this study reveals
314
that mild pressure and heat shock cell disruption evidenced 27.7% elevated energy output,
315
which could reduce the total cost of energy consumption and increase the lipid recovery from
316
the selected strain.
317
Acknowledgement
14
318
The authors sincerely acknowledge National Natural Science Foundation of China (No.
319
51676091), China Postdoctoral Science Foundation (2019T120408, 2018M630529),
320
Foundation of State Key Laboratory of Coal Combustion (FSKLCCA1904), the Six talent
321
peaks project in Jiangsu Province (XNY-007, 2018) for the financial support. Authors also
322
thank Jiangsu University, Zhenjiang, China and Sri Paramakalyani Centre of Excellence in
323
Environmental Sciences, Manonmaniam Sundaranar University for providing facilities
324
required to carry out this work.
325
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Figures and tables captions
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Figure 1: Growth curve of Chlorella vulgaris at different percentages of CO2 with confocal
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microscopy images stained with Nile red to visualize the intracellular lipids.
510
Figure 2: Microalgal biomass and total lipid production (a) and productivity (b) of C.
511
vulgaris MSU AGM 14 at different percentages of CO2. The same series with the same letter
512
showed insignificant difference (at P<0.05).
22
513
Figure 3: Optimized extraction of total lipids from C. vulgaris MSU AGM 14 by suggested
514
mild pressure and heat treated method. The horizontal dashed line represents the lipid
515
recovery by the conventional extraction method.
516
Figure 4: Cultivation, harvest and extraction process cost and energy output for biodiesel
517
produced from microalgae C. vulgaris MSU AGM 14.
518
Table 1: Selection of suitable photoperiod lengths for optimizing the growth and lipid
519
productivity.
520
Table 2: Composition of biodiesel produced from different lipid extraction process.
521
Table 3: Resemblance between calculated properties of conventional and suggested
522
extraction processed biodiesel with biodiesel standards.
23
TABLES
Table 1. Selection of suitable photoperiod lengths for optimizing the growth and lipid productivity. Illumination time
Biomass CO2
(Hrs at 100 µmol -2 -1
photons m s )
18L+6D
12L+12D
24L
dry
Total lipid
weight
(gg-1 dw)
-1
Biomass
Lipid
productivity (gL-
Productivity (gL-
1
day-1)
1
day-1)
(%)
(gL )
Control
0.66±0.02a
0.12±0.02a
0.052±0.003a
0.006±0.002a
4%
0.69±0.05a
0.15±0.04ab
0.054±0.004ab
0.008±0.003a
8%
0.79±0.04b
0.19±0.02b
0.064±0.003b
0.012±0.002b
Control
0.49±0.05a
0.14±0.03a
0.038±.004a
0.005±.002a
4%
0.93±0.02b
0.17±0.05ab
0.075±.003b
0.013±.004b
8%
1.37±0.03c
0.2±0.02b
0.111±.002c
0.022±.003c
Control
0.38±0.05a
0.13±0.02a
0.029±.002a
0.004±.002a
4%
0.91±0.02b
0.16±0.04ab
0.074±.003b
0.012±.003b
8%
1.44±0.02c
0.18±0.02b
0.117±.003c
0.021±.003c
L- Light; D- Dark, values are mean ± SD, n=3. The same parameter at the same light regime with the same letter showed insignificant difference (at P<0.05).
Table 2. Composition of biodiesel produced from different lipid extraction process. Fatty Acid Methyl Esters
Relative content Control
Mild pressure and heat treated
Myristic acid (C14:0)
-
1.40
Palmitic acid (C16:0)
7.82
6.78
Palmitoleic acid (C16:1n-7)
15.11
14.53
Stearic acid (C18:0)
12.57
12.08
Oleic acid (C18:1n-9)
50.66
51.62
α - Linolenic acid (C18:3n-3)
10.18
10.24
γ - Linolenic acid (C18:3n-6)
-
0.04
Arachidic acid (C20:0)
2.98
3.27
Saturated fatty acids (%)
23.37
23.53
Monounsaturated fatty acids (%)
65.77
66.15ns
Polyunsaturated fatty acids (%)
10.18
10.28ns
Total FAMEs (%)
99.32
99.96ns
Unidentified (%)
0.68
0.04
ns
Showed insignificant difference with the corresponding control (at P<0.05).
Table 3.Resemblance between calculated properties of conventional and suggested extraction processed biodiesel with biodiesel standards. Biodiesel standards Properties
ASTM D6751-02
Degree of unsaturation
Mild pressure Conventional
EN 14214
and heat treated
-
-
86.13
86.71
-
-
201.48
203.04
Iodine value (I2100g )
NA
≤120
88.50
89.06
Cetane number
≥47
≥51
53.48
53.14
-
-
10.05
9.99
NA
≤5/≤-20
15.1
14.91
-
-
71.02
72.18
-
-
26.32
27.10
-
≤12
10.18
10.28
1.9-6.0
3.5-5.0
1.35
1.35
Density (g cm-3)
NA
0.86-0.90
0.86
0.87
Higher heating value (MJ kg-1)
NA
NA
39.22
39.46
-1
Saponification value (mg KOHg ) -1
Long chain saturated factor Cold filter plugging point (ºC) Allylic position equivalents (APE) Bis- allylic position equivalents (BAPE) C18:3 (%) Kinematic viscosity (mm2s-1)
FIGURES
Figure 1. Growth curve of Chlorella vulgaris at different percentages of CO2.
Figure 2. Microalgal biomass and total lipid production (a) and productivity (b) of C. vulgaris MSU AGM 14 at different percentages of CO2. The same series with the same letter showed insignificant difference (at P<0.05).
0.25
5 min 15 min
10 min
-1
Lipid recovery (g g dw)
0.20
0.15
0.10
0.05
o
70C +C N 70C +1 70C +1. 5 70C +2 70C +2. 5
60C +C N 60C +1 60C +1. 5 60C +2 60C +2. 5
50C +C N 50C +1 50C +1. 5 50C +2 50C +2. 5
0.00
2
Temperature ( C) + Pressure (kg/cm ) Figure 3. Optimized extraction of total lipids from C. vulgaris MSU AGM 14 by suggested mild pressure and heat-treated method. The horizontal dashed line represents the lipid recovery by the conventional extraction method.
Figure 4.Cultivation, harvest and extraction process cost and energy output for biodiesel produced from microalgae C. vulgaris MSU AGM 14.
Highlights: •
Elevated CO2 (8%) promoted biomass (23%) and lipid productivity (94%).
•
Mild pressure with heat shock evidenced 12.5% additional recovery of total lipids.
•
Suggested extraction increased 1.96% of PUFA and 0.58% of MUFA recovery.
•
FAMEs of suggested process proved 27.8% elevated energy output.
Declaration of interests ☐The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
No conflict of interest to declare