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Biophotonic perception on Desmodesmus sp. VIT growth, lipid and carbohydrate content
Accepted Manuscript Biophotonic perception on Desmodesmus sp. VIT growth, lipid and carbohydrate content Sriram Srinivasan, Seenivasan Ramasubbu PII: DOI: Reference:
S0960-8524(15)01344-9 http://dx.doi.org/10.1016/j.biortech.2015.09.065 BITE 15574
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Bioresource Technology
Received Date: Revised Date: Accepted Date:
23 July 2015 16 September 2015 18 September 2015
Please cite this article as: Srinivasan, S., Ramasubbu, S., Biophotonic perception on Desmodesmus sp. VIT growth, lipid and carbohydrate content, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech. 2015.09.065
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Biophotonic perception on Desmodesmus sp. VIT growth, lipid and carbohydrate
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content
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SRIRAM Srinivasan, SEENIVASAN Ramasubbu*
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School of Bio Sciences and Technology, VIT University, Vellore – 632014, Tamil
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Nadu, India.
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*Corresponding author
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Dr. R. Seenivasan,
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Associate professor,
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School of Biosciences and Technology,
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VIT University,
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Vellore – 632014.
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Tamil Nadu,
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India.
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Telephone: +91-416-2202131
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Email: [email protected]
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1 2
Abstract Constant and fluctuating light intensity significantly affects the growth and
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biochemical composition of microalgae and it is essential to identify
4
suitableillumination conditions for commercialmicroalgae biofuel production. In the
5
present study, effects of light intensities, light: dark cycles, incremental light intensity
6
strategies and fluctuating light intensities simulating different sky conditions in indoor
7
photobioreactoron Desmodesmus sp. VIT growth, lipid and carbohydrate content were
8
analysed in batch culture.The results revealed that Desmodesmus sp. VIT obtained
9
maximum lipid content (22.5%) and biomass production (1.033 g/L) under incremental
10
light intensity strategy. The highest carbohydrate content of 25.4% was observed under
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constant light intensity of 16,000 lx and 16:08 h light: dark cycle. The maximum
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biomass productivity of Desmodesmus sp. VIT (53.38 mg/L/d) was occurred under
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fluctuating light intensity simulating intermediate overcastsky condition.
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Keywords: Desmodesmussp. VIT; Light; Lipid; Carbohydrate; Photobioreactor.
16 17 18 19 20 21 22 2
1
1. Introduction
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Commercial production of biofuel for transportation (biodiesel andbioethanol)
3
from soybean, sugarcane, corn, and wheat faced severe setback due to excessive land
4
use and competition with global food supply, whereasbiofuel production from
5
lignocellulosic biomass and waste cooking oil desires high initial investment and
6
improvement in extraction technologies. Alternative to crop based feedstocks,
7
microscopic photosynthetic organisms called microalgae wereconsidered as sustainable
8
raw material for biofuel production (lipid rich microalgaefor biodiesel production and
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carbohydrate rich algaefor bioethanol production). Hurdles in large scale microalgae
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biofuel production are costly upstream process and high energy consuming downstream
11
process (Rawat et al., 2013).
12
Manipulation of numerous factors such as nitrogen, salt, pH, phosphorus
13
(chemical) and light (physical) could alter the growth and biochemical composition of
14
microalgae (George et al., 2014). Among those factors, light as a primary energy source
15
for photoautotrophs have significantimpact on microalgae growth, biochemical
16
composition and cost of commercial indoor biofuel production (He at al., 2015b).
17
Supplying constant light intensity for entire cultivation cycle is themost common light
18
supplystrategy used for commercial microalgae cultivation in indoor photobioreactor.
19
However, microalgae light requirements in batch cultivation may differ for each growth
20
phase (Han et al., 2015). For example, microalgae requireless light intensity at initial lag
21
phase to avoid photoinhibitionand on the other hand, high light intensity at exponential
22
phase to prevent cell shading (photolimitation) in batch culture (Lee et al., 2006). It is
23
essential to use appropriate light intensity for each growth phase or incremental light
24
intensity strategy as proposed by Yan et al. (2013) to improve algal growth with less
25
power consumption in indoor cultivation. 3
1
Incase of outdoor cultivation, microalgae utilize sunlight as energy source to
2
carry out photochemical reaction for protein, carbohydrate and lipid synthesis.
3
Fluctuating light intensity, temperature, and pH in outdoor environment significantly
4
affects the microalgae growth. Biomass productivity of Chlorella ellipsoidea under
5
controlled environmental parameters in indoor cultivation was higher than the biomass
6
productivity occurred under uncontrolled outdoor condition (Wang et al.,
7
2014).Outdoor environmental factors such as light, temperature and pH are greatly
8
affected by weather condition. Among those factors, light intensity variation was largely
9
influenced by weather (Xia et al., 2014). Feng et al. (2011) reported that sky conditions
10
(cloudy sky in autumn resulted in less irradiance and less cloudy days in spring caused
11
slightly high irradiance) observed in spring and autumn greatly affected the cell
12
concentration of Chlorella zofingiensis in respective seasons. Hence, it is very important
13
to study microalgae biomass and low value products (lipid and carbohydrate)
14
production in fluctuating light intensities under different sky conditions (sunny sky,
15
intermediate overcast and quasi overcast). Tamburic et al. (2014) reported that
16
simulating the fluctuating light intensities of anticipated location in indoor
17
photobioreactor would be the cost effective way to carry out preliminary screening to
18
analyse the feasibility of microalgae growth in changing light and temperaturecompared
19
to costly outdoor field trails.
20
Effect of different light intensities and photoperiod regimes onmicroalgae
21
growth and biochemical composition was studied elaborately. However, no reports
22
regarding the impact of incremental light intensity strategy and light/dark photoperiod
23
regimeson Desmodesmus sp. lipid and carbohydrate content was reported. The current
24
study was carried out to analyze the effect of different light intensities, photoperiod
25
regimes, incremental light intensity strategies and fluctuating light intensities simulating 4
1
sunny sky, intermediate overcast and quasi overcast sky conditions in indoor
2
photobioreactor on Desmodesmus sp. VIT biomass production, lipid and carbohydrate
3
content.
4
2. Materials and Methods
5
2.1. Microalgae isolation
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Microalga was isolated from the freshwater pond in VIT University, Vellore,
7
India (12.9692° N, 79.1559° E). Algal enrichment culture was prepared by inoculating 5
8
mL of water sample into 95 mL sterilized Bold Basal Medium (BBM) with pH 6.86 and
9
illuminated under cool white fluorescent tube light at 4000 lx light intensity under 12:12
10
h light/dark photoperiod regime at room temperature. Axenic single clone was purified
11
from the mixed algae culture using serial dilution, spread plate and streak plate
12
techniques.
13
2.2. Molecular identification of microalgae
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Algal DNA was isolated by modified CTAB method of Varela-alvarezet al.
15
(2007). Exponentially growing algal cells were centrifuged at 8,000 rpm for 10 min to
16
concentrate the cells from the liquid medium. Algal pellet was homogenized in 500 µl
17
of prewarmed lysis buffer (2% CTAB, 1 M NaCl, 100mM Tris HCl (pH 8.0), 20mM
18
EDTA (pH 8.0), 100 µg/mL proteinase K and 2% β- mercaptoethanol) and incubated at
19
60 ºC for 50 min. After cell lysis, 500 µl of phenol:chloroform:isoamyl alcohol
20
(25:24:1) was added, vortexed for 10 min andcentrifuged at 13,000 rpm for 20 min at 25
21
ºC to separate the DNA. After centrifugation, upper aqueous phase containing DNA was
22
transferred to the new tube and two volumes of ice cold isopropanol was added and
23
incubated at -20 ºC for 20 min followed by centrifugation at 13,000 rpm for 20 min at
5
1
25 ºC to precipitate the DNA. 70% ethanol wash was given to the DNA pellet and Tris-
2
EDTA was used to dissolve the DNA for PCR reaction. The PCR amplification of the
3
DNA was carried out as per Dahlkild et al. (2001) protocol.
4
2.3. Polyethylene bag photobioreactor (PBR)
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2 L transparent polyethylene bag (30 X 25.5 cm) was used as photobioreactor
6
for culturing microalgae. The bag reactor was sterilized by autoclaving at 121 ºC for 20
7
min. Bottom end of the polyethylene bag was heat sealed and the upper end was tied
8
using nylon tag after the addition of cell suspension (600 mL working volume). Air was
9
bubbled into the photobioreactor from aerator through 0.2 µm sterile filter. Constant
10
aeration was maintained throughout the batch cultivation. In addition to aeration port,
11
gas exchange port and sampling port were present in culture bags.
12
2.4. Experimental design
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2.4.1. Effect of light intensityand photoperiod regimeon growth, lipid and carbohydrate
14
content
15
Microalgae growing in late exponential phase were inoculated into polyethylene
16
bag photobioreactor (PBR) containing 600 mL of modified BBM medium containing
17
NaNO3 1 g/L. Initial concentration of biomass in PBR at day 0 was set to 0.020 g/L.
18
Different light intensities viz.,8,000 lx, 16,000 lx and 32,000 lx were illuminated on the
19
surface of PBR for 24:00 h, 20:04 h, 16:08 h and 12:12 h light: dark cycles by adjusting
20
the distance between light source and PBR for establishing the respective light intensity.
21
Fluorescent tube light was manually switched on/off at required timeintervals for
22
achieving corresponding light: dark cycles. The light intensity on the surface of the
6
1
PBR was measured using Lux meter (Sigma Instruments). Each experiment was
2
performed in duplicate at room temperature for 15 days.
3
2.4.2. Effect of fluctuating light intensity on growth, lipid and carbohydrate content
4
Daily light intensity fluctuation for 12 days under three different sky conditions
5
of Lyon, France was simulated in indoor PBR by manually increasing or decreasing the
6
distance between the fluorescent tube light and PBR on hourly basis. Daily light
7
intensity was increased from 10,000 lx to 90,000 lx in midday and then gradually
8
decreased to 10,000 lx at the end of 12 h light cycle for replicating sunny sky condition
9
in indoor PBR. Similarly, for replicating intermediate overcast sky condition, light
10
intensity was increased from 5,000 lx to 60,000 lx in midday and then gradually
11
decreased to 5,000 lx at the end of the light cycle and for quasi overcast condition,
12
maximum of 20,000 lx light intensity was maintained in midday. The light: dark cycle
13
of about 12:12 h was maintained for different fluctuating light intensities and
14
experiments were performed in duplicate at room temperature in modified BBM
15
(NaNO3 1 g/L). The irradiance data for different sky conditions of Lyon, France was
16
obtained from Satel-Light database.
17
2.4.3. Incremental light intensity strategy (ILIS)
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Fifteen days of microalgae batch cultivation was divided into two phase. Phase I
19
(1-6 days) is of first six days which includes lag and mid-exponential phase and phase II
20
includes late exponential and stationary phase of batch cultivation cycle (7-15 days). All
21
the experiments were performed in duplicates in modified BBM.
22
Control (Constant light intensity): Cultures were illuminated under 8,000 lx light
23
intensityand 16:08 h light: dark cyclefor 15 days (Phase I and Phase II).
7
1
Strategy I (ILIS-1): Cultures were illuminated under 8,000 lx light intensity for first six
2
days (Phase I) and from 7th day onwards light intensity was increased from 8,000 lx to
3
16,000 lx light intensity (Phase II).
4
Strategy II (ILIS-2): Cultures were illuminated under 8,000 lx light intensity for first six
5
days (Phase I) and then light intensity was increased to 32,000 lx light intensity (Phase
6
II).
7
2.5. Analytical methods
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2.5.1. Dry cell weight and biomass productivity determination
9
Cells retained on preweighed whatman glass fiber filter paper (47 mm x 0.45
10
µm) after filtering 10 mL of algae culture (OD683 = 1.00) was dried in hot air oven at
11
105 ºC for 24 h and weighed again after cooling to room temperature. The difference
12
between the weight of the blank filter and filter loaded with cells provided the dry cell
13
weight of Desmodesmus sp. VIT. The experiment was performed in triplicate.
14
To determine the dry cell weight of algae cultured in PBR, 2 mL cell suspension
15
from photobioreactor was collected and optical density was measured at 683 nm
16
(OD683) using spectrophotometer (U.V 1800, Shimadzu, Japan). The absorbance at 683
17
nm was converted to dry cell weight through predetermined conversion factor (1 OD683
18
= 0.400 g DCW/L).
19
Biomass Productivity (P) was obtained by P (mg/L/d) = (X1 - X0)/(t1-t0). X1and X0 are
20
the microalgae dry cell weight (mg/L) at time t1and t0.
21
2.5.2. Lipid extraction
8
1
Lipid was extracted from algal biomass using modified method of Folch et al.
2
(1956). Briefly, freeze dried algae was extracted with 4 mL of chloroform: methanol
3
(2:1,v/v) for 1 h at room temperature with occasional vortexing. The cell debris was
4
separated from the supernatant (solvent) by centrifugation at 7,000 rpm for 7 min. Then
5
0.2 volumes of water was added to the supernatant and centrifuged at 2,500 rpm for 10
6
min to extract lipid into chloroform phase and non-lipid content into aqueous methanol
7
phase. The top aqueous phase was carefully removed and the bottom chloroform phase
8
containing lipid was evaporated at 60ºC to gravimetrically weigh the lipid content. The
9
lipid content was reported as percent of dry cell weight (DCW).
10 11
2.5.3. Total carbohydrate determination Carbohydrate determination in microalgae was carried out based on the modified
12
method of Wychen and Laurens (2013). Freeze dried algal biomass was initially
13
hydrolysed with 500 µl of 72% (w/w) sulphuric acid for 1 h at 30ºC in water bath for
14
primary hydrolysis and then deionised water was added to decrease the acid
15
concentration to 4% (w/w) and autoclaved at 121ºC for 20 min for secondary
16
hydrolysis. The carbohydrate content present in the hydrolysate was determined
17
according to Dubois et al. (1956) using glucose as standard. Carbohydrate content was
18
reported as percent of dry cell weight (DCW).
19
2.5.4. Determination of nitrate concentration
20
The nitrate concentration in the medium was determined using calorimetric
21
method (Xie et al., 2013). The cell suspension was collected from PBR and filtered
22
through whatman glass fiber filter paper. The filtrate was diluted and the optical density
23
of the filtrate at 220 nm (OD220) was measured using spectrophotometer. Sodium nitrate
24
was used as standard. 9
1 2
2.6. Statistical analysis The results were expressed as mean ± standard deviation. The values were
3
analysed by one-way ANNOVA using GraphPad Prism. The statistically significant
4
variation was considered at the level of p< 0.05.
5
3. Results and Discussion
6
3.1. Identification of microalgae
7
The phylogenetic position shown in the phylogenetic tree indicates that the
8
microalgae isolate is closely related to Desmodesmussp.S7 with 99% sequence
9
similarity (Fig. 1). The sequence was submitted to GENBANK (Accession number:
10
KP720618) and was designated as Desmodesmus sp. VIT.
11 12
3.2. Biomass production of Desmodesmus sp. VIT cultured at different light intensities
13
and photoperiod regimes
14
Viability of photoautotrophic microalgae in photobioreactor largely depends on
15
light quality, duration and quantity used for culturing microalgae. Supplying favourable
16
light intensity and light: dark cycle is highly essential for energy efficient biomass and
17
biofuel production in commercial indoor microalgae cultivation.
18
Dry cell weight (DCW) of Desmodesmus sp. VIT cultured under four different
19
photoperiod regimes (12:12 h, 16:08 h, 20:04 h and 24:00 h light: dark cycles) at three
20
different light intensities (8,000 lx, 16,000 lx and 32,000 lx) for 15 days was illustrated
21
in Fig.2A. In the current study, maximum biomass production was observed incultures
22
exposed to16:08 h light: dark cycle at 8,000 lx, 16,000lx and 32,000 lx light intensities.
23
The decrease in biomass production was observed when the cultures were exposed to
10
1
20:04 h, 24:00 hand 12:12 h light: dark cyclesat three different light intensities.The
2
cultures exposed to 12:12 h light: dark cycle yielded significantly lower biomass
3
compared to cells under 16:08 h light: dark cycles at16,000 lx and 32,000 lx light
4
intensities. In the case of biomass productivity, cells grown under 16:08 h light: dark
5
cycle obtained highest biomass productivity, whereas cells grown under 12:12 h light:
6
dark cycle yielded lowest biomass productivity at 16,000 lx and 32,000 lx light
7
intensities (Table 1). The results obtained in present studyhave confirmed that 6:08 h
8
light: dark cycle as the optimum photoperiod regime for Desmodesmus sp. VIT growth
9
at 8,000 lx, 16,000 lx and 32,000 lx light intensities. The growth of microalgae under
10
different light intensities is the sum of damage and repair mechanism. Continuous
11
illumination leads to continuous damage to photosystem II, whereas introducing short
12
dark period (light: dark cycles) might reduce the light induced damage to photosystem
13
II and enhance the growth in the next light period. This phenomenon might be the major
14
reason for the maximum biomass production attained under 16:08 h light: dark
15
cycle.The results of the current study are in accordance with Ji et al. (2013) report, who
16
suggested 14:10 h light: dark cycle as optimum photoperiod regime for Desmodesmus
17
sp. EJ15-2 biomass production.On the contrary, Wahidin et al. (2013) reported that
18
Nannochloropsis sp. illuminated under high light intensity for shorter photoperiod
19
produced maximum biomass compared to cultures illuminated at high light intensity for
20
longer photoperiod.
21
Light quantity significantly affects the growth of microalgae through
22
photoinhibition (excessive light intensity) or photolimitation (less light intensity) (Ho et
23
al., 2012).The dry cell weight of Desmodesmus sp. VIT increases with increase in light
24
intensity from 8,000 lx to 32,000 lx under four different light: dark cycles(12:12 h,
25
16:08 h, 20: 04 h and 24:00 h ) (Fig. 2A). In addition, biomass productivity of 11
1
Desmodesmus sp. VIT increased with increase in light intensity under four different
2
photoperiod regimes (Table 1). Xie et al. (2013); George et al. (2014) reports also stated
3
that Desmodesmus sp. F51 (150 – 750 µmol m-2 s-1) and Ankistrodesmus falcatus (30 –
4
150 µmol m-2 s-1) biomass production increases with increase in light intensity. In
5
contrast, Desmodesmus sp. EJ15-2 biomass production increases with increase in light
6
intensity upto 80 µmol m-2 s-1, further increase in light intensity reduced the biomass
7
production (Ji et al., 2013). The maximum biomass production of Desmodesmus sp.
8
VIT (0.999 g/L) obtained at 32,000 lx light intensity under 16:08 h light: dark cycle in
9
the present study was higher than the biomass production of Desmodesmus sp. EJ15-2
10
(0.569 g/L), Ankistrodesmus falcatus (0.224 g/L) (Ji et al., 2013 and George et al.,
11
2014) and comparable to Chlorella sp. (1.26 g/L) and Phaeodactylum tricornutum
12
(1.212 g/L) biomass production (Nogueira et al., 2015; Guo et al., 2015). Whereas,
13
Desmodesmus sp. F2 biomass production was higher than the present study due to
14
higher inoculum size and carbon dioxide (2.5%) used by Ho et al. (2014) for cultivation.
15
3.3. Biomass productivity of Desmodesmus sp. VIT under fluctuating light intensities
16
simulating different sky conditions
17
Microalgae tolerance to fluctuating light intensities and temperature variations
18
under different sky conditions are essential characteristics required for biofuel
19
production using sunlight. Feng et al. (2011) reported that the biomass productivity of
20
C. zofingiensis was higher in spring (intermediate clouds) than in autumn (cloudy sky).
21
Similarly, maximum biomass productivity (53.3 mg/L/d) of Desmodesmus sp. VIT was
22
obtained under fluctuating light intensities simulating intermediate overcast sky
23
condition in indoor photobioreactor. The biomass productivity was decreased to 49.8
24
mg/L/d and 47.6 mg/L/d under fluctuating light conditions replicating quasi overcast
25
and sunny sky conditions. The high light intensity (90,000 lx) maintained in fluctuating 12
1
light condition simulating sunny sky might be the reason for decrease in biomass
2
productivity of Desmodesmus sp. VIT.Maximum biomass productivity obtained in the
3
current study was higher than the C. zofingiensis biomass productivity (36 mg/L/d)
4
obtained under outdoor fluctuating irradiance (slightly higher than 60,000 lx during
5
midday) in 10 L photobioreactor (Feng et al., 2012).The biomass production of
6
Desmodesmus sp. VIT (0.592 - 0.660 g/L) observed under three different skyconditions
7
simulated in indoor photobioreactor was higher than the Fistulifera sp. biomass
8
production (0.210 -0.570 g/L) obtained in three different seasons (Sato et al., 2014)
9
(Fig. 3). The result obtained in the present study are consistent with Olofsson et
10
al.(2014) report, that Nannochloropsis oculata cultured outdoor in batch cultivation
11
produced maximum biomass in spring than in autumn. However, biomass productivity
12
of Desmodesmus sp. VIT obtained under different fluctuating light intensities was less
13
compared to the biomass productivity of Desmodesmus sp. in outdoor cultivation
14
reported by Xia et al. (2014). High biomass productivity of Desmodesmus sp. in outdoor
15
environmental condition was attained with 0.1 g/L initial inoculum size (Xia et al.,
16
2014). In the present study, 0.020 g/L was the initial inoculum concentration, which
17
could be the major reason for lower biomass production of Desmodesmus sp. VIT under
18
different fluctuating light conditions. Chen et al. (2014) reported that, the increase in
19
inoculum concentration from 0.25 g/L to 0.70 g/L almost doubled the biomass
20
production of Chlorella vulgaris ESP-31. The biomass productivity of Desmodesmus
21
sp. VIT could be improved further by increasing the initial inoculum density from 0.020
22
g/L.
23
3.4. Biomass production of Desmodesmus sp. VIT underincremental light intensity
24
strategies
13
1
Optimization of light, nutrients and temperature for microalgae biomass
2
production have been analysed widely by maintaining a constant growth condition
3
throughout the cultivation cycle. However, effect of changing growth condition along
4
with different growth state in batch cultivation on microalgae biomass production has
5
been seldom studied. In the present study, cultures illuminated using ILIS-2
6
illumination strategy produced higher biomass (1.033 ± 0.04 g/L) compared to control
7
(0.794 ± 0.06 g/L) in batch cultivation (15 days).The biomass productionof cultures
8
grown (0.854 ± 0.01 g/L) under ILIS-1 illumination strategy was slightly lower than the
9
biomass production occurred under ILIS-2. However, biomass production obtained
10
using incremental light intensity strategy (ILIS-2) was significantly higher than the
11
biomass production observed in cells grown under control (constant light intensity
12
strategy). In addition, maximum biomass (1.033 g/L) obtained in incremental light
13
intensity strategy was equivalent to the maximum biomass (0.999 g/L) obtained in
14
common light intensity optimization experiment (Section 3.2.).Similar to biomass
15
production, cells in ILIS-2 yielded highest biomass productivity (67.5 ± 3.2 mg/L/d).
16
Maximum biomass productivity achieved in ILIS was comparable with maximum
17
biomass productivity obtained in constant light intensity optimization experiment
18
(Section 3.2.). Reduction in photoinhibition at lag phase and photolimitation at late
19
exponential phase using incremental light intensity strategy might be the major reason
20
for the better performance of microalgae in batch cultivation. Yan et al. (2013) reported
21
that three phase increase in the light intensity throughout the cultivation cycle produced
22
maximum biomass similar to the maximum cell concentration occurred under constant
23
light intensity strategy, which is consistent with the results obtained in the current study.
24
Contrary to the results of the present study, Han et al. (2015) reported that cultures
25
illuminated with high light intensity of 10,000 lx for first six days of cultivation cycle
14
1
and then decreased to 6,000 lx light intensity promoted the Chlorella sp. growth. The
2
main advantage of the present study was that the light intensity was increased in two
3
phase compared to three or four phase of light intensity increase in batch cultivation.
4
3.5. Effects of different light intensities and photoperiod regimes on lipid and
5
carbohydrate production
6
Microalgae lipid accumulationwas increased due to energy imbalance created in
7
stressfulconditions (Klok et al., 2013). Maximum lipid content of Desmodesmus sp. VIT
8
was obtained at 32,000 lx light intensity under 12:12 h, 16:08 h, 20:04 h and 24:00 h
9
light: dark cycles. Reduction in lipid content was observed with the decrease in light
10
intensity from 32,000 lx to 16,000 lx under four different light: dark cycles (Table1).In
11
addition, maximum biomass obtained at 32,000 lx leads to higher lipid productivity at
12
respective light intensity compared to cultures grown at 16,000 lx under 16:08 h, 20:04
13
h and 24:00 h light: dark cycles (Table 1). The results obtained in the present study are
14
in agreement with Guo et al. (2015) report, which suggests high light intensity as
15
suitable condition for Chlorella sp. to achieve high lipid content. Similarly,
16
Botryococcus braunii KMITL 2 exposed to high light intensity accumulated maximum
17
lipid content compared to cultures grown at low light intensity (Ruangsomboon, 2012).
18
Harwood (1998) reported that lipid metabolism in microalgae was greatly altered by
19
light intensities and light durations of photoperiod regimes. The results of the present
20
study demonstrated that, Desmodesmus sp. VIT exposed to 16:08 h light/dark cycle at
21
16,000 and 32,000 lx light intensities attained maximum lipid content and lipid
22
productivity. The lipid content and lipid productivity was reduced with increase in light
23
duration of the light: dark cycle from 16 h to20 h and 24 h at 16,000 and 32,000 lx light
24
intensities, while decrease in light duration from 16 h to 12 h also reduced the lipid
25
content and lipid productivity of Desmodesmussp. VIT (Table 1). Ruangsomboon 15
1
(2012) also reported that Botryococcus braunii KMITL 2 cultivated under different
2
light: dark cycles produced maximum lipid content under 16:08 h light: dark cycle. The
3
results of the current study concluded that maximum lipid content of 17.5% and 11.4
4
mg/L/d of lipid productivity was obtained at 32,000 lx light intensity under 16:08 h
5
light: dark cycle.
6
In addition to lipid content, different light: dark cycles and light intensities
7
exhibited remarkable changes in photosynthetic efficiency, chemical composition and
8
pigment content of microalgae (Richardson et al., 1983). In the present study, maximum
9
carbohydrate content (25.4%) was obtained in Desmodesmus sp. VIT after 15 days of
10
batch cultivation at 16,000 lx light intensity under four different photoperiod regimes
11
(Table1). Carbohydrate content of Desmodesmus sp. VIT reduced with increase in light
12
intensity from 16,000 lx to 32,000 lx under 12:12 h, 16:08 h, 20:04 h and 24:00 h light:
13
dark cycles. The higher carbohydrate accumulation in cells at 16,000 lx leads to
14
maximum carbohydrate productivity at respective light intensity compared to cultures
15
grown at 32,000 lx under 16:08 h and 12:12 h light: dark cycles (Table 1).The results of
16
the present study are in agreement with He et al. (2015b), who reported that Chlorella
17
sp. L1 and M. dybowskii Y2 cultured under high irradiance attained less carbohydrate
18
and high lipid content in contrast to low light intensity grown cultures. During high light
19
intensity stress, lipid can act as sink for excess light energy and carbon due to high
20
energy demand for lipid biosynthesis compared to carbohydrate and this phenomenon
21
leads to more lipid and less carbohydrate production in high light intensity exposed
22
microalgae (He et al., 2015b). In case of photoperiod regimes effect on carbohydrate
23
content, Desmodesmus sp. VIT exposed to 16:08 h light: dark cycleshowed maximum
24
carbohydrate accumulation at 16,000 lx and 32,000 lx light intensities. The
25
carbohydrate content reduced in the cultures grown under 12:12 h, 20:04 h and 24:00 h 16
1
light: dark cyclesrespectively for 16,000 and 32,000 lx light intensities. George et al.
2
(2014) reported 6:18 h light: dark cycle as suitable condition for A. falcatusmaximum
3
carbohydrate accumulation. The results obtained in this study are in agreement with
4
Krzeminska et al. (2015) report, who stated that C. protothecoides cultivated with 5%
5
carbon dioxide and 16:08 h light: dark cycle produced maximum carbohydrate content
6
compared to 12:12 h and 24:00 h light: dark cycles. Moreover, conversion of starch to
7
sucrose in dark hours might be the reason for presence of high sugar content in
8
microalgae grown under 16:08 h light: dark cycle compared to cultures grown under
9
continuous illumination (Iglesias and Podest, 2005). The results of the present study
10
concluded that, 16:08 h light: dark cycle and 16,000 lx light intensity as optimum
11
condition for maximum carbohydrate accumulation in Desmodesmus sp. VIT.
12
Light intensity, nitrogen concentration and cultivation period significantly affect
13
the lipid and carbohydrate accumulation in microalgae (He et al., 2015b; Ho et al.,
14
2012). In the present study, Desmodesmus sp. VIT was cultivated in BBM for 15 days
15
at 16,000 lx and 32,000 lx light intensity and the changes in nitrate concentrationof
16
BBM, lipid and carbohydrate content of Desmodesmus sp. VIT cultivated at respective
17
light intensities were monitored at regular time intervals. As depicted in Fig. 2B and C,
18
changes in nitrate concentration of BBM were similar at two different light intensities
19
and the initial nitrate concentration (11.8 mM) was depleted after 9 days at 16,000 lx
20
and 32,000 lx. In the case of changes in biomolecule content, carbohydrate content of
21
cells at 16,000 lx increased (12.9% to 25.4%) with increase in cultivation time, whereas
22
relatively stable level of carbohydrate content was maintained in cells grown at 32,000
23
lx (Fig. 2B and C). In contrast, lipid content of Desmodesmus sp. VIT (7.6% to 17.5%)
24
grown at 32,000 lx increased with the increase in cultivation time, whereas slight
25
increase in lipid content was observed in cells (7.6% to 12.5%) grown at 16,000 lx (Fig. 17
1
2C and B). He et al. (2015b) reported that Chlorella sp. L1 and Monoraphidium
2
dybowskii Y2 carbohydrate content increased with increase in cultivation time under
3
low light intensity, which is consistent with the results obtained in the present study.
4
The current study results concluded that 15 days cultivation time is optimum for
5
maximum lipid and carbohydrate accumulation in Desmodesmus sp. VIT. at 32,000 lx
6
and 16,000 lx light intensities. In addition, variation in DCW of Desmodesmus sp. VIT
7
at 16,000 lx and 32,000 lx indicated that similar DCW was observed till 15 days of
8
cultivation period at respective light intensities (Fig. 2B and C).
9
3.6. Effects of fluctuating light intensities replicating sunny sky, intermediate overcast
10 11
and quasi overcast sky conditionson lipid and carbohydrate production Desmodesmus sp. VIT cultured under fluctuating light intensities simulating
12
sunny sky condition attained highest lipid content (13.7%). Same amount of lipid
13
content was observed under fluctuating light intensities replicating intermediate
14
overcast and quasi overcast sky conditions (Fig. 4A). The results observed in the current
15
study are in agreement with the He et al. (2015a) report, who concluded that high
16
fluctuating light intensity in outdoor cultivation was the suitable condition to achieve
17
maximum lipid content in microalgae. However, lipid content of Desmodesmus sp.
18
T28–1 (24.6%,), Desmodesmus sp. NMX451(25.5%), Scenedesmus obtusus XJ-15 (31.3
19
%) cultivated in 5 L flasks outdoor using autoclaved water was higher than the lipid
20
content of Desmodesmus sp. VIT (Xia et al., 2014). The major reason for the less lipid
21
content of Desmodesmus sp. VIT could be the high initial nitrogen concentration used
22
for cultivation compared to 0.2 g/L of urea used byXia et al. (2014). Feng et al. (2011)
23
reported enhancement in lipid content of C. zofingiensis was attained using nitrogen
24
limited condition in outdoor cultivation. Consequently, the lipid content of
18
1
Desmodesmus sp. VIT under fluctuating light intensities could be increased further by
2
implementing nitrogen limitation strategy.
3
Maximum carbohydrate content and carbohydrate productivity were observed in
4
Desmodesmus sp. VIT (16.1% and 8.6 mg/L/d) cultivated for 12 days under fluctuating
5
light intensities simulating intermediate overcast sky condition (Fig. 4A). Cultures
6
exposed to fluctuating light intensities simulating sunny sky and quasi overcast sky
7
conditions obtained almost same amount of carbohydrate. However, there was no
8
significant variation in lipid and carbohydrate content was observed in cells grown
9
under fluctuating light intensities simulating different sky conditions.The carbohydrate
10
content observed in the current study was comparable with Phaeodactylum tricornutum
11
UTEX 640 cultivated during summer and Chlorella zofingiensis G1 cultivated in
12
wastewater with 5-6% carbon dioxide during winter in outdoor environmental condition
13
(Benavides et al., 2013; Huo et al., 2012).
14
Variations in lipid, carbohydrate, DCW and nitrate concentration monitored
15
during the growth of cells under fluctuating light intensities simulating intermediate
16
overcast sky condition was illustrated in Fig. 4B. Desmodesmus sp. VIT carbohydrate
17
accumulation was gradually increased with increase in cultivation period, whereas
18
relatively stable level of lipid content was maintained during 12 days culture. In the case
19
of nitrate variation, 3.3 mM of nitrate was still left at the end of the cultivation period
20
(Fig. 4B). The cultivation time of 9 days was optimum for maximum lipid content and
21
12 days of cultivation time was optimum for maximum carbohydrate accumulation in
22
Desmodesmus sp. VIT under intermediate overcast sky condition.
23
3.7. Effects of incremental light intensity strategy on lipid and carbohydrate production
19
1
The carbohydrate and lipid content of Desmodesmus sp. VIT cultivated for 15
2
days under 16:08 h light: dark cycle in constant light intensity strategy and incremental
3
light intensity strategies was illustrated in Fig. 5A. Significantly higher lipid content
4
(22.5%) was obtained in cultures illuminated under ILIS-2. High biomass at ILIS-2
5
leads to maximum lipid productivity (15.4 mg/L/d) at respective light strategy.
6
However, same lipid productivity was obtained in cells under control and ILIS-1 (Fig.
7
5A). The lipid content and lipid productivity obtained using incremental light intensity
8
strategies was higher than the maximum lipid content and lipid productivityobtained in
9
constant light optimization experiment (Section 3.5.). The high light intensity at phase II
10 11
of ILIS-2 might be the major reason for high lipid accumulation in microalgae. Maximum carbohydrate content (18.4%) and carbohydrate productivity (10.2
12
mg/L/d) was observed in cells grown under ILIS-1compared to control and ILIS-2 (Fig.
13
5A). Cells grown under ILIS-1 and control attained similar amount of carbohydrate
14
productivity. The Carbohydrate content and carbohydrate productivity observed under
15
incremental light intensity strategy were less compared with results obtained in constant
16
light optimization experiments (Section 3.5.).
17
Changes in nitrate, lipid and carbohydrate concentration of Desmodesmus sp.
18
VIT under ILIS were illustrated in Fig. 5B and C. The similar levels of lipid and
19
carbohydrate content were maintained during first 6 days of cultivation period. The shift
20
in light intensity from 8,000 lx to 16,000 lx under ILIS-1 stimulated rapid carbohydrate
21
accumulation in cells from 6th to 15th day (Fig. 5C). On the other hand, rapid increase
22
in Desmodesmus sp. VIT lipid content under ILIS-2 was observed from 9th to 15th day
23
(Fig. 5B). However, changes in nitrate concentration was similar in different
24
incremental light intensity strategies and nitrate depleted condition was observed after
25
12 days (Fig. 5C). The 15 days of cultivation time was optimum for maximum lipid and 20
1
carbohydrate accumulation at respective ILIS-2 and ILIS-1. In the case of biomass
2
variation, cells under ILIS-2 attained higher DCW from 6th to 15th day of cultivation
3
period compared to cells grown under ILIS-1 (Fig. 5B).
4
4. Conclusions
5
Optimizationof different light supply strategies are important factors for
6
commercial microalgae biofuel production. Desmodesmus sp. VIT maximum biomass
7
(1.033 g/L) and lipid (22.5%) was observed in incremental light intensity strategy
8
(8,000 to 32,000 lx under 16:08 h light: dark cycle). Highest carbohydrate content
9
(25%) was obtained at 16,000 lx light intensity for 16:08 h light: dark cycle.
10
Desmodesmus sp. VIT produced maximum biomass and lipid underlight intensities
11
simulating intermediate overcast and sunny sky conditions.Based on the present study,
12
it is concluded incremental light intensity strategy as the suitable light supply method
13
for indoor microalgaecultivation.
14
Acknowledgement
15
The authors are thankful to the management of VIT University, Vellore for the financial
16
support to carry out the research work and necessary laboratory facilities.
17 18 19
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microalgae. Biomass Bioenerg. 72, 280-287.
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Algal Biomass. http://www.nrel.gov/docs/fy14osti/60957.pdf. Accessed on 22-
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07-2015.
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Bioresource. Technol. 174, 274–280.
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33. Xie, Y., Ho, S.H., Chen, C.N.N., Chen, C.Y., Ng, I.S., Jing, K.J., Chang, J.S., Lu, Y., 2013. Phototrophic cultivation of a thermo-tolerant Desmodesmus sp. for
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wavelengths and light intensity supply strategies on synthetic high-strength
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Cultivation of Chlorella protothecoides in photobioreactors: The combined
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9 10 11 12 13 14 15 16 17 18 19 20 21 26
1
Figure captions
2 3
Fig. 1. Phylogenetic tree showing the position of Desmodesmus sp. VIT.
4
Fig. 2. Different light intensities and light: dark cycles effect on Desmodesmus sp. VIT
5
dry cell weight at the end of cultivation period (A) and time-course profiles of nitrate
6
concentration, DCW, lipid and carbohydrate content during the growth of
7
Desmodesmus sp. VIT at (B) 16,000 lx light intensity and (C) 32,000 lx light intensity.
8
Fig. 3. Dry cell weight (DCW) of Desmodesmus sp. VIT under the fluctuating light
9
intensities simulating different sky conditions in indoor photobioreactor.
10
Fig. 4. Lipid and carbohydrate content of Desmodesmus sp. VIT under fluctuating light
11
intensities simulating different sky conditions in indoor photobioreactor (A) and the
12
changes in nitrate concentration, biomass, lipid and carbohydrate content during the
13
growth of Desmodesmus sp. VIT under fluctuating light intensity simulating
14
intermediate overcast sky condition (B)
15
Fig. 5. Lipid, carbohydrate content, lipid productivity (LP) and carbohydrate
16
productivity (CP) of Desmodesmus sp. VIT under incremental light intensity strategies
17
(A) and changes in biomolecule composition and cell growth under incremental light
18
intensity strategies: (B) lipid content and DCW, (C) carbohydrate content and nitrate
19
concentration.
20 21
27
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
1 2
Table 1 Effect of different light intensities and light: dark cycles on Desmodesmus sp.
3
VIT lipid content (LC), biomass productivity (BP), lipid productivity (LP),
LC (% DCW)
BP (mg/L/d)
LP (mg/L/d)
CC (% DCW)
CP (mg/L/d)
16,000 lx 12:12 h 32,000 lx 12:12 h
8 10
36.6 ± 3.2 38.1 ± 3.4
2.9 ± 0.2 3.8 ± 0.3
19.4 ± 0.5 13.6 ± 0.3
7.1 ± 0.4 5.2 ± 0.3
16,000 lx 16:08 h 32,000 lx 16:08 h
12.5 17.5
60.3 ± 0.6 65.2 ± 5
7.5 11.4 ± 0.8
25.4 ± 0.7 16.3 ± 1.4
15.3 ± 0.6 10.6 ± 0.1
16,000 lx 20:04 h 32,000 lx 20:04 h
7.5 13.3
50.9 ± 4.9 57.5 ± 1.7
3.8 ± 0.3 7.6 ± 0.2
18.8 ± 0.1 14.6 ± 0.5
9.6 ± 0.8 8.4
7.5 16,000 lx 24:00 h 48.6 ± 1.9 3.6 ± 0.2 13.7± 1.7 32,000 lx 24:00 h 57.2 ± 3.1 7.8 ± 0.5 carbohydrate content (CC) and carbohydrate productivity (CP)
17.9 ± 0.7 14.8 ± 1.3
8.7 ± 0.2 8.5 ± 1.2
Light intensities /light: dark cycles
4 5 6
28
1 2 3 4 5
Highlights
•
6
Light affected the growth, lipid and carbohydrate content of Desmodesmus sp. VIT.
7
•
Biomass and lipid content were enhanced by incremental light intensity strategy.
8
•
16:08 h light/dark cycle was optimum for growth, lipid and carbohydrate
9 10
content. •
High lipid and less carbohydrate content observed under high light intensity.
11
29
S0960-8524(15)01344-9 http://dx.doi.org/10.1016/j.biortech.2015.09.065 BITE 15574
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Bioresource Technology
Received Date: Revised Date: Accepted Date:
23 July 2015 16 September 2015 18 September 2015
Please cite this article as: Srinivasan, S., Ramasubbu, S., Biophotonic perception on Desmodesmus sp. VIT growth, lipid and carbohydrate content, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech. 2015.09.065
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1
Biophotonic perception on Desmodesmus sp. VIT growth, lipid and carbohydrate
2
content
3
SRIRAM Srinivasan, SEENIVASAN Ramasubbu*
4
School of Bio Sciences and Technology, VIT University, Vellore – 632014, Tamil
5
Nadu, India.
6 7 8 9 10 11 12 13 14
*Corresponding author
15
Dr. R. Seenivasan,
16
Associate professor,
17
School of Biosciences and Technology,
18
VIT University,
19
Vellore – 632014.
20
Tamil Nadu,
21
India.
22
Telephone: +91-416-2202131
23
Email: [email protected]
1
1 2
Abstract Constant and fluctuating light intensity significantly affects the growth and
3
biochemical composition of microalgae and it is essential to identify
4
suitableillumination conditions for commercialmicroalgae biofuel production. In the
5
present study, effects of light intensities, light: dark cycles, incremental light intensity
6
strategies and fluctuating light intensities simulating different sky conditions in indoor
7
photobioreactoron Desmodesmus sp. VIT growth, lipid and carbohydrate content were
8
analysed in batch culture.The results revealed that Desmodesmus sp. VIT obtained
9
maximum lipid content (22.5%) and biomass production (1.033 g/L) under incremental
10
light intensity strategy. The highest carbohydrate content of 25.4% was observed under
11
constant light intensity of 16,000 lx and 16:08 h light: dark cycle. The maximum
12
biomass productivity of Desmodesmus sp. VIT (53.38 mg/L/d) was occurred under
13
fluctuating light intensity simulating intermediate overcastsky condition.
14 15
Keywords: Desmodesmussp. VIT; Light; Lipid; Carbohydrate; Photobioreactor.
16 17 18 19 20 21 22 2
1
1. Introduction
2
Commercial production of biofuel for transportation (biodiesel andbioethanol)
3
from soybean, sugarcane, corn, and wheat faced severe setback due to excessive land
4
use and competition with global food supply, whereasbiofuel production from
5
lignocellulosic biomass and waste cooking oil desires high initial investment and
6
improvement in extraction technologies. Alternative to crop based feedstocks,
7
microscopic photosynthetic organisms called microalgae wereconsidered as sustainable
8
raw material for biofuel production (lipid rich microalgaefor biodiesel production and
9
carbohydrate rich algaefor bioethanol production). Hurdles in large scale microalgae
10
biofuel production are costly upstream process and high energy consuming downstream
11
process (Rawat et al., 2013).
12
Manipulation of numerous factors such as nitrogen, salt, pH, phosphorus
13
(chemical) and light (physical) could alter the growth and biochemical composition of
14
microalgae (George et al., 2014). Among those factors, light as a primary energy source
15
for photoautotrophs have significantimpact on microalgae growth, biochemical
16
composition and cost of commercial indoor biofuel production (He at al., 2015b).
17
Supplying constant light intensity for entire cultivation cycle is themost common light
18
supplystrategy used for commercial microalgae cultivation in indoor photobioreactor.
19
However, microalgae light requirements in batch cultivation may differ for each growth
20
phase (Han et al., 2015). For example, microalgae requireless light intensity at initial lag
21
phase to avoid photoinhibitionand on the other hand, high light intensity at exponential
22
phase to prevent cell shading (photolimitation) in batch culture (Lee et al., 2006). It is
23
essential to use appropriate light intensity for each growth phase or incremental light
24
intensity strategy as proposed by Yan et al. (2013) to improve algal growth with less
25
power consumption in indoor cultivation. 3
1
Incase of outdoor cultivation, microalgae utilize sunlight as energy source to
2
carry out photochemical reaction for protein, carbohydrate and lipid synthesis.
3
Fluctuating light intensity, temperature, and pH in outdoor environment significantly
4
affects the microalgae growth. Biomass productivity of Chlorella ellipsoidea under
5
controlled environmental parameters in indoor cultivation was higher than the biomass
6
productivity occurred under uncontrolled outdoor condition (Wang et al.,
7
2014).Outdoor environmental factors such as light, temperature and pH are greatly
8
affected by weather condition. Among those factors, light intensity variation was largely
9
influenced by weather (Xia et al., 2014). Feng et al. (2011) reported that sky conditions
10
(cloudy sky in autumn resulted in less irradiance and less cloudy days in spring caused
11
slightly high irradiance) observed in spring and autumn greatly affected the cell
12
concentration of Chlorella zofingiensis in respective seasons. Hence, it is very important
13
to study microalgae biomass and low value products (lipid and carbohydrate)
14
production in fluctuating light intensities under different sky conditions (sunny sky,
15
intermediate overcast and quasi overcast). Tamburic et al. (2014) reported that
16
simulating the fluctuating light intensities of anticipated location in indoor
17
photobioreactor would be the cost effective way to carry out preliminary screening to
18
analyse the feasibility of microalgae growth in changing light and temperaturecompared
19
to costly outdoor field trails.
20
Effect of different light intensities and photoperiod regimes onmicroalgae
21
growth and biochemical composition was studied elaborately. However, no reports
22
regarding the impact of incremental light intensity strategy and light/dark photoperiod
23
regimeson Desmodesmus sp. lipid and carbohydrate content was reported. The current
24
study was carried out to analyze the effect of different light intensities, photoperiod
25
regimes, incremental light intensity strategies and fluctuating light intensities simulating 4
1
sunny sky, intermediate overcast and quasi overcast sky conditions in indoor
2
photobioreactor on Desmodesmus sp. VIT biomass production, lipid and carbohydrate
3
content.
4
2. Materials and Methods
5
2.1. Microalgae isolation
6
Microalga was isolated from the freshwater pond in VIT University, Vellore,
7
India (12.9692° N, 79.1559° E). Algal enrichment culture was prepared by inoculating 5
8
mL of water sample into 95 mL sterilized Bold Basal Medium (BBM) with pH 6.86 and
9
illuminated under cool white fluorescent tube light at 4000 lx light intensity under 12:12
10
h light/dark photoperiod regime at room temperature. Axenic single clone was purified
11
from the mixed algae culture using serial dilution, spread plate and streak plate
12
techniques.
13
2.2. Molecular identification of microalgae
14
Algal DNA was isolated by modified CTAB method of Varela-alvarezet al.
15
(2007). Exponentially growing algal cells were centrifuged at 8,000 rpm for 10 min to
16
concentrate the cells from the liquid medium. Algal pellet was homogenized in 500 µl
17
of prewarmed lysis buffer (2% CTAB, 1 M NaCl, 100mM Tris HCl (pH 8.0), 20mM
18
EDTA (pH 8.0), 100 µg/mL proteinase K and 2% β- mercaptoethanol) and incubated at
19
60 ºC for 50 min. After cell lysis, 500 µl of phenol:chloroform:isoamyl alcohol
20
(25:24:1) was added, vortexed for 10 min andcentrifuged at 13,000 rpm for 20 min at 25
21
ºC to separate the DNA. After centrifugation, upper aqueous phase containing DNA was
22
transferred to the new tube and two volumes of ice cold isopropanol was added and
23
incubated at -20 ºC for 20 min followed by centrifugation at 13,000 rpm for 20 min at
5
1
25 ºC to precipitate the DNA. 70% ethanol wash was given to the DNA pellet and Tris-
2
EDTA was used to dissolve the DNA for PCR reaction. The PCR amplification of the
3
DNA was carried out as per Dahlkild et al. (2001) protocol.
4
2.3. Polyethylene bag photobioreactor (PBR)
5
2 L transparent polyethylene bag (30 X 25.5 cm) was used as photobioreactor
6
for culturing microalgae. The bag reactor was sterilized by autoclaving at 121 ºC for 20
7
min. Bottom end of the polyethylene bag was heat sealed and the upper end was tied
8
using nylon tag after the addition of cell suspension (600 mL working volume). Air was
9
bubbled into the photobioreactor from aerator through 0.2 µm sterile filter. Constant
10
aeration was maintained throughout the batch cultivation. In addition to aeration port,
11
gas exchange port and sampling port were present in culture bags.
12
2.4. Experimental design
13
2.4.1. Effect of light intensityand photoperiod regimeon growth, lipid and carbohydrate
14
content
15
Microalgae growing in late exponential phase were inoculated into polyethylene
16
bag photobioreactor (PBR) containing 600 mL of modified BBM medium containing
17
NaNO3 1 g/L. Initial concentration of biomass in PBR at day 0 was set to 0.020 g/L.
18
Different light intensities viz.,8,000 lx, 16,000 lx and 32,000 lx were illuminated on the
19
surface of PBR for 24:00 h, 20:04 h, 16:08 h and 12:12 h light: dark cycles by adjusting
20
the distance between light source and PBR for establishing the respective light intensity.
21
Fluorescent tube light was manually switched on/off at required timeintervals for
22
achieving corresponding light: dark cycles. The light intensity on the surface of the
6
1
PBR was measured using Lux meter (Sigma Instruments). Each experiment was
2
performed in duplicate at room temperature for 15 days.
3
2.4.2. Effect of fluctuating light intensity on growth, lipid and carbohydrate content
4
Daily light intensity fluctuation for 12 days under three different sky conditions
5
of Lyon, France was simulated in indoor PBR by manually increasing or decreasing the
6
distance between the fluorescent tube light and PBR on hourly basis. Daily light
7
intensity was increased from 10,000 lx to 90,000 lx in midday and then gradually
8
decreased to 10,000 lx at the end of 12 h light cycle for replicating sunny sky condition
9
in indoor PBR. Similarly, for replicating intermediate overcast sky condition, light
10
intensity was increased from 5,000 lx to 60,000 lx in midday and then gradually
11
decreased to 5,000 lx at the end of the light cycle and for quasi overcast condition,
12
maximum of 20,000 lx light intensity was maintained in midday. The light: dark cycle
13
of about 12:12 h was maintained for different fluctuating light intensities and
14
experiments were performed in duplicate at room temperature in modified BBM
15
(NaNO3 1 g/L). The irradiance data for different sky conditions of Lyon, France was
16
obtained from Satel-Light database.
17
2.4.3. Incremental light intensity strategy (ILIS)
18
Fifteen days of microalgae batch cultivation was divided into two phase. Phase I
19
(1-6 days) is of first six days which includes lag and mid-exponential phase and phase II
20
includes late exponential and stationary phase of batch cultivation cycle (7-15 days). All
21
the experiments were performed in duplicates in modified BBM.
22
Control (Constant light intensity): Cultures were illuminated under 8,000 lx light
23
intensityand 16:08 h light: dark cyclefor 15 days (Phase I and Phase II).
7
1
Strategy I (ILIS-1): Cultures were illuminated under 8,000 lx light intensity for first six
2
days (Phase I) and from 7th day onwards light intensity was increased from 8,000 lx to
3
16,000 lx light intensity (Phase II).
4
Strategy II (ILIS-2): Cultures were illuminated under 8,000 lx light intensity for first six
5
days (Phase I) and then light intensity was increased to 32,000 lx light intensity (Phase
6
II).
7
2.5. Analytical methods
8
2.5.1. Dry cell weight and biomass productivity determination
9
Cells retained on preweighed whatman glass fiber filter paper (47 mm x 0.45
10
µm) after filtering 10 mL of algae culture (OD683 = 1.00) was dried in hot air oven at
11
105 ºC for 24 h and weighed again after cooling to room temperature. The difference
12
between the weight of the blank filter and filter loaded with cells provided the dry cell
13
weight of Desmodesmus sp. VIT. The experiment was performed in triplicate.
14
To determine the dry cell weight of algae cultured in PBR, 2 mL cell suspension
15
from photobioreactor was collected and optical density was measured at 683 nm
16
(OD683) using spectrophotometer (U.V 1800, Shimadzu, Japan). The absorbance at 683
17
nm was converted to dry cell weight through predetermined conversion factor (1 OD683
18
= 0.400 g DCW/L).
19
Biomass Productivity (P) was obtained by P (mg/L/d) = (X1 - X0)/(t1-t0). X1and X0 are
20
the microalgae dry cell weight (mg/L) at time t1and t0.
21
2.5.2. Lipid extraction
8
1
Lipid was extracted from algal biomass using modified method of Folch et al.
2
(1956). Briefly, freeze dried algae was extracted with 4 mL of chloroform: methanol
3
(2:1,v/v) for 1 h at room temperature with occasional vortexing. The cell debris was
4
separated from the supernatant (solvent) by centrifugation at 7,000 rpm for 7 min. Then
5
0.2 volumes of water was added to the supernatant and centrifuged at 2,500 rpm for 10
6
min to extract lipid into chloroform phase and non-lipid content into aqueous methanol
7
phase. The top aqueous phase was carefully removed and the bottom chloroform phase
8
containing lipid was evaporated at 60ºC to gravimetrically weigh the lipid content. The
9
lipid content was reported as percent of dry cell weight (DCW).
10 11
2.5.3. Total carbohydrate determination Carbohydrate determination in microalgae was carried out based on the modified
12
method of Wychen and Laurens (2013). Freeze dried algal biomass was initially
13
hydrolysed with 500 µl of 72% (w/w) sulphuric acid for 1 h at 30ºC in water bath for
14
primary hydrolysis and then deionised water was added to decrease the acid
15
concentration to 4% (w/w) and autoclaved at 121ºC for 20 min for secondary
16
hydrolysis. The carbohydrate content present in the hydrolysate was determined
17
according to Dubois et al. (1956) using glucose as standard. Carbohydrate content was
18
reported as percent of dry cell weight (DCW).
19
2.5.4. Determination of nitrate concentration
20
The nitrate concentration in the medium was determined using calorimetric
21
method (Xie et al., 2013). The cell suspension was collected from PBR and filtered
22
through whatman glass fiber filter paper. The filtrate was diluted and the optical density
23
of the filtrate at 220 nm (OD220) was measured using spectrophotometer. Sodium nitrate
24
was used as standard. 9
1 2
2.6. Statistical analysis The results were expressed as mean ± standard deviation. The values were
3
analysed by one-way ANNOVA using GraphPad Prism. The statistically significant
4
variation was considered at the level of p< 0.05.
5
3. Results and Discussion
6
3.1. Identification of microalgae
7
The phylogenetic position shown in the phylogenetic tree indicates that the
8
microalgae isolate is closely related to Desmodesmussp.S7 with 99% sequence
9
similarity (Fig. 1). The sequence was submitted to GENBANK (Accession number:
10
KP720618) and was designated as Desmodesmus sp. VIT.
11 12
3.2. Biomass production of Desmodesmus sp. VIT cultured at different light intensities
13
and photoperiod regimes
14
Viability of photoautotrophic microalgae in photobioreactor largely depends on
15
light quality, duration and quantity used for culturing microalgae. Supplying favourable
16
light intensity and light: dark cycle is highly essential for energy efficient biomass and
17
biofuel production in commercial indoor microalgae cultivation.
18
Dry cell weight (DCW) of Desmodesmus sp. VIT cultured under four different
19
photoperiod regimes (12:12 h, 16:08 h, 20:04 h and 24:00 h light: dark cycles) at three
20
different light intensities (8,000 lx, 16,000 lx and 32,000 lx) for 15 days was illustrated
21
in Fig.2A. In the current study, maximum biomass production was observed incultures
22
exposed to16:08 h light: dark cycle at 8,000 lx, 16,000lx and 32,000 lx light intensities.
23
The decrease in biomass production was observed when the cultures were exposed to
10
1
20:04 h, 24:00 hand 12:12 h light: dark cyclesat three different light intensities.The
2
cultures exposed to 12:12 h light: dark cycle yielded significantly lower biomass
3
compared to cells under 16:08 h light: dark cycles at16,000 lx and 32,000 lx light
4
intensities. In the case of biomass productivity, cells grown under 16:08 h light: dark
5
cycle obtained highest biomass productivity, whereas cells grown under 12:12 h light:
6
dark cycle yielded lowest biomass productivity at 16,000 lx and 32,000 lx light
7
intensities (Table 1). The results obtained in present studyhave confirmed that 6:08 h
8
light: dark cycle as the optimum photoperiod regime for Desmodesmus sp. VIT growth
9
at 8,000 lx, 16,000 lx and 32,000 lx light intensities. The growth of microalgae under
10
different light intensities is the sum of damage and repair mechanism. Continuous
11
illumination leads to continuous damage to photosystem II, whereas introducing short
12
dark period (light: dark cycles) might reduce the light induced damage to photosystem
13
II and enhance the growth in the next light period. This phenomenon might be the major
14
reason for the maximum biomass production attained under 16:08 h light: dark
15
cycle.The results of the current study are in accordance with Ji et al. (2013) report, who
16
suggested 14:10 h light: dark cycle as optimum photoperiod regime for Desmodesmus
17
sp. EJ15-2 biomass production.On the contrary, Wahidin et al. (2013) reported that
18
Nannochloropsis sp. illuminated under high light intensity for shorter photoperiod
19
produced maximum biomass compared to cultures illuminated at high light intensity for
20
longer photoperiod.
21
Light quantity significantly affects the growth of microalgae through
22
photoinhibition (excessive light intensity) or photolimitation (less light intensity) (Ho et
23
al., 2012).The dry cell weight of Desmodesmus sp. VIT increases with increase in light
24
intensity from 8,000 lx to 32,000 lx under four different light: dark cycles(12:12 h,
25
16:08 h, 20: 04 h and 24:00 h ) (Fig. 2A). In addition, biomass productivity of 11
1
Desmodesmus sp. VIT increased with increase in light intensity under four different
2
photoperiod regimes (Table 1). Xie et al. (2013); George et al. (2014) reports also stated
3
that Desmodesmus sp. F51 (150 – 750 µmol m-2 s-1) and Ankistrodesmus falcatus (30 –
4
150 µmol m-2 s-1) biomass production increases with increase in light intensity. In
5
contrast, Desmodesmus sp. EJ15-2 biomass production increases with increase in light
6
intensity upto 80 µmol m-2 s-1, further increase in light intensity reduced the biomass
7
production (Ji et al., 2013). The maximum biomass production of Desmodesmus sp.
8
VIT (0.999 g/L) obtained at 32,000 lx light intensity under 16:08 h light: dark cycle in
9
the present study was higher than the biomass production of Desmodesmus sp. EJ15-2
10
(0.569 g/L), Ankistrodesmus falcatus (0.224 g/L) (Ji et al., 2013 and George et al.,
11
2014) and comparable to Chlorella sp. (1.26 g/L) and Phaeodactylum tricornutum
12
(1.212 g/L) biomass production (Nogueira et al., 2015; Guo et al., 2015). Whereas,
13
Desmodesmus sp. F2 biomass production was higher than the present study due to
14
higher inoculum size and carbon dioxide (2.5%) used by Ho et al. (2014) for cultivation.
15
3.3. Biomass productivity of Desmodesmus sp. VIT under fluctuating light intensities
16
simulating different sky conditions
17
Microalgae tolerance to fluctuating light intensities and temperature variations
18
under different sky conditions are essential characteristics required for biofuel
19
production using sunlight. Feng et al. (2011) reported that the biomass productivity of
20
C. zofingiensis was higher in spring (intermediate clouds) than in autumn (cloudy sky).
21
Similarly, maximum biomass productivity (53.3 mg/L/d) of Desmodesmus sp. VIT was
22
obtained under fluctuating light intensities simulating intermediate overcast sky
23
condition in indoor photobioreactor. The biomass productivity was decreased to 49.8
24
mg/L/d and 47.6 mg/L/d under fluctuating light conditions replicating quasi overcast
25
and sunny sky conditions. The high light intensity (90,000 lx) maintained in fluctuating 12
1
light condition simulating sunny sky might be the reason for decrease in biomass
2
productivity of Desmodesmus sp. VIT.Maximum biomass productivity obtained in the
3
current study was higher than the C. zofingiensis biomass productivity (36 mg/L/d)
4
obtained under outdoor fluctuating irradiance (slightly higher than 60,000 lx during
5
midday) in 10 L photobioreactor (Feng et al., 2012).The biomass production of
6
Desmodesmus sp. VIT (0.592 - 0.660 g/L) observed under three different skyconditions
7
simulated in indoor photobioreactor was higher than the Fistulifera sp. biomass
8
production (0.210 -0.570 g/L) obtained in three different seasons (Sato et al., 2014)
9
(Fig. 3). The result obtained in the present study are consistent with Olofsson et
10
al.(2014) report, that Nannochloropsis oculata cultured outdoor in batch cultivation
11
produced maximum biomass in spring than in autumn. However, biomass productivity
12
of Desmodesmus sp. VIT obtained under different fluctuating light intensities was less
13
compared to the biomass productivity of Desmodesmus sp. in outdoor cultivation
14
reported by Xia et al. (2014). High biomass productivity of Desmodesmus sp. in outdoor
15
environmental condition was attained with 0.1 g/L initial inoculum size (Xia et al.,
16
2014). In the present study, 0.020 g/L was the initial inoculum concentration, which
17
could be the major reason for lower biomass production of Desmodesmus sp. VIT under
18
different fluctuating light conditions. Chen et al. (2014) reported that, the increase in
19
inoculum concentration from 0.25 g/L to 0.70 g/L almost doubled the biomass
20
production of Chlorella vulgaris ESP-31. The biomass productivity of Desmodesmus
21
sp. VIT could be improved further by increasing the initial inoculum density from 0.020
22
g/L.
23
3.4. Biomass production of Desmodesmus sp. VIT underincremental light intensity
24
strategies
13
1
Optimization of light, nutrients and temperature for microalgae biomass
2
production have been analysed widely by maintaining a constant growth condition
3
throughout the cultivation cycle. However, effect of changing growth condition along
4
with different growth state in batch cultivation on microalgae biomass production has
5
been seldom studied. In the present study, cultures illuminated using ILIS-2
6
illumination strategy produced higher biomass (1.033 ± 0.04 g/L) compared to control
7
(0.794 ± 0.06 g/L) in batch cultivation (15 days).The biomass productionof cultures
8
grown (0.854 ± 0.01 g/L) under ILIS-1 illumination strategy was slightly lower than the
9
biomass production occurred under ILIS-2. However, biomass production obtained
10
using incremental light intensity strategy (ILIS-2) was significantly higher than the
11
biomass production observed in cells grown under control (constant light intensity
12
strategy). In addition, maximum biomass (1.033 g/L) obtained in incremental light
13
intensity strategy was equivalent to the maximum biomass (0.999 g/L) obtained in
14
common light intensity optimization experiment (Section 3.2.).Similar to biomass
15
production, cells in ILIS-2 yielded highest biomass productivity (67.5 ± 3.2 mg/L/d).
16
Maximum biomass productivity achieved in ILIS was comparable with maximum
17
biomass productivity obtained in constant light intensity optimization experiment
18
(Section 3.2.). Reduction in photoinhibition at lag phase and photolimitation at late
19
exponential phase using incremental light intensity strategy might be the major reason
20
for the better performance of microalgae in batch cultivation. Yan et al. (2013) reported
21
that three phase increase in the light intensity throughout the cultivation cycle produced
22
maximum biomass similar to the maximum cell concentration occurred under constant
23
light intensity strategy, which is consistent with the results obtained in the current study.
24
Contrary to the results of the present study, Han et al. (2015) reported that cultures
25
illuminated with high light intensity of 10,000 lx for first six days of cultivation cycle
14
1
and then decreased to 6,000 lx light intensity promoted the Chlorella sp. growth. The
2
main advantage of the present study was that the light intensity was increased in two
3
phase compared to three or four phase of light intensity increase in batch cultivation.
4
3.5. Effects of different light intensities and photoperiod regimes on lipid and
5
carbohydrate production
6
Microalgae lipid accumulationwas increased due to energy imbalance created in
7
stressfulconditions (Klok et al., 2013). Maximum lipid content of Desmodesmus sp. VIT
8
was obtained at 32,000 lx light intensity under 12:12 h, 16:08 h, 20:04 h and 24:00 h
9
light: dark cycles. Reduction in lipid content was observed with the decrease in light
10
intensity from 32,000 lx to 16,000 lx under four different light: dark cycles (Table1).In
11
addition, maximum biomass obtained at 32,000 lx leads to higher lipid productivity at
12
respective light intensity compared to cultures grown at 16,000 lx under 16:08 h, 20:04
13
h and 24:00 h light: dark cycles (Table 1). The results obtained in the present study are
14
in agreement with Guo et al. (2015) report, which suggests high light intensity as
15
suitable condition for Chlorella sp. to achieve high lipid content. Similarly,
16
Botryococcus braunii KMITL 2 exposed to high light intensity accumulated maximum
17
lipid content compared to cultures grown at low light intensity (Ruangsomboon, 2012).
18
Harwood (1998) reported that lipid metabolism in microalgae was greatly altered by
19
light intensities and light durations of photoperiod regimes. The results of the present
20
study demonstrated that, Desmodesmus sp. VIT exposed to 16:08 h light/dark cycle at
21
16,000 and 32,000 lx light intensities attained maximum lipid content and lipid
22
productivity. The lipid content and lipid productivity was reduced with increase in light
23
duration of the light: dark cycle from 16 h to20 h and 24 h at 16,000 and 32,000 lx light
24
intensities, while decrease in light duration from 16 h to 12 h also reduced the lipid
25
content and lipid productivity of Desmodesmussp. VIT (Table 1). Ruangsomboon 15
1
(2012) also reported that Botryococcus braunii KMITL 2 cultivated under different
2
light: dark cycles produced maximum lipid content under 16:08 h light: dark cycle. The
3
results of the current study concluded that maximum lipid content of 17.5% and 11.4
4
mg/L/d of lipid productivity was obtained at 32,000 lx light intensity under 16:08 h
5
light: dark cycle.
6
In addition to lipid content, different light: dark cycles and light intensities
7
exhibited remarkable changes in photosynthetic efficiency, chemical composition and
8
pigment content of microalgae (Richardson et al., 1983). In the present study, maximum
9
carbohydrate content (25.4%) was obtained in Desmodesmus sp. VIT after 15 days of
10
batch cultivation at 16,000 lx light intensity under four different photoperiod regimes
11
(Table1). Carbohydrate content of Desmodesmus sp. VIT reduced with increase in light
12
intensity from 16,000 lx to 32,000 lx under 12:12 h, 16:08 h, 20:04 h and 24:00 h light:
13
dark cycles. The higher carbohydrate accumulation in cells at 16,000 lx leads to
14
maximum carbohydrate productivity at respective light intensity compared to cultures
15
grown at 32,000 lx under 16:08 h and 12:12 h light: dark cycles (Table 1).The results of
16
the present study are in agreement with He et al. (2015b), who reported that Chlorella
17
sp. L1 and M. dybowskii Y2 cultured under high irradiance attained less carbohydrate
18
and high lipid content in contrast to low light intensity grown cultures. During high light
19
intensity stress, lipid can act as sink for excess light energy and carbon due to high
20
energy demand for lipid biosynthesis compared to carbohydrate and this phenomenon
21
leads to more lipid and less carbohydrate production in high light intensity exposed
22
microalgae (He et al., 2015b). In case of photoperiod regimes effect on carbohydrate
23
content, Desmodesmus sp. VIT exposed to 16:08 h light: dark cycleshowed maximum
24
carbohydrate accumulation at 16,000 lx and 32,000 lx light intensities. The
25
carbohydrate content reduced in the cultures grown under 12:12 h, 20:04 h and 24:00 h 16
1
light: dark cyclesrespectively for 16,000 and 32,000 lx light intensities. George et al.
2
(2014) reported 6:18 h light: dark cycle as suitable condition for A. falcatusmaximum
3
carbohydrate accumulation. The results obtained in this study are in agreement with
4
Krzeminska et al. (2015) report, who stated that C. protothecoides cultivated with 5%
5
carbon dioxide and 16:08 h light: dark cycle produced maximum carbohydrate content
6
compared to 12:12 h and 24:00 h light: dark cycles. Moreover, conversion of starch to
7
sucrose in dark hours might be the reason for presence of high sugar content in
8
microalgae grown under 16:08 h light: dark cycle compared to cultures grown under
9
continuous illumination (Iglesias and Podest, 2005). The results of the present study
10
concluded that, 16:08 h light: dark cycle and 16,000 lx light intensity as optimum
11
condition for maximum carbohydrate accumulation in Desmodesmus sp. VIT.
12
Light intensity, nitrogen concentration and cultivation period significantly affect
13
the lipid and carbohydrate accumulation in microalgae (He et al., 2015b; Ho et al.,
14
2012). In the present study, Desmodesmus sp. VIT was cultivated in BBM for 15 days
15
at 16,000 lx and 32,000 lx light intensity and the changes in nitrate concentrationof
16
BBM, lipid and carbohydrate content of Desmodesmus sp. VIT cultivated at respective
17
light intensities were monitored at regular time intervals. As depicted in Fig. 2B and C,
18
changes in nitrate concentration of BBM were similar at two different light intensities
19
and the initial nitrate concentration (11.8 mM) was depleted after 9 days at 16,000 lx
20
and 32,000 lx. In the case of changes in biomolecule content, carbohydrate content of
21
cells at 16,000 lx increased (12.9% to 25.4%) with increase in cultivation time, whereas
22
relatively stable level of carbohydrate content was maintained in cells grown at 32,000
23
lx (Fig. 2B and C). In contrast, lipid content of Desmodesmus sp. VIT (7.6% to 17.5%)
24
grown at 32,000 lx increased with the increase in cultivation time, whereas slight
25
increase in lipid content was observed in cells (7.6% to 12.5%) grown at 16,000 lx (Fig. 17
1
2C and B). He et al. (2015b) reported that Chlorella sp. L1 and Monoraphidium
2
dybowskii Y2 carbohydrate content increased with increase in cultivation time under
3
low light intensity, which is consistent with the results obtained in the present study.
4
The current study results concluded that 15 days cultivation time is optimum for
5
maximum lipid and carbohydrate accumulation in Desmodesmus sp. VIT. at 32,000 lx
6
and 16,000 lx light intensities. In addition, variation in DCW of Desmodesmus sp. VIT
7
at 16,000 lx and 32,000 lx indicated that similar DCW was observed till 15 days of
8
cultivation period at respective light intensities (Fig. 2B and C).
9
3.6. Effects of fluctuating light intensities replicating sunny sky, intermediate overcast
10 11
and quasi overcast sky conditionson lipid and carbohydrate production Desmodesmus sp. VIT cultured under fluctuating light intensities simulating
12
sunny sky condition attained highest lipid content (13.7%). Same amount of lipid
13
content was observed under fluctuating light intensities replicating intermediate
14
overcast and quasi overcast sky conditions (Fig. 4A). The results observed in the current
15
study are in agreement with the He et al. (2015a) report, who concluded that high
16
fluctuating light intensity in outdoor cultivation was the suitable condition to achieve
17
maximum lipid content in microalgae. However, lipid content of Desmodesmus sp.
18
T28–1 (24.6%,), Desmodesmus sp. NMX451(25.5%), Scenedesmus obtusus XJ-15 (31.3
19
%) cultivated in 5 L flasks outdoor using autoclaved water was higher than the lipid
20
content of Desmodesmus sp. VIT (Xia et al., 2014). The major reason for the less lipid
21
content of Desmodesmus sp. VIT could be the high initial nitrogen concentration used
22
for cultivation compared to 0.2 g/L of urea used byXia et al. (2014). Feng et al. (2011)
23
reported enhancement in lipid content of C. zofingiensis was attained using nitrogen
24
limited condition in outdoor cultivation. Consequently, the lipid content of
18
1
Desmodesmus sp. VIT under fluctuating light intensities could be increased further by
2
implementing nitrogen limitation strategy.
3
Maximum carbohydrate content and carbohydrate productivity were observed in
4
Desmodesmus sp. VIT (16.1% and 8.6 mg/L/d) cultivated for 12 days under fluctuating
5
light intensities simulating intermediate overcast sky condition (Fig. 4A). Cultures
6
exposed to fluctuating light intensities simulating sunny sky and quasi overcast sky
7
conditions obtained almost same amount of carbohydrate. However, there was no
8
significant variation in lipid and carbohydrate content was observed in cells grown
9
under fluctuating light intensities simulating different sky conditions.The carbohydrate
10
content observed in the current study was comparable with Phaeodactylum tricornutum
11
UTEX 640 cultivated during summer and Chlorella zofingiensis G1 cultivated in
12
wastewater with 5-6% carbon dioxide during winter in outdoor environmental condition
13
(Benavides et al., 2013; Huo et al., 2012).
14
Variations in lipid, carbohydrate, DCW and nitrate concentration monitored
15
during the growth of cells under fluctuating light intensities simulating intermediate
16
overcast sky condition was illustrated in Fig. 4B. Desmodesmus sp. VIT carbohydrate
17
accumulation was gradually increased with increase in cultivation period, whereas
18
relatively stable level of lipid content was maintained during 12 days culture. In the case
19
of nitrate variation, 3.3 mM of nitrate was still left at the end of the cultivation period
20
(Fig. 4B). The cultivation time of 9 days was optimum for maximum lipid content and
21
12 days of cultivation time was optimum for maximum carbohydrate accumulation in
22
Desmodesmus sp. VIT under intermediate overcast sky condition.
23
3.7. Effects of incremental light intensity strategy on lipid and carbohydrate production
19
1
The carbohydrate and lipid content of Desmodesmus sp. VIT cultivated for 15
2
days under 16:08 h light: dark cycle in constant light intensity strategy and incremental
3
light intensity strategies was illustrated in Fig. 5A. Significantly higher lipid content
4
(22.5%) was obtained in cultures illuminated under ILIS-2. High biomass at ILIS-2
5
leads to maximum lipid productivity (15.4 mg/L/d) at respective light strategy.
6
However, same lipid productivity was obtained in cells under control and ILIS-1 (Fig.
7
5A). The lipid content and lipid productivity obtained using incremental light intensity
8
strategies was higher than the maximum lipid content and lipid productivityobtained in
9
constant light optimization experiment (Section 3.5.). The high light intensity at phase II
10 11
of ILIS-2 might be the major reason for high lipid accumulation in microalgae. Maximum carbohydrate content (18.4%) and carbohydrate productivity (10.2
12
mg/L/d) was observed in cells grown under ILIS-1compared to control and ILIS-2 (Fig.
13
5A). Cells grown under ILIS-1 and control attained similar amount of carbohydrate
14
productivity. The Carbohydrate content and carbohydrate productivity observed under
15
incremental light intensity strategy were less compared with results obtained in constant
16
light optimization experiments (Section 3.5.).
17
Changes in nitrate, lipid and carbohydrate concentration of Desmodesmus sp.
18
VIT under ILIS were illustrated in Fig. 5B and C. The similar levels of lipid and
19
carbohydrate content were maintained during first 6 days of cultivation period. The shift
20
in light intensity from 8,000 lx to 16,000 lx under ILIS-1 stimulated rapid carbohydrate
21
accumulation in cells from 6th to 15th day (Fig. 5C). On the other hand, rapid increase
22
in Desmodesmus sp. VIT lipid content under ILIS-2 was observed from 9th to 15th day
23
(Fig. 5B). However, changes in nitrate concentration was similar in different
24
incremental light intensity strategies and nitrate depleted condition was observed after
25
12 days (Fig. 5C). The 15 days of cultivation time was optimum for maximum lipid and 20
1
carbohydrate accumulation at respective ILIS-2 and ILIS-1. In the case of biomass
2
variation, cells under ILIS-2 attained higher DCW from 6th to 15th day of cultivation
3
period compared to cells grown under ILIS-1 (Fig. 5B).
4
4. Conclusions
5
Optimizationof different light supply strategies are important factors for
6
commercial microalgae biofuel production. Desmodesmus sp. VIT maximum biomass
7
(1.033 g/L) and lipid (22.5%) was observed in incremental light intensity strategy
8
(8,000 to 32,000 lx under 16:08 h light: dark cycle). Highest carbohydrate content
9
(25%) was obtained at 16,000 lx light intensity for 16:08 h light: dark cycle.
10
Desmodesmus sp. VIT produced maximum biomass and lipid underlight intensities
11
simulating intermediate overcast and sunny sky conditions.Based on the present study,
12
it is concluded incremental light intensity strategy as the suitable light supply method
13
for indoor microalgaecultivation.
14
Acknowledgement
15
The authors are thankful to the management of VIT University, Vellore for the financial
16
support to carry out the research work and necessary laboratory facilities.
17 18 19
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9 10 11 12 13 14 15 16 17 18 19 20 21 26
1
Figure captions
2 3
Fig. 1. Phylogenetic tree showing the position of Desmodesmus sp. VIT.
4
Fig. 2. Different light intensities and light: dark cycles effect on Desmodesmus sp. VIT
5
dry cell weight at the end of cultivation period (A) and time-course profiles of nitrate
6
concentration, DCW, lipid and carbohydrate content during the growth of
7
Desmodesmus sp. VIT at (B) 16,000 lx light intensity and (C) 32,000 lx light intensity.
8
Fig. 3. Dry cell weight (DCW) of Desmodesmus sp. VIT under the fluctuating light
9
intensities simulating different sky conditions in indoor photobioreactor.
10
Fig. 4. Lipid and carbohydrate content of Desmodesmus sp. VIT under fluctuating light
11
intensities simulating different sky conditions in indoor photobioreactor (A) and the
12
changes in nitrate concentration, biomass, lipid and carbohydrate content during the
13
growth of Desmodesmus sp. VIT under fluctuating light intensity simulating
14
intermediate overcast sky condition (B)
15
Fig. 5. Lipid, carbohydrate content, lipid productivity (LP) and carbohydrate
16
productivity (CP) of Desmodesmus sp. VIT under incremental light intensity strategies
17
(A) and changes in biomolecule composition and cell growth under incremental light
18
intensity strategies: (B) lipid content and DCW, (C) carbohydrate content and nitrate
19
concentration.
20 21
27
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
1 2
Table 1 Effect of different light intensities and light: dark cycles on Desmodesmus sp.
3
VIT lipid content (LC), biomass productivity (BP), lipid productivity (LP),
LC (% DCW)
BP (mg/L/d)
LP (mg/L/d)
CC (% DCW)
CP (mg/L/d)
16,000 lx 12:12 h 32,000 lx 12:12 h
8 10
36.6 ± 3.2 38.1 ± 3.4
2.9 ± 0.2 3.8 ± 0.3
19.4 ± 0.5 13.6 ± 0.3
7.1 ± 0.4 5.2 ± 0.3
16,000 lx 16:08 h 32,000 lx 16:08 h
12.5 17.5
60.3 ± 0.6 65.2 ± 5
7.5 11.4 ± 0.8
25.4 ± 0.7 16.3 ± 1.4
15.3 ± 0.6 10.6 ± 0.1
16,000 lx 20:04 h 32,000 lx 20:04 h
7.5 13.3
50.9 ± 4.9 57.5 ± 1.7
3.8 ± 0.3 7.6 ± 0.2
18.8 ± 0.1 14.6 ± 0.5
9.6 ± 0.8 8.4
7.5 16,000 lx 24:00 h 48.6 ± 1.9 3.6 ± 0.2 13.7± 1.7 32,000 lx 24:00 h 57.2 ± 3.1 7.8 ± 0.5 carbohydrate content (CC) and carbohydrate productivity (CP)
17.9 ± 0.7 14.8 ± 1.3
8.7 ± 0.2 8.5 ± 1.2
Light intensities /light: dark cycles
4 5 6
28
1 2 3 4 5
Highlights
•
6
Light affected the growth, lipid and carbohydrate content of Desmodesmus sp. VIT.
7
•
Biomass and lipid content were enhanced by incremental light intensity strategy.
8
•
16:08 h light/dark cycle was optimum for growth, lipid and carbohydrate
9 10
content. •
High lipid and less carbohydrate content observed under high light intensity.
11
29