Razmig Kandilian*, Jeremy Pruvost†, Jack Legrand†, and Laurent

Experimental Determination of Radiation
Characteristics of Nannochloropsis oculata
During Nitrogen Starvation
Razmig Kandilian*, Jeremy Pruvost†, Jack Legrand†, and Laurent Pilon*
*University of California, Los Angeles – Mechanical and Aerospace Engineering Department, Los
Angeles, CA
†Université de Nantes, CNRS, GEPEA, UMR6144, Saint-Nazaire, France
Abstract: The marine microalgae Nannochloropsis oculata is known to accumulate lipids, mainly in the form of triglycerides making them an
attractive species for renewable biodiesel production. Lipid productivity can be increased by nitrogen starvation of cells causing them to
progressively decrease their replication rate in favor of accumulating lipids (up to 70% in dry weight). In addition to nitrogen starvation, recent
studies have shown light availability in photobioreactors (PBR) and light received per cell to be important factors in the lipid productivity of the
microalgae. These depend on the incident photon flux density, microalgae cell concentration, and the radiation characteristics of the microalgae.
The latter complicates the optimization of the lipid production protocols as nitrogen deprivation also causes the cells to progressively decrease
their pigmentation thus changing their radiation characteristics. These effects were quantified by the progressive changes in the average cell
absorption and scattering cross-sections caused by modifications in specific chlorophyll and carotenoid concentrations, and cell size distribution.
Motivations
Results
Nitrogen starvation in Nannochloropsis oculata leads to large percentage of lipid accumulation2,3,4 in form
of triglycerides (TAG) ideal for biodiesel production3
"  Previous studies have shown that lipid accumulation strongly depends on the light availability and fluence
rate inside the photobioreactor (PBR)
"  Specific pigment concentration decreases.
"  Carotenoid to chlorophyll a ratio increases.
"  Microalgae size increases with lipid accumulation
Fig. 3: A 1L airlift reactor used
to cultivate N.oculata showing
light attenuation inside the PBR
1.8
7
1.6
1.4
56% total lipids
by dry weight
1.2
1
0.8
0.6
0.4
0
0
2
4
6
8
Time, t (days)
2
! Steady State radiative transfer equation
∫
! Scattering Phase Function Φλ(ŝ, ŝi)
PC
aluminum dish with
45o banked sides
Miniature Gershun tube
0.36o
half acceptance
angle
2
4
lens 1 lens 2
He-Ne laser
chopper
rotary stage
miniature gershun tube
0.8
(day 0)
λ
−1
Φ (Θ ) =
2 I (Θ )[U (Θ )]
π
−1
∫ I (Θ)[U (Θ)]
w / sin Θ
U (Θ) =
∫
0
300 µm
aperture
Laser inlet
window
Capabilities of the setup
(1)  Low signal detection using lock-in amplifier.
(2)  Small acceptance angle (1.4o) fiber-optic detector.
(3)  Sample holder has 45o banked sides to eliminate internal reflection.
(4)  Magnetic stirrer ensures homogeneous and random orientation of microorganisms.
(5)  High precision motorized rotary stage for accurate angular positioning.
(6)  Computer controlled operation.
Phase function:
fiber jacket
Optical path to detector
z
0
0
w
w )# &
&
#&
,
, w
)#
1
+
β
c
tan
Θ
−
β
L
cos
Θ
1
−
β
r
−
1
−
β
−
L
*
'
*
'! dL
$
!
$
$%
!"
2
2
sin
Θ
sin
Θ
+
(
+
("
%
"%
and
β N ,λ =
ln Fλ ( z 2 ) / Fλ ( z1 ) + ln z12 / z 22
z1 − z 2
! Absorption and Scattering Cross-sections
integrating sphere
t
transmitted beam
It,λ
incident beam
I0,λ
detector
2ΘA
transmitted
signal Ih,λ
sampl
e
detector
χ h ,λ
1 & T
= − ln$$ h,λ , X
t % Th,λ , PBS
#
!
!
"
•  Corrected absorption coefficient
κ λ = χ h ,λ − χ h ,λ
0
χ λ − χ h ,λ
χ λ − χ h ,λ
0
0.4
0.2
0
400
500
•  Absorption cross-section
1.5
600
700
1
0.5
0
400
450
500
C Abs ,λ =
κλ
N
550
600
650
700
Wavelength, λ (nm)
Wavelength, λ (nm)
4
6
8
10
1 Θa
Φ λ (Θ )sin ΘdΘ
2 ∫0
1 & T
χ λ = − ln$$ λ , X
t % Tλ , PBS
1000
White Light, 2,000 lux (exp.)
Red LEDs 2,000 lux (exp.)
Lorentz-Mie theory (white light)
Lorentz-Mie theory (red LEDs)
100
10
Θ"
1
0.1
0.01
0.001
20
40
60
80
100
120
140
160
180
(degrees)
1.2
1
Day 0
Day 1
Day 3
Day 4
Day 5
Day 6
day 7
Day 8
0.8
0.6
0.4
0.2
0
400
500
600
700
4
3
2
1
0
0
b
a
ds
As,ellipsoid=As,sphere
2
4
6
8
10
Time, t (days)
Csca ,λ =
3.  Briassoulis et al. Bioresource Technology, 101(17), 6768–6777, 2010
4.  Van Vooren et al. Bioresource Technology, 124,421-432, 2012
•  Extinction and scattering cross-sections
and
1.4
±2σ
optical fiber
(600 µm)
2.  Rodolfi et al., Biotech. and Bioeng., 102(1),100–112, 2009.
χ − ε nκ λ
βλ = λ
= κ λ + σ s ,λ
1− εn
N
1.6
5
1.  Pilon et al., Journal of Quantitative Spectroscopy and Radiative Transfer, 112(17),2639-2660, 2011.
#
!
!
"
•  Corrected extinction coefficient
C Ext ,λ =
1.8
! Scattering phase function and size distribution
References
•  Apparent extinction coefficient
βλ
x 10
Wavelength, λ (nm)
t
εn represents the portion of the light
scattered in the forward direction and
detected by the spectrometer in
directions other than the normal
direction due to the finite size of the
acceptance angle:
εn =
2
Time, t (days)
2
Day 0
Day 1
Day 3
Day 4
Day 5
Day 6
day 7
day 8
(b) normal-normal transmittance
0
0
0
10
aperture
(a) normal-hemispherical transmittance
•  Apparent absorption coefficient
0.4
•  This work accurately characterized the evolution of radiation characteristics of N.oculata during progressive
nitrogen starvation. These included:
•  Biomass, pigments, and lipid concentration, as well as lipid profile
•  Lipid content in cells increased from 8 %dw. to 56 %dw.
•  Specific pigment concentration decreased
•  Absorption and scattering cross-sections
•  Absorption decreased but scattering cross-section increased
•  This correlates with the pigment decrease and the size increase respectively.
•  Size distribution
•  Average equivalent diameter decreased in the cell division phase and increased during lipid
accumulation.
•  The results obtained will be used to optimize lipid production and design reactors for large scale lipid
production.
sin ΘdΘ
sample
0.6
!  Summary
where
monochromatic
incident beam
I0,λ
8
abs,
0.6
Polar scattering angle,
plastic
window
magnetic
miniature
stirrer
Gershun tube
magnetic bar
0.8
Scattering cross-section, Csca, λ (m )
1
0
microorganism
suspension
magnetic stirrer
6
2
0.0001
container
pinhole
1
-11
PMT
fiber optic cable
1.2
0.2
abs,
1.2
/C
λ
1.4
)
Notations:
βλ: Extinction coefficient (1/m)
Cabs,λ: Absorption cross-section (m2/
Iλ"
Out-scattering Iλ"
kg)
Cext,λ: Extinction cross-section (m2/
kg)
σ s,λ
Iλ Φ λ dΩ
Csca,λ: Scattering cross-section (m2/
∫
4π 4π
kg)
In-scattering
Θ: Scattering angle (rad)
Iλ: Spectral intensity (W/m2 µm·sr)
κλ: Absorption coefficient (1/m)
λ: Wavelength (nm)
σ s ,λ
3)
N:
Number
density
(#/m
−κ λ I λ (rˆ, sˆ) − σ s ,λ I λ (rˆ, sˆ) +
I λ (rˆ, si )Φ λ ( sˆ, si )dΩi σ : Scattering coefficient (1/m)
s,λ
4π 4π
Φλ: Scattering phase function
Ωλ: Solid Angle (sr)
1.6
T,633(
Absorption Iλ"
Day 0
Day 2
Day 3
Day 4
Day 5
Day 6
day 7
Day 8
Total scattering phase function,
Measuring Radiation Characteristics
1.8
x 10
Normalized absorption, C
2
Absorption cross-section, Cabs,λ (m )
Fig. 2: Microscope image of N.oculata
during the later stages of progressive
nitrogen starvation
"  The purpose of this study is to experimentally characterize optical and
radiative properties of N.oculata during nitrogen starvation in order to
link lipid productivity to light absorption and light availability in the PBR.
chopper controller
1.4
!  Absorption and scattering cross-sections
Fig. 1: An illustration of the difference in pigmentation and absorption of
nitrogen starved (left) and nitrogen repelete (right) N.oculata cells.
lock-in amplifier
3
Decreasing
fluence rate
Nitrogen
replete
cells
high voltage
power supply
4
1.6
Time, t (days)
-12
sˆ ⋅ ∇I λ =
5
Average equivalent diameter, ds (µm)
Nitrogen
starved
cells
6
0
0
10
Chlorophyll a
Carotenoids
1.8
1
0.2
! Changes in radiation characteristics that need to be quantified
2
Chlorophyll a
Carotenoids
Specific pigment concentration (wt.%)
"  Dramatic variations in radiation properties during nitrogen starvation
Biomass concentration
Lipid concentration
Pigment concentration (kg/m3)
"  To optimize lipid production and design large scale PBRs we need to perform radiative analysis inside the
PBR requiring accurate quantitative measurements of the optical and radiation properties of the
microalgae1.
8
2
Lipid or biomass concentration (kg/m3)
"  Incident light intensity2,4
"  Biomass concentration4
"  PBR thickness4
!  Biomass and pigment concentration
σ s ,λ
N
5.  Kandilian et al. Bioresource Technology, 2013.