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Feb 12, 2013 (4 years and 6 months ago)

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Applied Microbiology and Biotechnology

Non-destructive monitoring of carotenogenesis in Haematococcus pluvialis via whole-
cell optical density spectra
--Manuscript Draft--

Manuscript Number:
AMAB-D-12-01674R1
Full Title:
Non-destructive monitoring of carotenogenesis in Haematococcus pluvialis via whole-
cell optical density spectra
Article Type:
Original Paper
Section/Category:
Applied microbial and cell physiology
Corresponding Author:
A. E. Solovchenko, Ph.D.
Moscow State University, Faculty of Biology
Moscow, RUSSIAN FEDERATION
Corresponding Author Secondary
Information:
Corresponding Author's Institution:
Moscow State University, Faculty of Biology
Corresponding Author's Secondary
Institution:
First Author:
A. E. Solovchenko, Ph.D.
First Author Secondary Information:
Order of Authors:
A. E. Solovchenko, Ph.D.
Claude Aflalo, Ph.D.
Alexander Lukyanov, Ph.D.
Sammy Boussiba, Prof.
Order of Authors Secondary Information:
Abstract:
We investigated the feasibility of rapid, non-destructive assay of carotenoid-to-
chlorophyll ratio (Car/Chl) and total carotenoids (Car) in cell suspensions of the
carotenogenic chlorophyte Haematococcus pluvialis Flotow under stressful conditions.
Whole cells spectra are characterized by variable non-linear contributions of
carotenoids (Car) and chlorophylls (Chl), with a strong influence of Car packaging and
sieve effect inherent to stressed H. pluvialis cells. Nevertheless, non-destructive assay
of Car/Chl in the range of 0.55-31.2 (Car content up to 188 mg * L-1; 5.4% of the cell
dry weight) turned to be achievable with a simple spectrophotometer lacking an
integrating sphere upon deposition of the cells on glass fiber filters. The scattering-
corrected optical density in the blue-green region of the whole-cell spectrum,
normalized to that in the red maximum of Chl absorption (OD500/OD678) was tightly
related (r2 = 0.96) with the Car/Chl ratio found in extracts. Some features such as the
amplitude and position of the first derivative optical density whole-cells spectra
exhibited also a strong (r2 > 0.90) non-linear correlation with Car/Chl. These spectral
indices were also tightly related with Car but the slope of the relationship varied with
the stressor intensity. The importance of calibration over the widest possible range of
pigment contents and a correct choice of biomass load per filter are emphasized. The
advantages and limitations of non-destructive monitoring of carotenogenesis in H.
pluvialis are discussed in view of its possible application in optical sensors for lab
cultivation and mass production systems of the algae.
Response to Reviewers:
{for the formatted text of the reply, see attachment}
Dear Dr. Schmidt-Dannert,
Powered by Editorial Manager® and Preprint Manager® from Aries Systems Corporation
Thank you very much indeed for evaluation of the paper and for its sending to two
referees who turned to be remarkably competent, critical, and constructive. In our
answer we would like to address the criticism expressed by the Referees. To make the
corrections apparent and to avoid compilation of lengthy lists, the changes were made
in the ‘Tracking Changes’ mode with lines numbered on each page. For the
convenience of the Editorial we also included the same file with changes accepted. We
hope that in the revised version we have answered the questions/points raised.
Sincerely yours
Dr. Alexei E. Solovchenko.
Dept. Bioengineering, Faculty of Biology
Moscow State University
GSP-1 Moscow 119234 Russia
Tel. +7(495)9392587
Fax: +7(495)9394309
Reviewer #1
…a direct correlation between optical absorption properties of the cells and their
cellular carotenoid (or astaxanthin) level is not established in this work, apparently
because the specific conditions of induction affected the relationship between
parameters. This also brings concern on the general validity of the procedure to
evaluate the carotenoid/chlorophyll ratio, since the evolution of carotenogenesis in
Haematococcus might depend upon the stress conditions or the specific stressor
applied.
Actually, a strong direct correlation with cellular Car content does exist but, as we note,
the relationships between absolute Car content and optical density in the blue-green
region of the spectrum were different under conditions differing in the intensity of
stress. There we found it essential to take into account the timing of the decrease of
Chl content occurring in parallel with accumulation of Car as an intrinsic marker of the
cell physiological condition. As a result, the relationships of Car/Chl ratio with optical
density of H. pluvialis cells deposited on a glass-fiber filter were uniform regardless of
the conditions employed, supporting the viability of the proposed approach.
Moreover, according to our tests, a model relating optical density of the cells deposited
on filter with absolute Car content, after calibration under specific conditions promoting
carotenogenesis, was valid under these conditions, further strengthening the possibility
of non-destructive Car assay in stressed H. pluvialis cells (though we refrained from
discussing this aspect in detail in the present work for the sake of brevity).
...Nevertheless, the results reported are considered of limited practical interest for a
standard use of the procedure in either basic research or applied uses. The
expectations to have at one's disposal a simple and straightforward spectrophotometric
procedure to determine cellular carotenoid (astaxanthin) level, that can replace current
analytical procedures, are not fulfilled…
We would like to note that we did not really intend to devise a technique which would
totally replace the traditional ‘wet’ methods. Though the proposed approach has its
merits even for the routine analysis (rapidity, resistance to artifacts relating with
completeness of the pigment extraction etc.), it seems that its potential would be better
implemented in real-time applications where the traditional pigment assay is of limited
applicability. As an example, we quote optical sensors for photobioreactors for large-
scale cultivation. The proof-of-concept development of such a sensor is under way at
the Biological Faculty (MSU).
Powered by Editorial Manager® and Preprint Manager® from Aries Systems Corporation
It has to be noted that not every regular simple spectrophotometer can be used for the
proposed measurements. The instrument should be designed as to allow the use of
solid samples (filters on which a cell layer is deposited) as well as have the capacities
to perform the required spectra corrections ( i) smooth the obtained 400-800 nm
spectrum with 3-point moving average algorithm and baseline shift to yield zero optical
density at 800nm; and ii) normalize the corrected spectrum to the red chlorophyll
optical density maximum at 678 nm). This is not just reading absorbance at 500 nm.
On the contrary, we emphasize that any modern conventional (i.e. lacking
sophisticated accessories such as integrating sphere or solid sample holder)
spectrophotometer could be used for implementation of the proposed approach. In
particular, we used the simplest member of Cary model line attaching the filters directly
on the vertical wall of cuvette compartment over the exit window (the wet filters behave
in a way like a self-adhesive tape). The (successful) tests obtained with other
spectrophotometric equipment are not described, again for the sake of brevity.
All spectral processing could be carried out with little effort using a spreadsheet like
Excel or Origin. Furthermore, the calculations could be stored as a template making
the subsequent processing as simple as pasting the spectral data into the spreadsheet
(in a real-time system, all the processing could be done on the fly in the microcode).
Taking the above-mentioned into account, one can see that the spectral data
processing steps employed in the work pose no limitation for the practical application
of our findings. Corresponding notes are added to the text of the revised version of the
paper.
Additional comments:
-Absorbance and optical density are interchangeable terms. Nevertheless, in the
manuscript they are used with different meanings.
-The alternative use of OD500/OD678 and ODN500 is confusing. For example
ODN500 is used in Figure 5, but the figure legend refers to "changes in optical density
at 500 nm normalized to the red chlorophyll maximum", whereas OD500/OD678 is the
term used in the corresponding text (page 8, line 29). Homogenization of notations and
concepts will facilitate understanding of the main points.
The Reviewer is right. ODN500 was replaced for OD500/OD678.
-Some statistical analysis of the data will be appreciated.
Since we do not have to differentiate mean values or solve a similar problem, the work
does not involve sophisticated statistical analysis. Still we were able to estimate the
significance of the correlations. In the revised version, we introduced the subsection
‘Statistical treatment’ into the Materials and Methods section.
Reviewer #2
…Additionally, this reviewer feels there's potential for additional impact in other algal
culture applications and possibly even in real-time or in-situ measurements. The
authors do not discuss these possibilities. If this were included, the impact would be
wider…
Actually we touched the mater briefly in the end of the Discussion section. In the
revised version, we tried to include the aspects suggested by the Reviewer into
conclusions, extending the utility of the approach to other systems under stress.
Additional comments:
1.Page 4, Line 59-60: The method described in this paper differs from that of
Solovchenko 2009 In that the cells are deposited on the filters rather than sandwiched
between the filters. These sentences are misleading and should be changed to
something like "a technique similar to that of Solovchenko 2009?"
We agree and follow the recommendation of the Reviewer.
2.The argument that the anomalies seen in the absorption spectra at high carotenoid
accumulations arise from a sieving effect or packing effect are certainly supported
however in several places the authors refer to the astaxanthin as accumulating in lipid
Powered by Editorial Manager® and Preprint Manager® from Aries Systems Corporation
bodies, or small globules (e.g. Page 10, lines 6 & 27) . While the 2011 PlosONE paper
by Collins et. al. shows some evidence for ast in lipid bodies at intermediate stages of
carotenogenesis, their work does not definitively show discrete packaging of ast in late
stage aplanospores. It is possible that the ast is packaged in bodies smaller than the
spatial resolution of the microscope used in Collins' work, though this reviewer would
have expected lipid bodies in close proximity to coalesce to form larger bodies as this
has been observed in other algae (several papers by Samek and Pilat 2012 J Applied
Phycology). Additional discussion is needed to address these observations.
We are sorry but we did not claim that the packaging is different in the oil bodies of
different size. By the way, Collins et al. used Raman spectroscopy whereas in our work
optical spectroscopy is used so the evidence on the extent of Ast packaging is hardly
comparable in these cases (at least directly). At the same time using the term
‘packaging’ we meant mostly the extent of mutual shading of Ast molecules. It may well
be that the effect of packaging is not directly related with the oil body size but with local
Ast content in the latter. We extended the discussion and included the references
suggested by the Reviewer.
Powered by Editorial Manager® and Preprint Manager® from Aries Systems Corporation






Dear Dr. Schmidt
-
Dannert,




Thank you very much indeed for evaluation of the paper and for its sending to two referees who
turned to be remarkably competent, critical, and constructive. In our answer we would like to
address the criticism expressed

by the Referees. To make the corrections apparent and to avoid
compilation of lengthy lists, the changes were made in the ‘Tracking Changes’ mode with lines
numbered on each page. For the convenience of the Editorial we also include
d

the same file with
ch
anges accepted. We hope that in the revised version we have answered the questions/points
raised.







Sincerely yours

Dr. Alexei E. Solovchenko.

Dept. Bioengineering, Fac
ulty

of Biology

Moscow State University

GSP
-
1 Moscow 119234 Russia

Tel. +7(495)93925
87

Fax: +7(495)9394309


Cover Letter
1




Reviewer #1


…a direct correlation between optical absorption properties of the cells and their cellular
carotenoid (or astaxanthin) level is not established in this work, apparently because the specific
conditions of induction affected the relationship

between parameters.

This also brings concern
on the general validity of the procedure to evaluate the carotenoid/chlorophyll ratio, since the
evolution of carotenogenesis in Haematococcus might depend upon the stress conditions or

the
specific stressor ap
plied.


Actually, a strong direct correlation with cellular Car content does exist but, as we
note, the relationships between absolute Car content and optical density in the blue
-
green region of the spectrum were different under conditions differing in the

intensity
of stress. There we found it essential to take into account the timing of the decrease of
Chl content occurring in parallel with accumulation of Car as an intrinsic marker of
the cell physiological condition. As a result, the relationships of Ca
r/Chl ratio with
optical density of
H. pluvialis

cells deposited on a glass
-
fiber filter were uniform
regardless of the conditions employed, supporting the viability of the proposed
approach.

Moreover, according to our tests, a model relating optical density of the cells
deposited on filter with absolute Car content, after calibration under specific
conditions promoting carotenogenesis, was valid under these conditions, further
strengthening th
e possibility of non
-
destructive Car assay in stressed
H. pluvialis

cells
(though we refrained from discussing this aspect in detail in the present work for the
sake of brevity).


...
Nevertheless, the results reported are considered of limited practical i
nterest for a standard
use of the procedure in either basic research or applied uses. The expectations to have at one's
disposal a simple and straightforward spectrophotometric procedure to determine cellular
carotenoid (astaxanthin) level, that can replac
e current analytical procedures, are not fulfilled





We would like to note that we did not really intend to devise a technique
which would totally replace the traditional ‘wet’ methods. Though the proposed
approach has its merits even for the routine ana
lysis (rapidity, resistance to artifacts
relating with completeness of the pigment extraction etc.), it seems that its potential
would be better implemented in real
-
time applications where the traditional pigment
assay is of limited applicability. As an ex
ample, we quote optical sensors for
photobioreactors for large
-
scale cultivation. The proof
-
of
-
concept development of
such a sensor is under way at the Biological Faculty (MSU).


It has to be noted that not every regular simple spectrophotometer can be use
d for the proposed
measurements. The instrument should be designed as to allow the use of solid samples (filters on
which a cell layer is deposited) as well as have the capacities to perform the required spectra
corrections ( i) smooth the obtained 400
-
800

nm spectrum with 3
-
point moving average
algorithm and baseline shift to yield zero optical density at 800nm; and ii) normalize the
corrected spectrum to the red chlorophyll optical density maximum at 678 nm). This is not just
reading absorbance at 500 nm
.


Authors' Response to Reviewers' Comments
Click here to download Authors' Response to Reviewers' Comments: Reply2Reviewers.docx
2



On the contrary, we emphasize that any modern conventional (i.e. lacking
sophisticated accessories such as integrating sphere or solid sample holder)
spectrophotometer could be used for implementation of the proposed approach. In
particular, we used th
e simplest member of Cary model line attaching the filters
directly on the vertical wall of cuvette compartment over the exit window (the wet
filters behave in a way like a self
-
adhesive tape). The (successful) tests obtained with
other spectrophotometric

equipment are not described, again for the sake of brevity.


All spectral processing could be carried out with little effort using a
spreadsheet like Excel or Origin. Furthermore, the calculations could be stored as a
template making the subsequent proces
sing as simple as pasting the spectral data into
the spreadsheet (in a real
-
time system, all the processing could be done on the fly in
the microcode). Taking the above
-
mentioned into account, one can see that the
spectral data processing steps employed in

the work pose no limitation for the
practical application of our findings
. Corresponding notes are added to the text of the
revised version of the paper.


Additional comments:

-

Absorbance and optical density are interchangeable terms. Nevertheless, in
the
manuscript they are used with different meanings.

-

The alternative use of OD500/OD678 and ODN500 is confusing. For example
ODN500 is used in Figure 5, but the figure legend refers to "changes in optical density at 500
nm normalized to the red chloroph
yll maximum", whereas OD500/OD678 is the term used in the
corresponding text (page 8, line 29). Homogenization of notations and concepts will facilitate
understanding of the main points.


The Reviewer is right. OD
N
500

was replaced for
OD
500
/OD
678
.


-

Some statistical analysis of the data will be appreciated.



Since we do not have to differentiate mean values or solve a similar problem,
the work does not involve sophisticated statistical analysis. Still we were able to
estimate the significance
of
t
he
correlations. In the revised version, we introduced the
subsection ‘Statistical

treatment’ into the Materials and Methods section.


Reviewer #2


…Additionally, this reviewer feels there's potential for additional impact in other algal culture
applicatio
ns and possibly even in real
-
time or in
-
situ measurements. The authors do not discuss
these possibilities. If this were included, the impact would be wider…



Actually we touched the mater briefly in the end of the Discussion section. In the
revised version, we tried
to include the aspects suggested by the Reviewer into
conclusions
, extending the utility of the approach to other systems under stress.


Additional

comments:

1.

Page 4, Line 59
-
60: The method described in this paper differs from that of
Solovchenko 2009 In that the cells are deposited on the filters rather than sandwiched between
the filters. These sentences are misleading and should be changed to so
mething like "a technique
similar to that of Solovchenko 2009?"


We agree and follow the recommendation of the Reviewer.


3



2.

The argument that the anomalies seen in the absorption spectra at high carotenoid
accumulations arise from a sieving effect or pac
king effect are certainly supported however in
several places the authors refer to the astaxanthin as accumulating in lipid bodies, or small
globules (e.g. Page 10, lines 6 & 27) . While the 2011 PlosONE paper by Collins et. al. shows
some evidence for as
t in lipid bodies at intermediate stages of carotenogenesis, their work does
not definitively show discrete packaging of ast in late stage aplanospores. It is possible that the
ast is packaged in bodies smaller than the spatial resolution of the microscop
e used in Collins'
work, though this reviewer would have expected lipid bodies in close proximity to coalesce to
form larger bodies as this has been observed in other algae (several papers by Samek and Pilat
2012 J Applied Phycology).
Additional discussio
n is needed to address these observations.



We are sorry but we did not claim that the packaging is different in the oil
bodies of different size. By the way, Collins et al. used Raman spectroscopy whereas
in our work optical spectroscopy is used so the evidence on the extent of Ast
packaging is har
dly comparable in these cases (at least directly). At the same time
using the term ‘packaging’ we meant mostly the extent of mutual shading of Ast
molecules. It may well be that the effect of packaging is not directly related with the
oil body size but wit
h local Ast content in the latter.
We extended
the
discussion and
included the references suggested by the Reviewer.


For the detailed list of amendments, see the text of manuscript in ‘tracking changes’ mode
below.
4


N
on
-
destructive
monitoring of carotenoge
nesis
in
Haematococcus pluvialis

via

whole
-
cell
optical
density

spectra


Alexei Solovchenko,
1,3
*

Claude Aflalo,

2

Alexander Lukyanov,

1

and Sammy Boussiba
2


1
Department of Bioengineering, Faculty of Biology, Moscow State University, 119
234
, GSP
-
1
Moscow,
Russia
.

Phone: +7(495)939
-
25
-
87

E
-
mail: solovchenko@mail.bio.msu.ru



2
Microalgal Biotechnology Laboratory, the Jacob Blaustein Institutes for Desert Research, Ben
-
Gurion University of the Negev, Sede
-
Boker Campus,
Midreshet Ben
-
Gurion
84990
, Israel


3
Timi
ryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya ul. 35,
Moscow, 127276 Russia



Running title: Non
-
destructive monitoring of carotenogenesis


*

To whom correspondence should be addressed

5


ABSTRACT

We investigated the
possi
bility
feasibility

of
rapid,
non
-
destructive
express
assay of carotenoid
-
to
-
chlorophyll ratio
(Car/Chl) and total carotenoids (Car) in cell suspensions of the carotenogenic chlorophyte
Haematococcus pluvialis

Flotow under stressful conditions. Whole cells spectra are characterized by variable non
-
linear contributions of
carotenoids (
Car
)

and chlorophylls (Chl), with a strong influence of
pigment
Car

packaging and sieve effect inherent
to stressed
H. pluvialis

cultures
cells
. Nevertheless, non
-
destructive assay of Car/Chl in the range of 0.55

31.2 (Car
content
up to

187
18
8

mg ∙ L

1
; 5.4% of the cell dry weight) turned to be
feasible
achievable

with a simple
spectrophotometer lacking an integrating sphere upon
deposition of the cells on glass fiber filters. The scattering
-
corrected optical density in the blue
-
green region of the

whole
-
cells

spectrum
,

normalized to that in the red
maximum of Chl absorption
(
OD
500
/
OD
678
)

was tightly related

(
r
2

= 0.96)

with
Car co
ntent and
the

Car/Chl ratio

found in extracts
.

A simple
OD
500
/
OD
678

ratio was tightly related with Car/Chl (
r
2

= 0.96) under our experimental
conditions
. Some features
such as

the

(
amplitude and position
)

of the
1
st

first

derivative optical density
whole
-
cells
spectra exhibited
also a
strong (
r
2

> 0.90) non
-
linear correlation with Car/Chl. These spectral indices were also
tightly related with Car but the slope of the relationship varied with the stressor intensity. The importance of
calibration over
the widest possible range of pigment contents and a correct choice of biomass load per filter are
emphasized. The advantages and limitations of non
-
destructive monitoring of carotenogenesis in
H. pluvialis

are
discussed in view of its possible application
in optical sensors for lab cultivation and mass production systems of the
algae.

Key words: astaxanthin, carotenogenesis, derivative spectroscopy,

Haematococcus,
non
-
invasive
assay, secondary carotenoids

INTRODUCTION

The chlorophyte
Haematococcus pluviali
s

Flotow is one of the most biotechnologically important
microalgal species
(
Del Campo et al. 2007
)
, the richest natural source
of
of the

ketocarotenoid
astaxanthin (Ast)
(
Sussela and Toppo 2006
)
.
The
ketocarotenoid
Ast exerts a plethora of beneficial effects on human health, it is also
a valuable feed additive in aquaculture
(
Guerin et al. 2003
;
Boussiba 2000
)
. In
H. pluvialis
, accumulation of Ast as a
maj
or (over 95% of total carotenoids
(
Lemoine and Schoefs 2010
)
) seconda
ry carotenoid (Car) takes place under
stressful conditions (high irradiance, nutrient limitation, high salinity, etc.), in parallel with
a
reduction
of
in
chlorophyll (Chl) content, and other cellular responses
(
Lemoine and Schoefs 2010
)
. It is believed that Ast, mainly
in the form of fatty acid esters
(
Zhekisheva et al. 2002
;
Boussiba 2000
)
, localized in cytoplasmic lipid globules

or
oil bod
ies
(
Peled et al. 2011
)

plays a role in protection of microalgal cells against photooxidative damage
via

optical screening and/or elimination of reactive oxygen species
(
Solovchenko 2011
;
Wang et al. 2003
;
Li et al.
2008
)
, although the extent of its involvement is s
ometimes difficult to assess
(
Fan et

al. 1998
)
. While the pattern of
the physiological responses to stress described above is conserved for various stress conditions, their extent
6


depend strongly on the stress intensity, as it is sensed by the algal cells. As a consequence, quantitative r
elations
between stress and response are often elusive and poorly reproducible
(
Hu et al. 2008
;
Sarada et al. 2002
;
Zhekisheva et al. 2005
)
.

The investigation of Car accumulation occurring in
H. pluvialis

on the background of Chl degradation is of
considerable importance for basic as well as
for
applied research. Studies of the relation between accumulation of
secondary Car, increase in Car/Chl ratio and change in light absorption by the microalgal culture provide valuable
information about acclimation of the photoautotrophic microorganisms to str
essors
(
Hu et al. 2008
;
Sarada et al.
2002
)
. In microalgal biotechnology, the information on the dynamics of Car content in biomass is essential for
timely and informed decisions on adjustment of illumination conditions, medium composition and on the time

for
biomass harvesting
.

Traditionally, the Car and Chl content is determined by ‘wet’ chemical methods with the use of
spectrophotometry or chromatography
(
Zhekisheva et al. 2005
)

which are time
-
consuming, expensive, and not
readil
y available in the field or at mass cultivation facilities. These considerations
make apparent the
demand
emphasize the need

for a reliable, rapid and preferably non
-
destructive technique for
express
fast
appraisal

assessment
of
the relative
Car content in
microalgal cultures

Remarkably, the
engagement
onset

of protective mechanisms based on the build
-
up of Ast
-
containing oil
bodies in the cell is accompanied by specific changes in the optical properties of the algal cells
(
Solo
vchenko 2011
)
.
Recent reports show that Ast presence and subcellular distribution
in vivo

could be characterized
(
Kaczor et al.
2011
)

and even distinguis
hed from β
-
carotene
(
Collins et al. 2011
)

using advanced spectral techniques such as
Raman spectroscopy. However, current understanding on the effects of Ast accumulation on the optical properties of

H. pluvialis

cell suspension is clearly insuffic
ient to develop non
-
destructive spectroscopic tools suitable for on
-
line
monitoring of Car in lab or industrial mass cultures of
H. pluvialis
.

A solution to this problem would require a better understanding of the spectroscopy of
H. pluvialis

cell
suspensi
ons and the relationships between whole
-
cell spectra and contributions of different pigments to overall light
absorption in the process of carotenogenesis. Recently, we successfully employed cell suspension optical density
measurements to develop an algori
thm for gauging high
-
light tolerance of
H. pluvialis
cultures
(
Solovchenko 2011
)
.
In the present work we show the possibility of obtaining of a quantitative description of carotenogenesis in stressed
H. pluvialis

culture
s with a simple spectrophotometer lacking integrating sphere. We also for the first time report the
relationships between Car/Chl and spectral features of
H. pluvialis

whole
-
cell optical density

which form the
foundation for the development of techniques f
or non
-
invasive assa
y of Car accumulation in
H. pluvialis

cells
.
It is
Formatted
Formatted
Formatted
Formatted
7


important to realize
that
t
h
is
e

technique
approach

is not intended to replace traditional pigment
s

determination
assay
,

but to
complement it
,

provid
ing

a
meaning
ful index for the extent of stress in
Haematococcus

cultures.


We also
believe that
which
I
i
t

c
w
ould

facilitate
also

be employed
extended

in the
further
the
development of
techniques for
non
-
destructive
algorithms for
non
-
destructive
real
-
time monitoring of
pigment assay
carotenogen
ic
other


micro
algae cultivated under stressful conditions
.

MATERIALS AND METHODS

Cultivation conditions

The unicellular chlorophyte
Haematococcus pluvialis

Flotow 1844 em. Wille K
-
0084 was obtained from the
Scandinavia Culture Center for Algae and Protozoa at the University of Copenhagen, Denmark. The algae were
cultivated on modified BG11 medium
(
Boussiba and

Vonshak 1991
)

in 0.4 L glass columns (5 cm ID) under constant
illumination provided by daylight fluorescent lamps. The exponentially growing cells (referred to as 'green' cells)
initially grown at the irradiance of 35
μ
E



m

2



s

1

were transferred to

nitrate
-
free medium at the irradiance of 350
μ
E



m

2



s

1

as measured by a LiCor 850 quantometer (LiCor, USA) in the center of an empty column. The cultures
were constantly bubbled with CO
2
: air mixture (2: 98, v/v) at 25 °C and the pH of the culture
under these
conditions was in the range 7.0

7.8.

Pigment
s

extraction and analysis

Total Chl and Car were extracted from microalgal cells pelleted by centrifugation with dimethyl sulfoxide
(DMSO) for 5 min at 70 °C with 5

mL per
ca
. 3.5 mg DW. The pigment c
oncentrations were routinely determined
spectrophotometrically
in dimethyl sulfoxide (DMSO) extracts
spectrophotometrically
with a Cary 50 Bio
spectrophotometer (Varian, USA)
(
Solovchenko et al. 2010
)
. The extracts were diluted such that the absorbance at
pigment maxima was kept in the range 0.2

0.8. In certain experiments the pigment content in
H. pluvialis

cells was
determined in acetone

extracts after separation using an HPLC chromatography system equipped with a
photodiode array detector (Varian Analytical Instruments, Walnut Creek, CA, USA) as previously described
(
Zhekisheva et al. 2005
;
Peled et al. 2011
)
.

Filter spectra measurement and processing

To reduce the influence of scattering and a rapid sedimentation of cells on a cuvette floor, a t
echnique for
measuring turbid microalgal samples
similar to that
described in
(
Solovchenko et al. 2009
)

was used. Cells were
8


deposited on 25
-
mm GF/F glass
-
wool filters (Schleicher & Schuell, Germany)
.
The wet filters were mounted with
cells facing the detector on the output window of the cuvette compartment.
The optical density of the filters,
OD
,
was expressed as

log
10

T
, where
T

is transmittance (Fig. 1a). The spectra were taken in the range 400

800 nm
with subtraction of the values of a base line provided by an empty filter soaked in the culture medium. Th
e
corrected spectra,
OD
(
λ
), were obtained by smoothing the measured spectra with 3
-
point moving average
algorithm and baseline shift to yield zero optical density at 800 nm. The
OD
(
λ
) spectra were normalized to the red
Chl optical density maximum at 678 nm

yielding
OD
N
(
λ
)
/
OD
678

spectra (Fig. 1b). Finally, the 1
st

derivative of the
OD
(
λ
)/
OD
678
OD
N
(
λ
)
spectra

(
OD
N
(
λ
)')

(Fig. 1c)
was calculated
using routine spreadsheet equation
(Fig. 1c)
to
estimate the steepness of the slope in the range
550
520
-
600 nm and
the position of the associated inflexion point.

This part of the

original spectra is
further
referred to as the

green edge
’.

The coefficient of variation on spectral
measurements was routinely under 5%.

Statistical treatment

The results of three
independent experiments are presented in the figures.

Where appropriate, averages
and standard errors of the mean were calculated and displayed.
All correlations are significant at P < 0.001 level.

RESULTS

The dynamics of biomass accumulation and pigment c
ontent

In routine experiments the cells of
H. pluvialis

cultures were incubated under conditions favoring
carotenogenesis (nitrogen
-
free medium, 350
μ
E



m

2



s

1

irradiance) for 6 days. At the beginning of cultivation
(
day
0

d
)
,

the
H. pluvialis

cells were typically characterized by a high Chl content (1.8

2.0% DW) and a
correspondingly low Car content (0.5

1.1% DW). The Car profile at this stage was dominated by lutein and
β
-
carotene, containing also neoxanthin, violaxanthin, and zeaxanthin as t
he minor Car, but only traces of Ast were
detectable (not shown). After 6 days under the stress conditions, the algae steadily accumulate biomass
(increasing by 4 fold), while the cell density remained relatively unchanged (Fig. 2a). The Chl content steadi
ly
decreased by more than 3 fold, while total carotenoid content increased by 17 fold (Fig. 2b), the same applies to
specific Car content (Fig. 2c). Consequently, the Car/Chl weight ratio increased spectacularly (by more than 31 fold;
Fig. 2b, curve
3
), in
dicating this is the most sensitive and unequivocal indicator of stress. The proportion of total
Formatted:

Font:

10

pt,

English

(U.S.)
9


Ast (in free and fatty acid
-
esterified forms), negligible before the onset of stress, increased sharply, reaching > 95%
of total Car by the end of the experime
nt (not shown).


Optical density spectra of whole
H. pluvialis

cells

Our preliminary attempts to record conventional optical density spectra of
H. pluvialis

cell suspensions in a
standard 1
-
cm cuvette gave unsatisfactory results due to rapid sedimentation
of the algal cells on the cuvette floor
and to the strong influence of light scattering (data not shown), stressing the need for means to correct the
contribution of the latter.

One may circumvent the use of an integrating sphere for the measurement of op
tical density in cell
suspension by acquiring spectra from cells deposited on a wet glass fiber filter using a conventional
spectrophotometer. The wet filter is positioned over the photomultiplier window, with cells away from the
incident beam. In this cas
e too, it is critical to correct for light scattering. Using the spectrum of a wet empty filter
for base line correction allowed obtaining reliable spectral data in a wide range of cell density and Car content of
the culture, and over the whole spectral ra
nge studied (see Materials and Methods). A further correction proved
to be necessary to eliminate the contribution of scattering by the cells, with the extent of the correction depending
on the amount of biomass loaded on the filter (see
e.g.

the 750

800 n
m range in Fig. 1a). The optical density at
800 nm (where no pigment absorbs) was used as a blank correction for this purpose. As a result, the corrected
OD
(
λ
) spectra were very close to baseline in the 750

800 nm range indicative of a great decrease in th
e
contribution of scattering.

The scattering
-
corrected optical density spectra of
H. pluvialis

cells deposited on a wet filter, acquired
without the use of integrating sphere, at different physiological states (from vegetative green cells to stressed red
c
ysts with high Car/Chl) are presented in Fig. 3, in comparison with the conventional spectra of the pigments
extracted from the same samples. The spectra of the whole cells (Fig. 3a) present profound differences with those
for the extracts (Fig. 3b), in wh
ich pigments are in dilute solution. In particular, the amplitudes of the pigment
absorption bands in the red and in the blue
-
green parts of the spectrum were lower, the bands were broader, and
the peaks were less resolved in the whole cell spectra than in

the spectra of the pigment extracts, apparently due
to a sieving effect and/or aggregation of the molecules of the pigments (see Discussion below). Remarkably, the
10


magnitude of the difference increased along with accumulation of Car and loss of Chl; the h
ighest difference
between whole
-
cells and extracts spectra was recorded for the cells with the highest Car content (
cf
. spectral
curves in Figs. 3a and 3b).

The data presented in Fig. 1 are representative of suspensions subjected to intense and prolonged stress
achieving very high Car content and Car/Chl to test the feasibility of the suggested approach. Different amounts of
biomass were loaded upon filters. T
he raw spectra (Fig. 1a) indicate that, similarly to spectra recorded with an
integrating sphere (see spectra in
(
Solovchenko 2011
)
), only the Chl absorption is well resolved (peak centered at
678 nm), while the combined

contribution of Car and Chl in the blue
-
green region of the spectrum appears as a
broad flat band in the range 400

550 nm. After subtraction of
OD
800

and normalization to
OD
678

the spectra
essentially coincided (see
e.g.

Fig. 1b), revealing the quasi
-
line
ar dependence between optical density and the
amount of biomass loaded on the filter in the range 0.4
-
4.5 mg DW/filter, corresponding to
OD
500

of 0.04

0.78.

The analysis of the relationship between
OD
(

)

and the amount of biomass applied to the filter with
different Car/Chl in the diluted suspensions showed that the relation ‘
OD
(

)

vs
. DW on filter’ was nearly linear in
the range 0.4

4.5 mg DW in all cases studied (see
e.g.

the data in Fig. 4). This relat
ionship departed from linearity
at higher
OD
500

(> 0.9) in the case of ‘green’ cell suspension (low Car/Chl), but less in the case of ‘red’ cells with
high Car/Chl, apparently due to stronger aggregation of Car and sieve effect in the latter case. Notably,

the higher
the Car/Chl ratio of the samples taken at different cultivation stages, the lower the slope of the linear part of the
relationships ‘
OD
500

vs
. DW on filter’ (Fig. 4). This effect was also apparent with
OD
678

(not shown).


Changes of the optical

density spectra of
H. pluvialis

cells in the course of
carotenogenesis

Cultivation of
H. pluvialis

under stressful conditions brought about profound changes in the optical density
spectra of the microalga (Fig. 3). The stress
-
induced carotenogenesis was a
ccompanied by the disappearance of
spectral details in the blue
-
green region resulting in formation of a broad (
ca.

150 nm wide), almost featureless
band with an abrupt longer
-
wavelength slope.
Interestingly, the amplitude of raw
OD
(

)

in this band apparently
possessed a weak relationship with Car or Chl (
r
2

< 0.5).
In particular
A
, a

considerable decrease of the amplitude in
the band of
the
specific

Chl absorption
optical density contributed by Chl
in the red (Fig. 3a) took place along with
dramatic
a
n

overall
increase
reduction

of
the
relative
absorption of light
that

in the blue
-
green region, where
Formatted
11


absorption by both Car and Chl occurs
, despite the increase in Car content
.
The latter phenomenon
On
the other
hand
, an increase in
Car
absorbance

was readily apparent in the extract spectra (Fig. 3b)
at intermediate Car/Chl
ratios; however, at higher Car/Chl, a saturation e
ffect
becomes obvious, equivalent to that expected from the Beer
law at high
chrom
ophore

concentrations.
whereas
in whole
-
cell spectra

it

was
revealed only

after normalization to
the red Chl maximum.

In order to rule out a possible interference from variable Chl background disturbing the relationship ‘
OD
(

)

vs.

pigment content or ratio’, we tried to relate the
OD
(

)

spectra normalized to Chl red absorption maximum,
OD
500
/
OD
678
OD
N
(

)
, with Car or Car/Chl. Normalization revealed, apart from a decline of Chl contribution to light
absorption by the microalgal
cells, a dramatic increase in the absorption in the 400

550 nm range with flattening
of the spectrum shape and an increase in the longer
-
wavelength slope of this band (
Fig. 3c,
which may be called

g
G
reen
e
E
dge


as an analog of
by analogy to

the
Red
‘red
Edge
edge’
(
Gitelson et al. 1996
)

in plants

(
Gitelson et al.
1996
)
; Fig. 3c
). This effect was even more pronounced in the extract spectra (Fig. 3d).


By contrast, t
T
he amplitude of
the
OD
500
/
OD
678


OD
N
(

) spectra
ratio

was
proportional
correlated
to
the Car
content
and
Car/Chl

mass ratio

and
to a lesser extent to
the Car content (not shown)
,
in the broad band 400

550
nm


(not shown)
. Thus, the ratio
OD
500
/
OD
678

was linearly related with
the
Car/Chl


ratio at Car/Chl < 20 (
r
2

= 0.95);
at higher values of the ratio
,

this relationship departed from linearity but remained uniform and tight under all
conditions investigated (
cf
. solid and broken lines in Fig. 5). The relationships ‘
OD
500
/
OD
678

vs
. Car’ w
ere
as

also
linear in the wh
ole range studied
,

(
r
2

> 0.96)
but the slope of the relationship
varied
was variable and
depending
dependent
on
the
stress intensity
experienced by the cultures

(data not shown)
,

presenting therefore a lesser
diagnostic value
.

Notably, due to stress
-
induced carotenogenesis, which brought about the broadening and flattening of
the
OD
500
/
OD
678
OD
N
(

)

spectra, the longer
-
wavelength slope of the broad absorption band in the blue
-
green
(
Green
green e
E
dge) became more abrupt and moved towards longer wavelengths (Fig. 6c). This effect was readily
apparent as a bathochromic shift and an increase in the amplitude of the characteristic minimum in the range
560
520

600 nm on the 1
st

derivative
OD
N
(

)

spectr
a
a,
OD
N
(

)


(Fig. 6a). The position
of
(
the inflection point
)

and
the amplitude of the

derivative

OD
N
(

)’
minimum (
i.e.

the inclination of the slope of the unresolved absorption
band governed by Car, Fig. 6) exhibited a uniform positive correlation with Car/Chl ratio under our experimental
conditions (Figs. 6b and 6c, respectively). As in the case of
OD
500
/
OD
678
, the
g
G
ree
n
E
e
dge parameters were tightly
12


related with Car content but the parameters of the relationships were different in different experiments,
depending of the stress intensity (data not shown).

DISCUSSION

Current literature on
H.

pluvialis

includes only a few
reports on systematic investigation of spectral
properties of whole
-
cell suspension (see
e.g.

(
Solovchenko 2011
)
), especially cysts with high Car content (or
Car/Chl). These circumstances obviously stem from the optical complexity of this system. In particular,
H.

pluvialis

cells contain high amounts of pigments which are localized in specific structures (thylakoid
membranes of
chloroplast or cytoplasmic oil bodies in case of Chl and primary Car or secondary Car, respectively) non
-
uniformly
distributed within the cell volume
(
Boussiba 2000
;
Peled et al. 2011
)
. Under stressful conditions
H.

pluvialis

cells
rapidly accumulate secondary Car concomitantly with a
dramatic
decline in Chl and primary (photosynthetic) Car.
A
The
drastic
increase in concentration of chromophore
(such as Ast) molecules
in
confined to
the
small volume of
oil bodies could lead to
aggregation of the Car molecules facilitating
broadening and bathochromic shifts of their
maxima
(
Zsila et al. 2001
)
. As a consequence, a number of serious obstacles for measurement of
optical density
spectra
in
H.

pluvialis

cell suspension
optical density spectra
arise including
rapid cell sedimentation, significant
influence of light scattering, strong pigment aggregation, and sieving effect.


Thus, the apparent paradox observed

in
raw
whole
-
cell spectra

(decrease in blue
-
green band upon
increasing Car content)

was
fully resolved

after normalization to the red Chl maximum

(Fig. 3c), indicating that the
increase in Car content contributes more than the decrease in Chl, in the course of stress
-
induced
carotenogenesis
.

Traditionally, integrating spheres are used to cope with
incomplete light collection due to light scattering
,

although a portion of light is lost in spite of all effort

(
Merzlyak et al. 2008
)
,
;

The
the
application of more
sophisticated approaches for light
-
scattering compensation requires more advanced and expensive
spectrophotometers and additional spectrum scan
s

for each sample
(
Merzlyak and Naqv
i 2000
)

and
is
being

therefore
is
less suitable for rapid estimation of Car or Car/Chl. In this work, we used an alternative approach
similar to the opal glass method developed by Shibata
(
Shibata 1973
)
. This method is based on deposition of
microalgal cells on glass
-
fiber filters prior to measurement and was previously developed to record optical density
spectra of the chlorophyte
Parietochl
oris incisa

(
Solovchenko et al. 2009
)
.
Remarkably, th
e
is

approach does not
13


require any special
sampling
acc
essories such as solid sample holder since the wet filters
are adhesive
enough to
be
mounted on the vertical wall of the cuvette comp
artment
over its exit
in front of the light
-
exit

window.
There is
no need for
special

software
and/or
processing
modules as well
since all
the steps of
spectral data
processing
are
carried out with

a

little effort
in
using

a
standard
spreadsheet

functions
.

Application of this technique made it
possible to obtain reliable spectral data on
H.

pluvialis

cell suspensions
,

compatible with those recorded with a
more advanced spectrophotometer fitted with an integrating sphere (see
e.g.

spectra in
(
Solovchenko 2011
)
).

The analysis of cell suspension spectra recorded using this approach revealed certain features characteristic
of stress
-
induced pigment changes, primarily

due to

carotenogenesis in
H.

pluvialis
cells. Noteworthy,
the
amplitude of the

raw

OD
(

)

spectra was not
always
directly
proportional
correlated
to their pigment content (Figs.
3a,b). Thus, the magnitude of the broad maximum in the blue
-
green region of the spectrum
often
was
often
lower
in the red cells with high

Car/Chl (> 3) than in samples with lower Car/Chl.
However
, th
is

apparent paradox
observed in raw whole
-
cell spectra (decrease in blue
-
green band upon increasing Car content) was fully resolved
after normalization to the red Chl maximum (Fig. 3c),
suggesting

that the increase in Car content contributes more
than the decrease in Chl, in the course of stress
-
induced carotenogenesis.

We believe that th
e
is

discrepancy

observed with raw whole
-
cells spectra

could
stem
s

from a sieving effect
which
may
coul
d
be
further
exacerbated by fusion of Ast
-
containing oil bodies

(
Pilát et al. 2012
)


at advanced
stages of carotenogenesis.
T
Alt
hough
there
is
are

some
evidence
indications

of
for

the bui
l
d
-
up of Ast
in
small and
moderate
medium

oil
bodies at intermediate stages of carotenogenesis

as well

(
Collins et al. 2011
)
,
it is difficult to
determine
infer

the relation
ships

between oil body size and
the extent of Ast packaging therein
.
F
urthermore,
the
manifestations of packaging
could
also
be
affected by
depend on
reflect

the

extension of
the
effective
optical path
,

due to light scattering

(
which
should be
inverse
ly

relat
ed

with
to

the
size of scattering particles

(oil bodies
in this
case
)
.

All
th
ese
is

possibility
circumstances
may
complicate the construction of
a robust
algorithm for direct
estimation of Car content
via

OD
(

)
. It is unlikely that
this effect
the above
-
mentioned discrepancy

resulted from
distortion of spectra due to deposition of the cells on glass fiber filters since
the
the

measurements of the diluted
suspension
normalized

spectra
confirmed that
are largely independent of

the
amount of
biomass
(Fig.
1b)
routinely
applied to
the filters in our experiments
;

was well within the
indeed, a quasi

linear
range of the
relation

relationships
exists in

OD
(

)

vs
. DW on filter’ (Fig. 4). On the other hand, one should carefully check the linearity of
this relationship in each particular experimental system since it is influenced, apart from the optical properties of
14


microalgal cells
per se
, by
the
scattering proper
ties of the filters and
the
spectrophotometer geometry.
Remarkably, the slope of ‘
OD
500
vs
. DW on filter’ relationship decreased with the onset of stress
-
induced
carotenogenesis
,

reflecting the increase in sieve effect due to build
-
up of local Ast concentr
ation and, possibly,
fusion of oil bodies containing the pigment.

It is well known that in
H.

pluvialis

cultures subjected to stressful conditions
(
Torzillo et al. 2003
;
Boussiba
2000
)
, carotenogenesis occurs in parallel with degradation of Chl manifesting the reduction of photosynthetic
apparatus in order to avoid photooxidative damage
(
Wang et al. 2003
;
Solovchenko 2011
)
. It was found recently

that
the
Car/Chl ratio, but not the absolute amount of Chl or Car correlates directly with high light
-
stress tolerance
in
H.

pluvialis

(
Solovchenko 2011
)

making Car/Chl an informative index of the cell physiological c
ondition. At the
same time an extra care should be exercised at very high Car/Chl since small errors in Chl assay in this case could
lead to a considerable inaccuracy

in the determination of the ratio. The normalization of
OD(

)

to the red Chl
maximum essentially equalized the contribution of Chl to light absorption making apparent the relative
contribution of Car which drastically increased in the course of carotenogenesis (Fig. 3c). Indeed,
OD
(

)/
OD
678

OD
N
(

)

exhibited a tight r
elationship with Car/Chl in the studied range (Fig. 5).

The relationships ‘
OD
(

)/
OD
678

OD
N
(

)
vs
. Car content’ and ‘
OD
(

)/
OD
678

OD
N
(

)

vs
. Car/Chl’ were linear in
a wide range of Car changes and began to depart from linearity only at high Car (see Fig. 5).

It is difficult to say
whether it is an effect of saturation or the change of effective absorption coefficient of Ast taking place along with
its accumulation. Plausible reasons include changes in Car composition (
e.g.,

increase in proportion of Ast from
<1% to >95%
(
Zhekisheva et al. 2005
)
) and in the degree of aggregation of Car molecules.

Analysis of the 1
st

derivative spectra revealed that the characteristic spectral changes accompanying
carotenogenesis in
H.

pluvialis
include a remarkable increase in
the
so called
g
G
reen
e
E
dge
effect
(the amplitude
of
OD
N
(

)′
the derivative

minimum

in the
560
520

600 nm range and its profound bathochromic shift, Fig. 6
b
a
). It
was found that the amplitude of
Green
green
Edge
edge
slope
and the magnitude of its shift towards longer
wavelengths are exponentially related with Car/Chl and these relationships are uniform under the experimental
conditions used in the present work (Figs. 6b and 6c). On the contrary, the relationships of Car con
tent with the
Green
green
Edge
edge
features (as well as with
OD
(

)/
OD
678
OD
N
(

)
) were tight (
r
2

>

0.95) but possessed different
slopes under different stress intensities. One may speculate that this phenomenon is caused by different trends of
15


Chl degradation significantly affecting
OD
(

)/
OD
678
OD
N
(

)

but not Car content. This further supports the
suggestion of Car/Chl ratio as a preferable marker of stress in
H. pluvialis
.

We would like to note in conclusion that, despite the strong influence of pigment packaging and sieve effect
inherent to the who
le
-
cell spectra of stressed
H. pluvialis

cultures, the non
-
destructive rapid assay of Car/Chl ratio
and Car content turned to be feasible in this system upon deposition of the cells on a glass fiber filter

with a simple
spectrophotometer without the use of

an integrating sphere. It was found that the dramatic changes in scattering
-
corrected optical density occurring in the blue
-
green region of the spectrum, upon normalization to variable Chl
absorption, are tightly related with changes in pigment content an
d composition. Simple
OD

ratios as well as the
1
st

derivative minimum amplitude and position in the range
560
520

600 nm
(so called Green Edge)
exhibited a
strong positive correlation with Car/Chl in the range 0.55

31.2 and Car up to
187
188
mg



L

1
. It should be noted
however that
due to the extremely high local Car concentrations

achieved in stressed
H. pluvialis
cells
,
the specific
parameters of
the relationship with absolute Car content varied depending on the intensity of the stress.

Nevertheles
s, in view of a similar
quasi
-
linear
evolution of the Car/Chl mass ratio
observed with other microalgae
under stress
(C.
Aflalo, unpublished)
, this methodology could be extended to study cellular response in other
systems.


The findings described in this work allow one to obtain a quantitative record of the development of stress
-
induced carotenogenesis in
H. pluvialis

non
-
destructively
via

optical density measurements. In particular, the
normalized optical density in the broa
d band
of
around
500

600

nm, as well as the
g
G
reen
e
E
dge features, could
be employed in the development of models for rapid assay of Car/Chl
or Car
in the algal cells suspensions
.
,

It
should be noted however that obtaining a calibration in the widest possible range of Car/Chl changes in any
particular culture system and careful control of the biomass load per filter is crucial for the robustness of
Car and
Car/Chl estimation.

The ad
vantages of optical density
-
based non
-
destructive monitoring of carotenogenesis in microalgae such
as rapidity

, simplicity, and affordability form a ground to believe that this approach could find
a potential
use in
the development of algorithms for
real
-
time monitoring of carotenogenesis
in
and physiological condition of the
microalga

in situ
.

Such algorithms could be used in
solid state optical sensor

design
s

for automation of large
-
scale
lab cultivation and mass production systems of
H. pluvialis
and, possibly, other
carotenogenic
microalgae
provided
that its limitations are taken into account.

16


ACKNOWLEDGEMENTS

This work was in part supported by Ministry of Science and Education of Russian Federation (contract Nr.
16.513.12.3028)

and ‘Skolkovo’ Sci
entific Fund
. Financial support by the European Commission's

Seventh
Framework Programme for Research and Technology Development (FP7), project SENSBIOSYN, Grant Nr.
232522, is

gratefully acknowledged. Dedicated technical assistance of Mrs. Larisa Lozovska
ya is much appreciated.

The authors declare no conflict of interests.

NOMENCLATURE

A
(

)



absorbance

value at wavelength
λ
;
we
used
this notation
for dissolved pigments in extracts
;

OD
(

)



scattering
-
corrected optical density
spectra
of
a
microalgal cells

sample deposited on a glass
-
fiber filter; .

OD




OD
(

)

value at wavelength
λ
;

OD
N
(

) =
OD
(

)/
OD
678



OD
(

)/
OD
678
OD(

)




spectrum normalized to red chlorophyll absorption maximum;

first derivative


OD
N
(

)’ =

[
OD
N
(

)
]/





partial

derivative
first derivative
of

OD
(

)/
OD
678
OD
N
(

)

spectra
.
:


[

OD
(

)/
OD
678
]/





REFERENCES

Boussiba S (2000) Carotenogenesis in the green alga

Haematococcus pluvialis
: cellular physiology and stress
response. Physiol Plant 108 (2):111
-
117.
doi:10.1034/j.1399
-
3054.2000.108002111.x

Boussiba S, Vonshak A (1991) Astaxanthin accumulation in the green alga
Haematococcus pluvialis
. Plant Cell
Physiol 32 (7):1077
-
1082

Collins AM, Jones HDT, Han D, Hu Q, Beechem TE, Timlin JA (2011) Carotenoid Distri
bution in Living Cells of
Haematococcus pluvialis

(Chlorophyceae). PLoS ONE 6 (9):e24302. doi:10.1371/journal.pone.0024302

Del Campo J, García
-
González M, Guerrero M (2007) Outdoor cultivation of microalgae for carotenoid production:
current state and
perspectives. Appl Microbiol Biotechnol 74 (6):1163
-
1174. doi:10.1007/s00253
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007
-
0844
-
9

Fan L, Vonshak A, Zarka A, Boussiba S (1998) Does astaxanthin protect
Haematococcus

against light damage?
Zeitschrift für Naturforschung C, Journal of biosciences 53 (1
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2):93

Gitelson A, Merzlyak M, Lichtenthaler H (1996) Detection of red edge position and chlorophyll content by
reflectance measurements near 700 nm. J Plant Physiol 148 (3
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4):501
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508. doi:10.1016/S0176
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1617(96)80285
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9

Guerin M, Huntley M, Olaizola M (2003
) Haematococcus astaxanthin: applications for human health and nutrition.
Trends Biotechnol 21 (5):210
-
216. doi:10.1080/09670260802227736

17


Hu Z, Li Y, Sommerfeld M, Chen F, Hu Q (2008) Enhanced protection against oxidative stress in an astaxanthin
-
overprodu
ction
Haematococcus
mutant (Chlorophyceae). Eur J Phycol 43 (4):365
-
376.
doi:10.1080/09670260802227736

Kaczor A, Turnau K, Baranska M (2011) In situ Raman imaging of astaxanthin in a single microalgal cell. Analyst
136 (6):1109
-
1112. doi:10.1039/C0AN00553C

Lemoine Y, Schoefs B (2010) Secondary ketocarotenoid astaxanthin biosynthesis in algae: a multifunctional
response to stress. Photosynthesis Res 106 (1):155
-
177. doi:10.1007/s11120
-
010
-
9583
-
3

Li Y, Sommerfeld M, Chen F, Hu Q (2008) Consumption of oxygen b
y astaxanthin biosynthesis: A protective
mechanism against oxidative stress in
Haematococcus pluvialis

(Chlorophyceae). J Plant Physiol 165
(17):1783
-
1797. doi:10.1016/j.jplph.2007.12.007

Merzlyak M, Chivkunova O, Maslova I, Naqvi K, Solovchenko A, Klyachk
o
-
Gurvich G (2008) Light absorption
and scattering by cell suspensions of some cyanobacteria and microalgae. Russ J Plant Physiol 55 (3):420
-
425. doi:10.1134/S1021443708030199

Merzlyak MN, Naqvi KR (2000) On recording the true absorption spectrum and the s
cattering spectrum of a turbid
sample: application to cell suspensions of the cyanobacterium
Anabaena variabilis
. Journal of
Photochemistry & Photobiology, B: Biology 58 (2
-
3):123
-
129. doi:10.1016/S1011
-
1344(00)00114
-
7

Peled E, Leu S, Zarka A, Weiss M, Pick U, Khozin
-
Goldberg I, Boussiba S (2011) Isolation of a novel oil globule
protein from the green alga
Haematococcus pluvialis

(Chlorophyceae). Lipids 46 (9):851
-
861.
doi:10.1007/s11745
-
011
-
3579
-
4

Pilát Z, Bernatová S,
Ježek J, Šerý M, Samek O, Zemánek P, Nedbal L, Trtílek M (2012) Raman microspectroscopy
of algal lipid bodies: β
-
carotene quantification. J Appl Phycol 24 (3):541
-
546. doi:10.1007/s10811
-
011
-
9754
-
4

Sarada R, Tripathi U, Ravishankar G (2002) Influence of st
ress on astaxanthin production in Haematococcus
pluvialis grown under different culture conditions. Process Biochem 37 (6):623
-
627. doi:10.1016/S0032
-
9592(01)00246
-
1

Shibata K (1973) Dual wavelength scanning of leaves and tissues with opal glass. Biochim B
iophys Acta 304
(2):249

Solovchenko A (2011) Pigment composition, optical properties, and resistance to photodamage of the microalga
Haematococcus pluvialis

cultivated under high light. Russ J Plant Physiol 58 (1):9
-
17.
doi:10.1134/S1021443710061056

Solovc
henko A, Khozin
-
Goldberg I, Cohen Z, Merzlyak M (2009) Carotenoid
-
to
-
chlorophyll ratio as a proxy for
assay of total fatty acids and arachidonic acid content in the green microalga
Parietochloris incisa
. J Appl
Phycol 21 (3):361
-
366. doi:10.1007/s10811
-
008
-
9377
-
6

Solovchenko A, Merzlyak M, Khozin
-
Goldberg I, Cohen Z, Boussiba S (2010) Coordinated carotenoid and lipid
syntheses induced in
Parietochloris incisa

(Chlorophyta, Trebouxiophyceae) mutant deficient in Δ5
desaturase by nitrogen starvation and high l
ight. J Phycol 46 (4):763
-
772. doi:10.1111/j.1529
-
8817.2010.00849.x

Sussela M, Toppo K (2006) Haematococcus pluvialis
-
a green alga, richest natural source of astaxanthin. Curr Sci 90
(12):1602
-
1603

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Font:

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pt,

Italic
Formatted:

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10

pt,

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18


Torzillo G, Goksan T, Faraloni C, Kopecky J, Masojídek J (
2003) Interplay between photochemical activities and
pigment composition in an outdoor culture of Haematococcus pluvialis during the shift from the green to
red stage. J Appl Phycol 15 (2):127
-
136. doi:10.1023/A:1023854904163

Wang B, Zarka A, Trebst A, Bou
ssiba S (2003) Astaxanthin accumulation in
Haematococcus pluvialis

(Chlorophyceae) as an active photoprotective process under high irradiance. J Phycol 39 (6):1116
-
1124.
doi:10.1111/j.0022
-
3646.2003.03
-
043.x

Zhekisheva M, Boussiba S, Khozin
-
Goldberg I, Zar
ka A, Cohen Z (2002) Accumulation of oleic acid in
Haematococcus pluvialis

(Chlorophyceae) under nitrogen starvation or high light is correlated with that of
astaxanthin esters. J Phycol 38 (2):325
-
331. doi:10.1046/j.1529
-
8817.2002.01107.x

Zhekisheva M, Za
rka A, Khozin
-
Goldberg I, Cohen Z, Boussiba S (2005) Inhibition of astaxanthin synthesis under
high irradiance does not abolish triacylglycerol accumulation in the green alga
Haematococcus pluvialis

(Chlorophyceae). J Phycol 41 (4):819
-
826. doi:10.1111/j.0
022
-
3646.2005.05015.x

Zsila F, Deli J, Simonyi M (2001) Color and chirality: carotenoid self
-
assemblies in flower petals. Planta 213
(6):937
-
942. doi:10.1007/s004250100569



19


FIGURE LEGENDS

Fig. 1.

Optical density spectra of red
H. pluvialis

cells (Car content =
130
1
88

mg



L

1
; 5.4% DW) measured upon
deposition on glass fiber filters at different biomass loads: (a) measured spectra, the amount of biomass on filter (mg
DW) is indicated near the respective curves; (b) measured spectra from pa
nel (a) corrected to
OD
800
= 0 and
normalized to
OD
678
; (c) the 1
st

derivative of the normalized optical density spectra from panel (b).

Fig. 2. Representative kinetics of (a) biomass accumulation,
1

and cell number,
2
, (b) changes in volumetric pigment
(Car,
1

and Chl,
2
) contents and their ratio,
3
, and (c) accumulation of Car in biomass (as DW percentage,
1

or per
cell,
2
) in a
H. pluvialis

culture under nitrogen starvation conditions employed in this work.

Fig. 3.

R
epresentative changes of optical density of

whole

H. pluvialis

cells (a, c) and the corresponding
absorbance of DMSO extracts (b, d) in the course of nitrogen starvation (see Fig. 2). The spectra before (a, b) and
(
b, c
c, d
) after normalization to Chl red
absorption maximum are shown. The carotenoid
-
to
-
chlorophyll ratio is
indicated. Biomass filter load was 1.33 mg DW (a
) while the average for variable loads (0.
4
-
4.5 mg DW
,
) for each
Car/Chl are presented in

(
c)
, together with

the standard errors of the me
an

(

n
=7
)

for the 3 upper curves
. Finally,
;

the

specific

values for optical density
(a)
and

absorbance

in panel


(b
)
)


were
was
calculated for

whole
-
cells and

extracted
pigments
, respectively,

extracted

from
equivalent to

1.33 mg DW
.

and dissolved in 1 mL

DMSO
.

Fig. 4.

Relationship between
scattering
-
corrected optical density of the filter at 500 nm and
the amount of biomass
loaded on filter
and scattering
-
corrected optical density of the filter at 500 nm
for
H. pluvialis

whole
-
cell
s

suspension
samples
with different Car/Chl ratio taken at different stages of nitrogen starvation (see Fig. 2).

Fig. 5.

Relationship between the changes in optical density at 500 nm normalized to the red chlorophyll maximum
and the Car/Chl ratio in the
H. pluvialis

cells in
the course of nitrogen starvation (see Figs. 2 and 3). The data of
three

independent experiments are shown by different symbols. Dashed line is the best fit function for the entire
dataset; solid line is that for Car/Chl < 20

(
n=23;
r
2
=0.9
x
6
)
.

Fig. 6.

The changes in first derivative of the normalized optical density spectra of whole cells on filter (a) and
relationships between the position (b) or the amplitude (c) of the ‘green edge’ feature (see hollow dots connected
with a broken line in panel a) wit
h Car/Chl ratio in the course of
H. pluvialis

nitrogen starvation (see Figs. 2 and 3c).
Dashed line is the best fit function

(
n=23;
r
2
=0.9
0
)
Formatted:

Font:

10

pt,

Highlight
Formatted:

Font:

10

pt,

Highlight
Formatted:

Font:

10

pt,

Highlight
20





1


N
on
-
destructive
monitoring of
1

carotenogenesis
in
Haematococcus pluvialis

2

via

whole
-
cell
optical density

spectra

3


4

Alexei Solovchenko,
1,3
*

Claude Aflalo,

2

Alexander Lukyanov,

1

and Sammy
5

Boussiba
2

6


7

1
Department of Bioengineering, Faculty of Biology, Moscow
State University,
8

119
234
, GSP
-
1 Moscow, Russia
.

9

Phone: +7(495)939
-
25
-
87

10

E
-
mail: solovchenko@mail.bio.msu.ru


11


12

2
Microalgal Biotechnology Laboratory, the Jacob Blaustein Institutes for Desert
13

Research, Ben
-
Gurion University of the Negev, Sede
-
Boker Campus,
M
idreshet
14

Ben
-
Gurion
84990
, Israel

15


16

3
Timiryazev Institute of Plant Physiology, Russian Academy of Sciences,
17

Botanicheskaya ul. 35, Moscow, 127276 Russia

18


19


20

Running title:
Non
-
destructive monitoring of carotenogenesis

21


22

*

To whom correspondence should be addre
ssed

23

24

*Manuscript
Click here to download Manuscript: Solovchenko-ea_2012_revised_changes-accepted.docx
Click here to view linked References
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2
3
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2


ABSTRACT

1

We investigated the
feasibility

of
rapid,
non
-
destructive assay of carotenoid
-
to
-
chlorophyll ratio
2

(Car/Chl) and total carotenoids (Car) in cell suspensions of the carotenogenic chlorophyte
3

Haematococcus pluvialis

Flotow under stressful conditions. Whole cells spectra are characterized
4

by variable non
-
linear contributions of
carotenoids (
Car
)

and chlorophylls (Chl), with a strong
5

influence of
Car

packaging and sieve effect inherent to stressed
H. pluvialis

cells
. N
evertheless,
6

non
-
destructive assay of Car/Chl in the range of 0.55

31.2 (Car content
up to

18
8

mg ∙ L

1
; 5.4%
7

of the cell dry weight) turned to be
achievable

with a simple spectrophotometer lacking an
8

integrating sphere upon deposition of the cells on glas
s fiber filters. The scattering
-
corrected
9

optical density in the blue
-
green region of the

whole
-
cell

spectrum
,

normalized to that in the red
10

maximum of Chl absorption
(
OD
500
/
OD
678
)

was tightly related

(
r
2

= 0.96) with
the
Car/Chl ratio

11

found in extracts
. S
ome features
such as the
amplitude and position of the
first

derivative optical
12

density
whole
-
cells
spectra exhibited
also a
strong (
r
2

> 0.90) non
-
linear correlation with Car/Chl.
13

These spectral indices were also tightly related with Car but the slope of
the relationship varied
14

with the stressor intensity. The importance of calibration over the widest possible range of pigment
15

contents and a correct choice of biomass load per filter are emphasized. The advantages and
16

limitations of non
-
destructive monitori
ng of carotenogenesis in
H. pluvialis

are discussed in view
17

of its possible application in optical sensors for lab cultivation and mass production systems of the
18

algae.

19

Key words: astaxanthin, carotenogenesis, derivative spectroscopy,

20

Haematococcus,
non
-
i
nvasive assay, secondary carotenoids

21

INTRODUCTION

22

The chlorophyte
Haematococcus pluvialis

Flotow is one of the most biotechnologically
23

important microalgal species
(
Del Campo et al. 2007
)
, the richest natural source
of the

24

ketocarotenoid astaxanthin (Ast)
(
Sussela and Toppo 2006
)
. Ast exerts a plethora of beneficial
25

effects on human health, it is also a valuable feed additive in
aquaculture
(
Guerin et al. 2003
;
26

Boussiba 2000
)
. In
H. pluvialis
, accumulation of Ast as a major (over 95% of total carotenoids
27

(
Lemoine and Schoefs 2010
)
) secondary carotenoid (Car) takes place under stressful conditions
28

(high irradiance, nutrient limitation, high salinity, etc.), in parallel with
a
reduction
in

chlorophyll
29

(Chl)
conten
t, and other cellular responses
(
Lemoine and Schoefs 2010
)
. It is bel
ieved that Ast,
30

mainly in the form of fatty acid esters
(
Zhekisheva et al. 2002
;
Boussiba 2000
)
, localized in
31

cytoplasmic lipid globules

or
oil bodies
(
Peled et al. 2011
)

plays a role in protection of
32

microalgal cells against photooxidative damage
via

optic
al screening and/or elimination of reactive
33

oxygen species
(
Solovchenko 2011
;
Wang et al. 2003
;
Li et al. 2
008
)
, although the extent of its
34

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3


involvem
ent is sometimes difficult to assess
(
Fan et al. 1998
)
. While the pattern of the
1

physiological responses to stress described above is conserved for various stress conditions, their
2

extent depend strongly on the stress intensity, as it is sensed by the algal cells. As a consequence,
3

quanti
tative relations between stress and response are often elusive and poorly reproducible
(
Hu et
4

al. 2008
;
Sarada et al. 2002
;
Zhekisheva et al. 2005
)
.

5

The investigation of Car accumulation occurring in
H. pluvialis

on the background of Chl
6

degradation is of considerable importance
for basic as well as applied research. Studies of the
7

relation between accumulation of secondary Car, increase in Car/Chl ratio and change in light
8

absorption by the microalgal culture provide valuable information about acclimation of the
9

photoautotrophic
microorganisms to stressors
(
Hu et al. 2008
;
Sarada et al. 2002
)
. In microalgal
10

biotechnology, the information on the dynamics of Car content in biomass is essential for timely
11

and informed decisions on adjustment of illumination conditions, medium composition and on the
12

time for b
iomass harvesting
.

Traditionally, the Car and Chl content is determined by ‘wet’
13

chemical methods with the use of spectrophotometry or chromatography
(
Zhekisheva et al. 2005
)

14

which are time
-
consuming, expensive, and not readily avai
lable in the field or at mass cultivation
15

facilities. These considerations
emphasize the need

for a reliable, rapid and preferably non
-
16

destructive technique for
fast

appraisal

of
the relative
Car content in microalgal cultures

17

Remarkably, the engagement of

protective mechanisms based on the build
-
up of Ast
-
18

containing oil bodies in the cell is accompanied by specific changes in the optical properties of the
19

algal cells
(
Solovchenko 2011
)
. Recent reports show that Ast p
resence and subcellular distribution
20

in vivo

could be characterized
(
Kaczor et al. 2011
)

and even distinguished from β
-
carotene
21

(
Collins et al. 2011
)

using advanced spectral techniques such as Raman spectroscopy. However,
22

current understanding on the effects of Ast accumulation on the optical properties of

H. pluvialis

23

cell suspension is clearly insufficient to develop non
-
destructive spectroscopic t
ools suitable for
24

on
-
line monitoring of Car in lab or industrial mass cultures of
H. pluvialis
.

25

A solution to this problem would require a better understanding of the spectroscopy of
H.
26

pluvialis

cell suspensions and the relationships between whole
-
cell sp
ectra and contributions of
27

different pigments to overall light absorption in the process of carotenogenesis. Recently, we
28

successfully employed cell suspension optical density measurements to develop an algorithm for
29

gauging high
-
light tolerance of
H. pluv
ialis
cultures
(
Solovchenko 2011
)
. In the present work we
30

show the possibility of obtaining of a quantitative description of carotenogenesis in stressed
H.
31

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4


pluvialis

cultures with a simple spectrophotometer lacking int
egrating sphere. We also for the first
1

time report the relationships between Car/Chl and spectral features of
H. pluvialis

whole
-
cell
2

optical density

which form the foundation for the development of techniques for non
-
invasive
3

assay of Car accumulation in
H. pluvialis

cells. It is important to realize that this approach is not
4

intended to replace traditional pigment assay, but to complement it, providing a meaningful index
5

for the extent of stress in
Haematococcus

cultures.

We also believe that it

w
ould

facilitate the
6

development of
algorithms for
non
-
destructive
real
-
time monitoring of other microalgae cultivated
7

under stressful conditions
.

8

MATERIALS AND METHODS

9

Cultivation conditions

10

The unicellular chlorophyte
Haematococcus pluvialis

Flotow 1844 em. W
ille K
-
0084 was
11

obtained from the Scandinavia Culture Center for Algae and Protozoa at the University of
12

Copenhagen, Denmark. The algae were cultivated on modified BG11 medium
(
Boussiba and
13

Vonshak 1991
)

in 0.4 L glass columns (5 cm ID) under constant illumination provided by daylight
14

fluorescent lamps. The exponentially growing cells (referred to as 'green' cells) initially grown at
15

the irradiance of 35 μE



m

2



s

1

were transferred to nitrate
-
free medi
um at the irradiance of 350
16

μE



m

2



s

1

as measured by a LiCor 850 quantometer (LiCor, USA) in the center of an empty
17

column. The cultures were constantly bubbled with CO
2

: air mixture (2

: 98, v/v) at 25 °C and the
18

pH of the culture under these condit
ions was in the range 7.0

7.8.

19

Pigment
s

extraction and analysis

20

Total Chl and Car were extracted from microalgal cells pelleted by centrifugation with
21

dimethyl sulfoxide (DMSO) for 5 min at 70 °C with 5

mL per
ca
. 3.5 mg DW. The pigment
22

concentrations were

routinely determined spectrophotometrically in
dimethyl sulfoxide (
DMSO
)

23

extracts with a Cary 50 Bio spectrophotometer (Varian, USA)
(
Solovchenko et al. 2010
)
. The
24

extracts were diluted such that the absorbance at pigment maxima was kept in the range 0.2

0.8. In
25

certain experiments the pigment content in
H. pluvialis

cells was determined in acetone extracts
26

after separation using an H
PLC chromatography system equipped with a photodiode array detector
27

(Varian Analytical Instruments, Walnut Creek, CA, USA) as previously described
(
Zhekisheva et
28

al. 2005
;
Peled et al. 2011
)
.

29

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Filter spectra measurement and processing

1

To reduce the influence of scattering and a rapid sedimentation of cells on a cuvette floor, a
2

technique for measuring turbid microalgal samples
similar

to that
described in
(
Solovchenko et al.
3

2009
)

was us
ed. Cells were deposited on 25
-
mm GF/F glass
-
wool filters (Schleicher & Schuell,
4

Germany)
.
The wet filters were mounted with cells facing the detector on the output window of the
5

cuvette compartment.
The o
ptical density of the filters,
OD
, was expressed as


log
10

T
, where
T

is
6

transmittance (Fig. 1a). The spectra were taken in the range 400

800 nm with subtraction of the
7

values of a base line provided by an empty filter soaked in the culture medium. The corrected
8

spectra,
OD
(
λ), were obtained by smoothing t
he measured spectra with 3
-
point moving average
9

algorithm and baseline shift to yield zero optical density at 800 nm. The
OD
(
λ) spectra were
10

normalized to the red Chl optical density maximum at 678 nm yielding
OD
(λ)
/
OD
678

spectra (Fig.
11

1b). Finally, the 1
st

derivative of the
OD
(λ)
/
OD
678

spectra (Fig. 1c) was calculated
using routine
12

spreadsheet equation
to estimate the steepness of the slope in the range 5
2
0
-
600 nm and the
13

position of the associated inflexion point.

This part of the

original spectra is fur
ther referred to as
14

the ‘green edge’.
The coefficient of variation on spectral measurements was routinely under 5%.

15

Statistical treatment

16

The results of three independent experiments are presented in the figures.

Where
17

appropriate, averages and standard e
rrors of the mean were calculated and displayed. All
18

correlations are significant at P < 0.001 level.

19

RESULTS

20

The dynamics of biomass accumulation and pigment content

21

In routine experiments the cells of
H. pluvialis

cultures were incubated under conditions
22

favoring carotenogenesis (nitrogen
-
free medium, 350 μE



m

2



s

1

irradiance) for 6 days. At the
23

beginning of cultivation (d
ay

0)
,

the
H. pluvialis

cells were
typically
characterized by a high Chl
24

content (
1.8

2.0% DW) and a correspondingly low Car content (
0.5

1.1% DW). The Car profile at
25

this stage was dominated by lutein and β
-
carotene, containing also neoxanthin, violaxanthin, and
26

zeaxanthin as the minor Car, but only traces of Ast were detectable (not shown
). After 6 days
27

under the stress conditions, the algae steadily accumulate biomass (increasing
by
4 fold), while the
28

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
6


cell density remained relatively unchanged (Fig. 2a). The Chl content steadily decreased by more
1

than 3 fold, while total carotenoid conten
t increased by 17 fold (Fig. 2b), the same applies to
2

specific Car content (Fig. 2c). Consequently, the Car/Chl weight ratio increased spectacularly (by
3

more than 31 fold; Fig. 2b, curve
3
), indicating this is the most sensitive and unequivocal indicator
4

o
f stress. The proportion of total Ast (in free and fatty acid
-
esterified forms), negligible before the
5

onset of stress, increased sharply, reaching > 95% of total Car by the end of the experiment (not
6

shown).

7


8

Optical density spectra of whole
H. pluvialis

cells

9

Our preliminary attempts to record conventional optical density spectra of
H. pluvialis

cell
10

suspensions in a standard 1
-
cm cuvette gave unsatisfactory results due to rapid sedimentation of
11

the algal cells on the cuvette floor and to the strong influ
ence of light scattering (data not shown),
12

stressing the need for means to correct the contribution of the latter.

13

One may circumvent the use of an integrating sphere for the measurement of optical
14

density in cell suspension by acquiring spectra from cell
s deposited on a wet glass fiber filter using
15

a conventional spectrophotometer.

The wet filter is positioned over the photomultiplier window,
16

with cells away from the incident beam.

In this case too, it is critical to correct for light scattering.
17

Using th
e spectrum of a wet empty filter for base line correction allowed obtaining reliable
18

spectral data in a wide range of cell density and Car content of the culture, and over the whole
19

spectral range studied (see Materials and Methods). A further correction p
roved to be necessary to
20

eliminate the contribution of scattering by the cells, with the extent of the correction depending on
21

the amount of biomass loaded on the filter (see
e.g.

the 750

800 nm range in Fig. 1a). The optical
22

density at 800 nm (where no pi
gment absorbs) was used as a blank correction for this purpose. As
23

a result, the corrected
OD
(
λ
) spectra were very close to baseline in the 750

800 nm range
24

indicative of a great decrease in the contribution of scattering.

25

The scattering
-
corrected optical
density spectra of
H. pluvialis

cells deposited on a wet
26

filter, acquired without the use of integrating sphere, at different physiological states (from
27

vegetative green cells to stressed red cysts with high Car/Chl) are presented in Fig. 3, in
28

comparison
with the conventional spectra of the pigments extracted from the same samples. The
29

spectra of the whole cells (Fig. 3a) present profound differences with those for the extracts (Fig.
30

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
7


3b), in which pigments are in dilute solution. In particular, the amplitu
des of the pigment
1

absorption bands in the red and in the blue
-
green parts of the spectrum were lower, the bands were
2

broader, and the peaks were less resolved in the whole cell spectra than in the spectra of the
3

pigment extracts, apparently due to a sievi
ng effect and/or aggregation of the molecules of the
4

pigments (see Discussion below). Remarkably, the magnitude of the difference increased along
5

with accumulation of Car and loss of Chl; the highest difference between whole
-
cell
s

and extract
s

6

spectra was
recorded
for

the cells with the highest Car content (
cf
. spectral curves in Figs. 3a and
7

3b).

8

The data presented in Fig. 1 are representative of suspensions subjected to intense and
9

prolonged stress achieving very high Car content and Car/Chl to test the
feasibility of the
10

suggested approach. Different amounts of biomass were loaded upon filters. The raw spectra (Fig.
11

1a) indicate that, similarly to spectra recorded with an integrating sphere (see spectra in
12

(
Solovchenko
2011
)
), only the Chl absorption is well resolved (peak centered at 678 nm), while
13

the combined contribution of Car and Chl in the blue
-
green region of the spectrum appears as a
14

broad flat band in the range 400

550 nm. After subtraction of
OD
800

and norm
alization to
OD
678

15

the spectra essentially coincided (see
e.g.

Fig. 1b), revealing the quasi
-
linear dependence between
16

optical density and the amount of biomass loaded on the filter in the range 0.4
-
4.5 mg DW/filter,
17

corresponding to
OD
500

of 0.04

0.78.

18

Th
e analysis of the relationship between
OD
(

)

and the amount of biomass applied to the
19

filter with different Car/Chl in the diluted suspensions showed that the relation ‘
OD
(

)

vs
. DW on
20

filter’ was nearly linear in the range 0.4

4.5 mg DW in all cases studi
ed (see
e.g.

the data in Fig.
21

4). This relationship departed from linearity at higher
OD
500

(> 0.9) in the case of ‘green’ cell
22

suspension (low Car/Chl), but less in the case of ‘red’ cells with high Car/Chl, apparently due to
23

stronger aggregation of Car a
nd sieve effect in the latter case. Notably, the higher the Car/Chl ratio
24

of the samples taken at different cultivation stages, the lower the slope of the linear part of the
25

relationships ‘
OD
500

vs
. DW on filter’ (Fig. 4). This effect was also apparent wit
h
OD
678

(not
26

shown).

27


28

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
8


Changes of the optical density spectra of
H. pluvialis

cells in the course of
1

carotenogenesis

2

Cultivation of
H. pluvialis

under stressful conditions brought about profound changes in the
3

optical density spectra of the microalga (Fig. 3). The stress
-
induced carotenogenesis was
4

accompanied by the disappearance of spectral details in the blue
-
green region resulting in
5

formation

of a broad (
ca.

150 nm wide), almost featureless band with an abrupt longer
-
wavelength
6

slope.
A
considerable decrease of the amplitude in the band of
the specific optical density

7

contributed by
Chl in the red (Fig. 3a) took place along with
an

overall red
uction

of
that

in the
8

blue
-
green region, where absorption by both Car and Chl occurs
, despite the increase in Car
9

content
.
On the other hand, an increase in Car
contribution to the
absorbance

was readily apparent
10

in the extract spectra (Fig. 3b)
at interme
diate Car/Chl ratios; however, at higher Car/Chl, a
11

saturation effect becomes obvious, equivalent to that expected from the
Lambert
-
Beer law at high
12

chromophore concentrations.

13

In order to rule out a possible interference from variable Chl background dist
urbing the
14

relationship ‘
OD
(

)

vs.

pigment content or ratio’, we tried to relate the
OD
(

)

spectra normalized
15

to Chl red absorption maximum,
OD
500
/
OD
678

, with Car or Car/Chl. Normalization revealed, apart
16

from a decline of Chl contribution to light
absorption by the microalgal cells, a dramatic increase
17

in the absorption in the 400

550 nm range with flattening of the spectrum shape and an increase in
18

the longer
-
wavelength slope of this band (Fig. 3c
,

which may be called
‘g
reen
e
dge


by analogy to

19

the

‘r
ed
e
dge


(
Gitelson et al. 1996
)

in plants). This effect was even more pronounced in the
20

extract spectra (Fig. 3d).


21

T
he amplitude of
the
OD
500
/
OD
678

ratio

was
correlated

to Car/Chl

mass ratio

and
,

to a lesser
22

extent
,

to
the Car content (not shown)
,
in the broad band 400

550 nm. Thus, the ratio
OD
500
/
OD
678

23

was linearly related with
the
Car/Chl

ratio at Car/Chl < 20 (
r
2

= 0.95); at higher values of the ratio
,

24

this relationship departed from linearity but remained uniform and tight under all conditions
25

investigated (
cf
. solid and broken lines in Fig. 5). The relationships ‘
OD
500
/
OD
678

vs
. Car’ w
ere

26

also
linear in the whole range studied
,

but the slope of the re
lationship
was variable and

depend
ent

27

on
the
stress intensity
experienced by the cultures
(data not shown)
, presenting therefore a lesser
28

diagnostic value
.

29

Notably, due to stress
-
induced carotenogenesis, which brought about the broadening and
30

flattening of

the
OD
500
/
OD
678

spectra, the longer
-
wavelength slope of the broad absorption band in
31

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
9


the blue
-
green (
g
reen
e
dge) became more abrupt and moved towards longer wavelengths (Fig. 6c).
1

This effect was readily apparent as a bathochromic shift and an increase i
n the amplitude of the
2

characteristic minimum in the range 5
2
0

600 nm on the 1
st

derivative spectr
a

(Fig. 6a). The
3

position
of
the inflection point and the amplitude of the

derivative
minimum (
i.e.

the inclination of
4

the slope of the unresolved absorption
band governed by Car, Fig. 6) exhibited a uniform positive
5

correlation with Car/Chl ratio under our experimental conditions (Figs. 6b and 6c, respectively).
6

As in the case of
OD
500
/
OD
678
, the
g
reen
e
dge parameters were tightly related with Car content but
7

the parameters of the relationships were different in different experiments, depending of the stress
8

intensity (data not shown).

9

DISCUSSION

10

Current literature on
H.

pluvialis

includes only a few reports on systematic investigation of
11

spectral properties of

whole
-
cell suspension (see
e.g.

(
Solovchenko 2011
)
), especially cysts with
12

high Car content (or Car/Chl). These circumstances obviously stem from the optical complexity of
13

this system. In particular,
H.

pluvialis

cells contain high amounts of pigments which are localized
14

in specific structures (thylakoid membranes of chloroplast or cytoplasmic oil bodies in case of Chl
15

and primary Car or secondary Car, respectively) non
-
uniformly distributed within the cell volume

16

(
Boussiba 2000
;
Peled et al. 2011
)
. Under stressful conditions
H.

pluvialis

cells rapidly accumulate
17

secondary Car concomitantly with a decline in Chl and
primary (photosynthetic) Car.
The

increase
18

in concentration of chromophore (such as Ast) molecules
confined to

the
small volume of oil
19

bodies could lead to broadening and bathochromic shifts of their maxima
(
Zsila et al. 2001
)
. As a
20

consequence, a number of serious obstacles for measurement of optical density spectra
in
21

H.

pluvialis

cell suspension arise including ra
pid cell sedimentation, significant influence of light
22

scattering, strong pigment aggregation, and sieving effect.

23

Traditionally, integrating spheres are used to cope with incomplete light collection due to
24

light scattering
(
Merzlyak et al. 2008
)
,

The
application of more sophisticated approaches for light
-
25

scattering compensation requires more advanced and exp
ensive spectrophotometers and additional
26

spectrum scan
s

for each sample
(
Merzlyak and Naqvi 2000
)

being

therefore less suitable for rapid
27

estimation of Car or Car/Chl. In this work, we used an alternative approach similar to the opal
28

gl
ass method developed by Shibata
(
Shibata 1973
)
. This method is based on deposition of
29

microalgal cells on glass
-
fiber filters prior to measure
ment and was previously developed to record
30

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
10


optical density spectra of the chlorophyte
Parietochloris incisa

(
Solovchenko et al. 2009
)
.
1

Remarkably, the approach does not require any special sampling accessories such as solid sample
2

holder since the wet filters are adhesive enough to be mounted on the vertical

wall of the cuvette
3

compartment in front of the light
-
exit window. There is no need for special software and/or
4

processing
modules as well since all the steps of
spectral
data processing are
carried out with little
5

effort
using

standard
spreadsheet

functi
ons.
Application of this technique made it possible to
6

obtain reliable spectral data on
H.

pluvialis

cell suspensions
,

compatible with those recorded with
7

a more advanced spectrophotometer fitted with an integrating sphere (see
e.g.

spectra in
8

(
Solovchenko 2011
)
).

9

The analysis of cell suspension spectra recorded using this approach revealed certain
10

features characteristic of stress
-
induced pigment changes, primarily

due to

carotenogenesis in
11

H.

pluvialis
cells. Notewort
hy, the amplitude of the

raw

OD
(

)

spectra was not directly
correlated

12

to their pigment content (Figs. 3a,b). Thus, the magnitude of the broad maximum in the blue
-
green
13

region of the spectrum was often lower in the red cells with high Car/Chl (> 3) than in

samples
14

with lower Car/Chl.
However, this apparent paradox observed

in
raw
whole
-
cell spectra

(decrease
15

in blue
-
green band upon increasing Car content)

was
fully resolved

after normalization to the red
16

Chl maximum

(Fig. 3c), suggesting that the increase i
n Car content contributes more than the
17

decrease in Chl, in the course of stress
-
induced carotenogenesis
.

18

We believe that th
e

discrepancy

observed with raw whole
-
cells spectra

stem
s

from a
19

sieving effect which
could

be
further
exacerbated by fusion of
Ast
-
containing oil bodies

(
Pilát et
20

al. 2012
)

at advanced stages of carotenogenesis.
Although there are some indications for the build
-
21

up of Ast
in
small and medium oil
bodies at intermediate st
ages of carotenogenesis

(
Collins et al.
22

2011
)
,
it is difficult to infer the relation between oil body size and the extent of Ast packaging
23

therein
.
Furthermore, the manifestations of packaging could also reflect the extension of the
24

effective opt
ical path, due to light scattering (inversely related to the size

of scattering particles).
25

All
th
ese

circumstances

complicate the construction of
a robust
algorithm for direct estimation of
26

Car content
via

OD
(

)
. It is unlikely that
the above
-
mentioned di
screpancy

resulted from
27

distortion of spectra due to deposition of the cells on glass fiber filters since
the

normalized

spectra
28

are largely independent of

the biomass
(Fig. 1b)
routinely applied to the filters in our experiments
;

29

indeed, a quasi

linear
relation

exists in

OD
(

)

vs
. DW on filter’ (Fig. 4). On the other hand, one
30

should carefully check the linearity of this relationship in each particular experimental system
31

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
11


since it is influenced, apart from the optical properties of microalgal cells
per

se
, by
the
scattering
1

properties of the filters and
the
spectrophotometer geometry. Remarkably, the slope of ‘
OD
500
vs
.
2

DW on filter’ relationship decreased with the onset of stress
-
induced carotenogenesis
,

reflecting
3

the increase in sieve effect due to b
uild
-
up of local Ast concentration and, possibly, fusion of oil
4

bodies containing the pigment.

5

It is well known that in
H.

pluvialis

cultures subjected to stressful conditions
(
Torzillo et al.
6

2003
;
Boussiba 2000
)
, carotenogenesis occurs in parallel with degradation of C
hl manifesting the
7

reduction of photosynthetic apparatus in order to avoid photooxidative damage
(
Wang et al. 2003
;
8

Solovchenko 2011
)
. It was found recently that
the
Car/
Chl ratio, but not the absolute amount of
9

Chl or Car correlates directly with high light
-
stress tolerance in
H.

pluvialis

(
Solovchenko 2011
)

10

making Car/Chl an informative index of the cell physiological condition. At t
he same time an extra
11

care should be exercised at very high Car/Chl since small errors in Chl assay in this case could
12

lead to a considerable inaccuracy

in the determination of the ratio. The normalization of
OD(

)

to
13

the red Chl maximum essentially equali
zed the contribution of Chl to light absorption making
14

apparent the relative contribution of Car which drastically increased in the course of
15

carotenogenesis (Fig. 3c). Indeed,
OD
(

)/
OD
678

exhibited a tight relationship with Car/Chl in the
16

studied range
(Fig. 5).

17

The relationships ‘
OD
(

)/
OD
678

vs
. Car content’ and ‘
OD
(

)/
OD
678

vs
. Car/Chl’ were
18

linear in a wide range of Car changes and began to depart from linearity only at high Car (see Fig.
19

5). It is difficult to say whether it is an effect of saturatio
n or the change of effective absorption
20

coefficient of Ast taking place along with its accumulation. Plausible reasons include changes in
21

Car composition (
e.g.,

increase in proportion of Ast from <1% to >95%
(
Zhekisheva et al. 2005
)
)
22

and in the degree of aggregation of Car molecules.

23

Analysis of the 1
st

derivative spectra revealed that the characteristic spectral changes
24

accompanying carotenogenesis in
H.

pluvialis
include a remarkable increase in
the g
reen
e
dge
25

effect
(the amplitud
e of
the derivative

minimum

in the 5
2
0

600 nm range and its profound
26

bathochromic shift, Fig. 6
a
). It was found that the amplitude of
g
reen
e
dge
slope
and the
27

magnitude of its shift towards longer wavelengths are exponentially related with Car/Chl and these
28

relationships are uniform under the experimental conditions used in the present work (Figs. 6b and
29

6c). On the contrary, the relationships of Car con
tent with the
g
reen
e
dge features (as well as with
30

OD
(

)/
OD
678
) were tight (
r
2

> 0.95) but possessed different slopes under different stress
31

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
12


intensities. One may speculate that this phenomenon is caused by different trends of Chl
1

degradation significantly
affecting
OD
(

)/
OD
678

but not Car content. This further supports the
2

suggestion of Car/Chl ratio as a preferable marker of stress in
H. pluvialis
.

3

We would like to note in conclusion that,
despite the strong influence of pigment packaging
4

and sieve effe
ct inherent to the whole
-
cell spectra of stressed
H. pluvialis

cultures, the non
-
5

destructive rapid assay of Car/Chl ratio and Car content turned to be feasible in this system upon
6

deposition of the cells on a glass fiber filter

with a simple spectrophotome
ter without the use of an
7

integrating sphere. It was found that the dramatic changes in scattering
-
corrected optical density
8

occurring in the blue
-
green region of the spectrum, upon normalization to variable Chl absorption,
9

are tightly related with changes

in pigment content and composition. Simple
OD

ratios as well as
10

the 1
st

derivative minimum amplitude and position in the range 5
2
0

600 nm exhibited a strong
11

positive correlation with Car/Chl in the range 0.55

31.2 and Car up to 18
8

mg



L

1
. It should be
12

noted however that
due to the extremely high local Car concentrations achieved in stressed
H.
13

pluvialis
cells,
the relationship with absolute Car content varied depending on the intensity of the
14

stress.

Nevertheless, in view of a similar quasi
-
linear evolu
tion of the Car/Chl mass ratio observed
15

with other microalgae under stress (C. Aflalo, unpublished
; see also
(
M
erzlyak et al. 2007
;
16

Solovchenko et al. 2009
)
), this methodology could be extended to study cellular response in other
17

systems.

18

The findings described in this work allow one to obtain a quantitative record of the
19

development of stress
-
induced carotenogenesis in
H. pluvialis

non
-
destructively
via

optical density
20

measurements. In particular, the normalized optical density in the broa
d band
around

500 nm, as
21

well as the
g
reen
e
dge features, could be employed in the development of models for rapid assay
22

of Car/Chl in the algal cells suspensions
.

It should be noted however that obtaining a calibration in
23

the widest possible range of Car/
Chl changes in any particular culture system and careful control
24

of the biomass load per filter is crucial for the robustness of Car/Chl estimation.

25

The advantages of optical density
-
based non
-
destructive monitoring of carotenogenesis in
26

microalgae such as

rapidity, simplicity, and affordability form a ground to believe that this
27

approach could find use in
the development of algorithms for real
-
time monitoring of
28

carotenogenesis and physiological condition of the microalga
in situ
. Such algorithms could be
29

used in
solid state optical sensor

design

for automation of large
-
scale lab cultivation and mass
30

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13


production systems of
H. pluvialis
and, possibly, other microalgae
provided that its limitations are
1

taken into account.

2

ACKNOWLEDGEMENTS

3

This work was in part

supported by Ministry of Science and Education of Russian Federation
4

(contract Nr. 16.513.12.3028)

and ‘Skolkovo’ Scientific Fund
. Financial support by the European
5

Commission's

Seventh Framework Programme for Research and Technology Development (FP7),
6

pr
oject SENSBIOSYN, Grant Nr. 232522, is

gratefully acknowledged. Dedicated technical
7

assistance of Mrs. Larisa Lozovskaya is much appreciated.

The authors declare no conflict of
8

interests.

9

APPENDIX

10

NOMENCLATURE

11

A
(

)



absorbance
value at wavelength
λ
; we
used
this notation
for dissolved pigments in
12

extracts
;

13

OD
(

)



scattering
-
corrected optical density
spectra
of
a
microalgal cell sample deposited on a
14

glass
-
fiber filter;

15

OD




OD
(

)

value at wavelength
λ
;

16

OD
(

)/
OD
678



spectrum normalized to red chloroph
yll absorption maximum;

17

first derivative




partial derivative
of

OD
(

)/
OD
678

spectra
:


[

OD
(

)/
OD
678
]/




18


19

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as a proxy for assay of total fatty acids and arachidonic acid content in the green
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microalga
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ppl Phycol 21 (3):361
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Goldberg I, Cohen Z, Boussiba S (2010) Coordinated
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carotenoid and lipid syntheses induced in
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(Chlorophyta,
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Sussela M, Toppo K (2006) Haematococcus pluvialis
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Torzillo
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Wang B, Zarka A, Trebst A, Boussiba S (2003) Astaxanthin accumulation in
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18

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Zhekisheva M, Boussiba S, Khozin
-
Goldberg I, Zarka A, Cohen Z (2002) Accumulation of oleic
21

acid in
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(Chlorophyceae) under nitrogen starvation or high light
22

is correlated with that of astaxanthin esters. J Phycol 38 (2):325
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331. doi:
10.1046/j.1529
-
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8817.2002.01107.x

24

Zhekisheva M, Zarka A, Khozin
-
Goldberg I, Cohen Z, Boussiba S (2005) Inhibition of astaxanthin
25

synthesis under high irradiance does not abolish triacylglycerol accumulation in the green
26

alga
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(Chlorop
hyceae). J Phycol 41 (4):819
-
826.
27

doi:10.1111/j.0022
-
3646.2005.05015.x

28

Zsila F, Deli J, Simonyi M (2001) Color and chirality: carotenoid self
-
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29

Planta 213 (6):937
-
942. doi:10.1007/s004250100569

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31


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16


FIGURE LEGENDS

1

Fig. 1.

Optical density spectra of red
H. pluvialis

cells (Car content = 1
88

mg



L

1
; 5.4% DW)
2

measured upon deposition on glass fiber filters at different biomass loads: (a) measured spectra,
3

the amount of biomass on filter (mg DW) is indicated near the respecti
ve curves; (b) measured
4

spectra from panel (a) corrected to
OD
800
= 0 and normalized to
OD
678
; (c) the 1
st

derivative of the
5

normalized optical density spectra from panel (b).

6

Fig. 2. Representative kinetics of (a) biomass accumulation,
1

and cell number,
2
, (b) changes in
7

volumetric pigment (Car,
1

and Chl,
2
) contents and their ratio,
3
, and (c) accumulation of Car in
8

biomass (as DW percentage,
1

or per cell,
2
) in a
H. pluvialis

culture under nitrogen starvation
9

conditions employed in this work.

10

Fig. 3.

Representative changes of optical density of

whole

H. pluvialis

cells (a, c) and the
11

corresponding absorbance of DMSO extracts (b, d) in the course of nitrogen starvation (see Fig.
12

2). The spectra before (a, b) and (
c, d
) after normalization to Chl red abs
orption maximum are
13

shown. The carotenoid
-
to
-
chlorophyll ratio is indicated. Biomass filter load was 1.33 mg DW (a
)
14

while the average for variable loads (0.4
-
4.5 mg DW) for each Car/Chl are presented in

(
c)
,
15

together with the standard errors of the mean (
n
=7) for the 3 upper curves. Finally,

the

specific
16

values for optical density (a) and

absorbance

(b)

were
calculated for

whole

cells and

extracted
17

pigments
, respectively,

equivalent to

1.33 mg DW
.

18

Fig. 4.

Relationship between scattering
-
corrected optical de
nsity of the filter at 500 nm and the
19

amount of biomass loaded on filter for
H. pluvialis

whole

cell
s

with different Car/Chl ratio taken at
20

different stages of nitrogen starvation (see Fig. 2).

21

Fig. 5.

Relationship between the changes in optical density a
t 500 nm normalized to the red
22

chlorophyll maximum and the Car/Chl ratio in the
H. pluvialis

cells in the course of nitrogen
23

starvation (see Figs. 2 and 3). The data of
three

independent experiments are shown by different
24

symbols. Dashed line is the best f
it function for the entire dataset; solid line is that for Car/Chl <
25

20

(
n

= 23,
r
2
=0.9
6
).

26

Fig. 6.

The changes in first derivative of the normalized optical density spectra of whole cells on
27

filter (a) and relationships between the position (b) or the amplitude (c) of the ‘green edge’ feature
28

(see hollow dots connected with a broken line in panel a) wit
h Car/Chl ratio in the course of
H.
29

pluvialis

nitrogen starvation (see Figs. 2 and 3c). Dashed line is the best fit
function

(
n

= 23,
30

r
2
=0.9
0
).
31

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
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17


17


1


2

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
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56
57
58
59
60
61
62
63
64
65
Fig. 1. Solovchenko et al., 2012
a) b) c)
400 500 600 700 800
0.0
0.2
0.4
0.6
0.8
4.5
3.0
0.9
1.3
2.0
0.6
OD(

)
Wavelength (nm)
0.4
400 500 600 700 800
0
1
2
3
4
5
OD(

)/OD
678
Wavelength (nm)
400 500 600 700 800
-0.015
-0.010
-0.005
0.000
4.5
3.0
2.0
1.3
0.9
0.6
0.4
First derivative of OD(

)/OD
678
Wavelength (nm)
Figure 1
Fig. 2. Solovchenko et al., 2012
a) b) c)
0 2 4 6
1
2
3
4
Starvation time (d)
2
1
DW (g ∙ L
-1
)
0.0
0.2
0.4
0.6
0.8
1.0
Cell density (10
9 ∙ L
-1
)
0 2 4 6 7
0
40
80
120
160
200
3
Starvation time (d)
2
1
Car (mg ∙ L
-1
)
0
5
10
15
20
25
30
Chl (mg ∙ L
-1
) or Car/Chl (wt:wt)
0 2 4 6 7
0
100
200
300
400
500
Starvation time (d)
2
1
Car (pg ∙ cell
-1
)
0
1
2
3
4
5
6
Car (% DW)
Figure 2
Fig. 3. Solovchenko et al., 2012
a) b)
400 500 600 700 800
0.0
0.1
0.2
0.3
0.4
0.5
Car/Chl (wt/wt):
0.55
0.67
3.01
4.50
10.7
15.1
31.2
Specific optical density (OD/mg DW)
Wavelength (nm)
400 500 600 700 800
0
2
4
6
8
10
Car/Chl (wt/wt):
0.55
0.67
3.01
4.50
10.7
15.1
31.2
Specific Absorbance (A L/g DW)
Wavelength (nm)
c) d)
400 500 600 700 800
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Car/Chl (wt/wt):
0.55
0.67
3.01
4.50
10.7
15.1
31.2
OD(

)/OD
678
Wavelength (nm)
400 500 600 700 800
0
1
2
20
40
60
Car/Chl (wt/wt):
0.55
0.67
3.01
4.50
10.7
15.1
31.2
A(

)/A666
Wavelength (nm)
Figure 3
Fig. 4 Solovchenko et al., 2012
0 1 2 3 4 5
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Car/Chl
0.55
0.67
3.01
4.50
10.7
15.1
31.2
Biomass (mg DW on filter)
OD
500
Figure 4
Fig. 5 Solovchenko et al., 2012
0 10 20 30
1
2
3
y = 1.10 + 0.088∙x
Car/Chl (wt:wt)
OD
500
/OD
678
y = 3.78 - 2.75∙e
(-x/22.05)
Figure 5
Fig.
6
a)
Fitditif
OD
(

)/
OD
6
. Solovchenko et
a
400 50
0
-0.07
-0.06
-0.05
-0.04
-0.03
-0.02
-0.01
0.00
0.01
0.02
0.03
Car/Chl (wt
/
0.55
0.67
3.01
4.50
9.18
15.1
31.2
Fi
rs
td
er
i
va
ti
ve o
f
OD
(

)/
OD
678
a
l., 2012
0
600 70
/
wt):
Wavelength (nm)
0
0 800
b)
c)
Figure 6