Algal Research 26 (2017) 294301

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Algal Research
journal homepage: www.elsevier.com/locate/algal

A new approach to quantify system eciency with dissolved oxygen MARK

isotopes during engineered growth of Galdieria sulphuraria
Michael Madera,, Philipp Schwernab, Rainer Buchholzb, Robert van Gelderna,
Johannes A.C. Bartha
a
GeoZentrum Nordbayern, Department of Geography and Geosciences, Friedrich-Alexander University Erlangen-Nuremberg, Schlossgarten 5, 91054 Erlangen, Germany
b
Institute of Bioprocess Engineering, Department of Chemical and Biological Engineering, Friedrich-Alexander University Erlangen-Nuremberg, Paul-Gordan-Strasse 3,
91052 Erlangen, Germany

A R T I C L E I N F O A B S T R A C T

Keywords: The microalgae Galdieria sulphuraria was grown in a rst technical approach in a closed system experiment for
Dissolved oxygen 29 days. In this time period, 6 separate asks were sampled for oxygen concentration and their stable isotope
Stable isotopes ratios (18O/16O) in dissolved and headspace phases. The oxygen isotope composition of the water was also
Algae growth analysed as an input for the transfer of its 18O/16O ratio to molecular oxygen via production by algae. This
Closed system
photosynthetic transfer of the isotope composition of water was counterbalanced by oxygen consumption that
Respiration
Galdieria sulphuraria
enriched both phases in 18O. For this reason, neither dissolved oxygen nor oxygen in the headspace reached the
18
O-depleted oxygen isotope ratio of H2O despite excessive photosynthesis. Oxygen that was produced by
photosynthesis accumulated with a yield of 13.96 mmol L 1 in the headspace and with 0.5 mmol L 1 in the
uid phase. This dierence was due to rapid degassing of the solution. It was further amplied by preferential
consumption of the dissolved O2 phase. In order to quantify oxygen production and its consumption we de-
termined photosynthesis/respiration (P/R) ratios with a formula that combined O2 concentrations and its iso-
tope ratios. It revealed a P/R ratio of 7.7 after 11 days. After this it decreased again and moved towards
dominance of respiration. With this work our results introduce a new method to monitor the growth and e-
ciency of algae in controlled experiments.

1. Introduction microalgae, optical density (OD) or dry biomass measurements are

Microalgae have huge potential to recycle waste products, such as
glycerin, and can be cultivated under photo-, mixo-, and heterotrophic
conditions. Such applications have received much attention in bio-
technology and associated research elds [14]. Here we investigated
Galdieria sulphuraria as a model algal strain because it has good back-
ground information and has been studied under various conditions such
as successive starvation stages [57]. It is a representative of eukaryotic
microalgae that can tolerate high temperatures and low pH-values. This
strain is also interesting for exploitation of thermo-stable enzymes [8].
Because of its exibility to various environments, this alga was also
used to exploit secondary metabolic products such as carotenoids, vi-
tamins and phycobiliproteins. All of these products found applications
in the food and feed market as natural occurring colorant or micro-
nutrient agents [9].
As a standard procedure to control growth and production of
usually applied as simple and robust measurements [10]. These mea-
surements provide reliable estimates for biomass changes of algae.
However, they also suer from possible artefacts that do not represent
the growth of algae alone. This includes turbidity and/or other biomass
types that may stem from bacterial growth. In addition, both mea-
surements may not account for oxygen consumption by respiration.
However, the latter process presents an important counterpart to algae
productivity and may oer an important source of CO2 for further
growth of algal communities.
Here we present additional investigations of natural abundance
18
O/16O ratios of the water molecule together with those of O2 in the
headspace and in solution under closed system conditions. Such isotope
techniques are new in algal research and bioengineering approaches
and only were sporadically applied with labelled substrates [11]. Pre-
vious studies of individual microorganisms in closed systems were de-
signed to outline fundamental principles and mainly focused on oxygen

Abbreviations: P/R ratio, photosynthesis/respiration ratio; OD, optical density; DO, dissolved oxygen; VSMOW, Vienna Standard Mean Ocean Water; IRMS, Isotope Ratio Mass
Spectrometer; DIC, dissolved inorganic carbon

Corresponding author.
E-mail address: michael.mader@fau.de (M. Mader).

http://dx.doi.org/10.1016/j.algal.2017.07.026
Received 27 February 2017; Received in revised form 19 June 2017; Accepted 20 July 2017
2211-9264/ 2017 Elsevier B.V. All rights reserved.
M. Mader et al.
isotope fractionation factors during respiration [1217] or photo-
synthesis [18,19]. Results from these studies were applied to natural
water systems often in the context of global or regional carbon and
oxygen cycling. Related studies in open systems also focused on oceanic
productivity [2029] and more recently on terrestrial waters [3035].
One advantage of investigating stable isotopes of molecular oxygen
together with its concentration changes is that it allows quantications
of photosynthesis/respiration (P/R) ratios. This parameter is critical to
determine at which stage an algae growth curve has its optimal pho-
tosynthetic activity. The aims of this study were, therefore, to apply
stable isotope measurements to monitor algal growth under closed
system conditions and to nd out if it oers further useful information
that may help to quantify and monitor growth eciencies of algae.
Photosynthetic oxygen production is continuously counteracted by re-
spiration, however, the interaction of both processes is dicult to
quantify. The underlying principle presented here is that 18O/16O ratios
of molecular oxygen and its deviations from the water isotope signal
can indicate when a closed system is in balance with respect to pho-
tosynthesis and respiration. This parameter also helps to establish re-
liable and easy to use photosynthesis/respiraton (P/R) ratios. This can
serve as an additional measure to complement photosynthetic e-
ciency during growth. With this, we introduce a new dynamic stable
isotope tracer as a tool in algae growth applications that helps to dene
when photosynthetic activity is optimal.
2. Principles of natural abundance stable isotope applications
Natural abundance stable isotope ratios are usually expressed in a -
notation that describes a deviation from a known standard. It is dened
as
R (sample)
= 1
R (standard) (1)
where R is the molar ratio of the number of heavy and light isotopes of
an element (e.g., n(18O)/n(16O)) in the sample and the standard [36].
This number is then multiplied by 1000 to express values in per mille
().
Oxygen stable isotope values are expressed versus the international
reference material Vienna Standard Mean Ocean Water (VSMOW) that
has an 18O/16O ratio of 0.0020052 [37]. Eq. (1) also denes that phases
that are relatively enriched in heavier isotopes with respect to VSMOW
have more positive -values. For instance, atmospheric oxygen consists
of 99.76% of 16O and 0.20% of 18O [37] and its stable isotope value is
+ 23.88 0.02 versus VSMOW [38]. Changes of stable isotope
ratios during and after phase changes are known as isotope fractiona-
tions and often occur during transition of physical states, such as from
gaseous to dissolved phases. In case of molecular oxygen the fractio-
nation during dissolution in water amounts to +0.7 at 20 C [39].
Consumption of oxygen mostly causes kinetic fractionations with a
relative stable isotope enrichment of the heavy isotope in the remaining
molecular O2. The oxygen molecule 16O16O is known to become con-
sumed faster than its heavier counterparts (18O16O, or 17O16O) during
respiration [15,40]. On the other hand, photosynthesis splits the water
molecule and transfers the isotope ratio of the substrate water to the
molecular oxygen pool without fractionation [19].
3. Materials and methods
For this study, six glass asks with two greased and tightly closing
faucets and a butyl septum with a volume of 1 L were prepared as
closed photo-bioreactors. In a rst step, all asks were ushed and
lled with CO2 (purity 99.995%). The asks were then lled via a
funnel with 200 mL of a mixture of a uid medium and juvenile grown
algae of the acido- and thermophilic red algae of Galdieria sulphuraria
(SAG 108.79). These were provided by the culture collection of algae at
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Algal Research 26 (2017) 294301
Goettingen University in Germany (SAG). They were added in an in-
itially sterile environment in order to guarantee monoseptic cultivation.
The uid medium for the algae was prepared according to Gross and
Schnarrenberger [41]. After this lling stage with the algae-bearing
medium, all asks were ushed again for a minimum of 6 min with CO2
at 500 mL min 1 before their nal sealing. This also marked the start of
the experiment. In addition, 6 asks was prepared with algae-free
medium as a blank control.
All samples were permanently shaken with a rotation speed of
35 rpm on a benchtop shaker (TR-250, Infors HT) in order to achieve a
homogenous algae suspension without sedimentation. The experi-
mental asks with the microalgae cultures were also illuminated con-
tinuously with a photon ux density of 75 mol m 2 s 1 that was
measured with a planar light sensor (LI190, FA LiCOR, Germany). The
temperature was kept nearly constant at (26 2) C during the whole
cultivation. This temperature range did not aect the quality of the data
because it can only cause small changes in oxygen solubilities
of 0.02 mmol L 1 O2.
After two days of equilibration between the gas and liquid phase,
the pH of the samples reached a value of 2 and the rst algae-bearing
ask and the blank sample were sampled as starting points to ensure
adequate starting conditions. Further samples were taken from in-
dividual, and up to this point unopened asks, after 7, 11, 18, and
29 days. As a further control to ensure that no atmospheric oxygen
entered the system, one of the asks was sampled for stable isotopes of
dissolved oxygen (DO) and oxygen concentration without addition of
the algae. Further 3 sets of duplicate asks with medium control were
kept separate of the experiment to ensure good repeatability of samples
between asks.
3.1. Sampling procedures
A volume of 1, 2, and 4 mL of each ask's headspace gas were
transferred with a gastight syringe into helium pre-ushed 12 mL Labco
Exetainers with butyl rubber septa (Labco Ltd., Lampeter, UK). Before
this transfer, the gastight syringe was ushed three times with sample
headspace from the experimental asks. These dierent volumes al-
lowed compensating for variable O2 concentrations in the headspace
and helped maintain appropriate signal heights on the mass spectro-
meter during the analyses. The subsampled headspace gas was then
analysed for its O2 concentrations and their stable 18O/16O ratios that
are expressed here as 18OO2.
In order to exclude any limitation of algae growth, samples for CO2
concentration were also collected and analysed from the asks' head-
space gas.
At each sampling event, samples of the uid medium were also
extracted from the asks' septum ports via disposable syringes. Aliquots
of the medium were measured for dissolved oxygen concentrations with
an optical sensor probe (Hach HQ40D, Loveland, Colorado, U.S.A.) that
uses an oxygen sensitive luminescent optode (luminescent dissolved
oxygen; LDO sensor). Cell concentrations were determined by optical
density measurements at a wavelength of 750 nm in a spectro-
photometer (Model SPECCORD by Analytic Jena). The corresponding
algal biomass was determined according to Schwerna et al. [10]:
X = 0.4705 OD750 nm + 0.0716 (2)
1
where X is the algal biomass in g L and OD750nm the optical density
at 750 nm wavelength.
In order to support this measure, a proxy of dry biomass was ltered
on 0.7 m pore size glass bre lter papers that were previously heated
at a temperature of 550 C overnight to make them sterile. After l-
tration the lters were dried at 60 C and the dry biomass was de-
termined by weight dierence. Results are listed in Table 3.
Samples for isotope measurements of dissolved oxygen (expressed
as 18ODO) were ltered through 0.45 m pore size polyethersulfone
(PES) disk lters and transferred into 12 mL Exetainers that were
M. Mader et al.
immediately capped and lled without any headspace or air bubbles.
Control experiments revealed no inuence on the oxygen isotope signal
by this sample treatment. Within 5 min after sampling, all Exetainer
vials for stable isotope measurements were poisoned with 50 L of a
saturated HgCl2 and CuSO4 solution with concentrations of 10 g L 1
and 100 g L 1, respectively. The vials were then wrapped into alumi-
nium foil to block o any further inuences of light and kept at 4 C in
the dark. Stable isotope analyses were performed within 12 h.
Samples for dissolved inorganic carbon (DIC) concentration were
lled into 40 mL poisoned brown glass vials in the same manner as
described for DO. From each ask, one additional Exetainer was lled
for the analyses of oxygen stable isotope ratios of water (18OH2O). It
was lled in the same manner as for the 18ODO analyses but without
poisoning. The latter step was not necessary because biological activity
does not change the water isotope composition if large amounts of
water molecules are present. Triplicate control measurements for water
isotopes were also carried out from separate Exetainers in order to
ensure good repeatability of the samples.
3.2. Measurement procedures
3.2.1. Stable isotope measurements
Water and headspace samples were analysed for their stable isotope
ratios of oxygen (18OO2 and 18ODO) and also for their oxygen con-
centrations by an automated continuous ow approach by Barth et al.
[42]. This method was adapted to an automated extraction unit (Gas-
bench II) that was coupled to a Delta V Advantage Isotope Ratio Mass
Spectrometer (IRMS; Thermo Fisher Scientic, Bremen, Germany). The
isolation of dissolved oxygen into a headspace relies on the helium
extraction technique described by Kampbell et al. [43] and Wassenaar
and Koehler [44].
All samples were measured in triplicates and the reported value here
is the mean value. Values are reported in the standard delta notation in
per mille () versus VSMOW according to Eq. (1). Further selected
duplicate asks with control media were also analysed with triplicate
shots in order to show good repeatability between asks.
The concentration of the O2 in the headspace gas was determined
from the peak area of the chromatogram of the IRMS. It was calibrated
with a series of air dilution standards with known amounts of oxygen
(from 20 to 1500 L air in 12 mL helium-ushed Exetainers).
CO2 concentration was determined from the peak areas of the
chromatogram by an automated equilibration unit (Gasbench 2;
Thermo Fisher Scientic, Bremen, Germany) coupled in continuous
ow mode to a Delta XP IRMS (Thermo Fisher Scientic, Bremen,
Germany). The calibration was performed as a 1 point calibration with
a gas mixture of 0.3% CO2 in a Helium ushed Exetainer. Due to the
large range of CO2 concentrations within the headspace and the sen-
sibility of this IRMS system, a series of diluted gas samples with variable
gas contents (2 and 4 mL in a Helium ushed Exetainer) were mea-
sured.
Concentration of DIC was determined with an Aurora 1030 W TIC/
TOC analyser (OI Analytical, College Station, Texas). The sample was
reacted with 0.5 mL of 5% phosphoric acid (H3PO4) at 70 C for 2 min
to release the dissolved inorganic carbon (DIC) as CO2. The latter was
measured by an internal nondispersive infrared sensor (NDIR) and a set
of calibration standard with known concentrations.
Fluid samples were analysed for 18OH2O by an automated equili-
bration unit (Gasbench II) in continuous ow mode coupled to a Delta
plus XP IRMS (Thermo Fisher Scientic, Bremen, Germany). All sam-
ples were measured in duplicates and the reported value is the mean.
These water isotope measurements are also reported according to Eq.
(1).
3.2.2. Determination of the photosynthesis to respiration ratio
The photosynthesis to respiration ratio (P/R) was determined ac-
cording to Eqs. (3) and (4) [45]. They are based on the inuences of
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Algal Research 26 (2017) 294301
photosynthesis, respiration, and air/water exchange [45,46]. The
equation was re-arranged for closed system conditions and uses the
measured 18OO2 isotope value of the headspace gas instead of the
isotope composition of the atmosphere.
18:16O 18:16 Og
r
P: R = 18:16O
w p 18:16 Og (3)
P:R is the photosynthesis to respiration ratio, 18:16
Ow represents the
oxygen isotopic composition of water, p is the fractionation factor due
to photosynthesis (p = 1 according to Guy et al. [19]), r is the iso-
tope fractionation factor due to respiration (r = 0.982 according to
Kiddon et al. [12]),18:16O is the measured dissolved oxygen isotope
composition of the sample and 18:16Og represents the ratio of the net air-
water uxes of 18:16O and 16:16O [45]. It is calculated according to the
following equation:
O2 18:16
18:16O =
g 18:16OHS s
( ) O2s
O
g
1 O2
O
( ) 2s (4)
g is the fractionation factor associated with gas transfer (g = 0.992
Knox et al. [47]), 18:16OHS is the measured oxygen isotopic composition
of the headspace gas, s is the fractionation factor associated with gas
dissolution (s = 1.007 Benson and Krause [39]), O2 is the dissolved
oxygen concentration of the sample and O2s is the dissolved oxygen
concentration at saturation.
3.3. Experimental replication and statistical treatment of data
Each ask served as one single photo-bioreactor. However each
sample was measured in triplicate for 18ODO and the values presented
here are the mean values. These triplicates had standard deviations that
were smaller than 0.2 vs. VSMOW except for samples with very
low oxygen content (blank uid with a standard deviation of 0.4)
and sample no. 5 (with a standard deviation of 0.9). For con-
rmation of the validity of the method multiple measurements of
18OO2 controls also had standard deviations smaller than 0.2 vs.
VSMOW. Moreover, each individual sample was injected three times
into the IRMS for isotope ratio determinations of dissolved and gaseous
O2. Also standard deviations of water stable isotope analyses (18OH2O)
were better than 0.1 as determined by multiple analyses of stan-
dards from dierent Exetainers (n = 5) and samples (n = 3). The data
sets for stable isotope measurements were corrected for a blank signal,
linearity (i.e., detector-related shifts of isotope ratios in response to
peak size) and instrumental drift during the run.
Specied errors of the optical oxygen probe by Hach for DO con-
centration were 0.003 mmol L 1 for concentrations of up to
0.25 mmol L 1 and 0.006 mmol L 1 for concentrations above
0.25 mmol L 1. Oxygen concentrations were also measured by cali-
brating the chromatogram of the IRMS in triplicate. The latter revealed
a standard deviation that was smaller than 0.07 mmol L 1. The
dierence between oxygen probe values and oxygen concentration
measured by IRMS was smaller than 0.01 mmol L 1. Multiple con-
centration measurements of oxygen gas samples at various concentra-
tions revealed a precision better than 0.05 mmol L 1.
Accuracy of analyses for CO2 concentration was monitored by re-
peated analysis of a standard with known concentration during the run.
Here the reproducibility was better than 0.05 mmol L 1 (1).
Higher numerical values of the calculated dilution factors may explain
additional errors during multiple transfer of samples. Multiple mea-
sured samples yielded a precision of about 1 mmol L 1 or 3% sa-
turation.
The DIC concentrations showed standard deviations
of 0.03 mmol L 1 (1).
Triplicates of optical density (OD) measurements showed standard
deviations that were smaller than 0.03. Errors for P/R ratios were
M. Mader et al. Algal Research 26 (2017) 294301

15 Table 1
[O2] mmolL-1 Headspace
A 0.5
Measured stable isotope ratios of oxygen and oxygen concentrations of the gas phase
during the experiment.
[DO] mmol L-1 Sample

Dissolved Oxygen mmol L-1

Days 18OO2 [O2](g)
[] VSMOW s.d. [mmol L1] s.d.1
0.4
[DO] mmol L-1 Control
10
O2(g) mmol L-1

CO2 container 23.2 0.5 0.10 0.01

Blank HS 0/2 23.0 0.2 0.16 0.01
0.3 Sample HS 1 2 11.8 0.2 0.06 0.01
Sample HS 2 7 4.5 0.1 1.08 0.01
Sample HS 3 11 2.3 0.1 3.62 0.01
Sample HS 4 18 3.0 0.1 5.27 0.02
0.2 Sample HS 5 29 3.5 0.2 13.96 0.04
5
VSMOW = Vienna Standard Mean Ocean Water; HS = Headspace.

0.1
value of the headspace blank sample only changed slightly to a value of
+ 23.0 (Table 1). Headspace O2(g) concentrations were below
0.0 0.2 mmol L 1. Both values conrmed a sterile environment as the
0
0 10 20 30 starting conditions of the experiments.
days This blank sample was then used to dene the starting point of the
experiment. The next sample (no. 1) was collected 2 days later and
30 showed only slight changes in oxygen concentration in the headspace
18
ODO Sample
B 18O Headspace
O2
that also remained below 0.2 mmol L 1. However, its 18OO2 signal
18OH2O
had already reached a value of +11.8. This value further declined to
20 18
ODO Medium
a minimum of 4.5 after 7 days (sample no. 2). After this, the
18O Control
DO
18OO2 values remained close to this minimum with values between
2.0 and 3.5 during the rest of the experiment. During the course
18OO2 []

of the experiment, oxygen concentrations in the headspace increased up

10 to 13.96 mmol L 1 (sample no. 5) after 29 days with a rate of about
+ 0.5 mmol per day.
One concern when conducting this experiment was leakage of at-
mospheric O2 into the experimental asks that would have masked the
0 oxygen produced by photosynthesis. This process could be excluded by
a mass balance calculation that applied the oxygen stable isotope values
of headspace oxygen after 29 days and the known oxygen isotope va-
lues of outside atmospheric oxygen (+ 23.9), as well as the measured
-10
isotope values of water. It revealed that enrichment in 18O by addition
of atmospheric O2 would produce an increase of 190 mmol of oxygen.
0 10 20 30 However, with maximum values of 13.9 mmol measured in the ex-
days periments this leakage could be neglected.

Fig. 1. A: O2 headspace gas concentrations (squares and left axis) and dissolved oxygen
concentrations (right axis) of samples (black diamonds) and medium controls from
multiple (transparent diamonds) during the experiment. B: headspace gas oxygen isotope
composition (squares), dissolved oxygen isotopic composition of samples (black diamond)
and medium controls from multiple asks (transparent diamonds) and oxygen isotopic
composition of water (circles). Standard deviations of multiple measurements are smaller
than symbol size except the last dissolved oxygen concentration and 18ODO sample. The
grey area marks the range of control medium standards.
calculated with minimum and maximum values of the input data and
are shown in Fig. 2 as dark grey areas bracketing the data points.
Reproducibilities among the asks of medium of 6 control asks
that were analysed in duplicate after 2, 7 and 11 days ranged between
22.1 and 20.4 vs. VSMOW for 18ODO and 0.04 to 0.08 mmol L 1
oxygen concentrations during the experiment. They are indicated as
grey horizontal bars in Fig. 1.
4. Results
4.1. Oxygen isotope values and concentrations
4.1.1. Gas phase
At the start of the experiment, the headspace gas in the ask was
almost oxygen free with a gas concentration of < 0.1 mmol L 1
(Fig. 1). This background-O2 had an isotope composition of +23.2
that was close to the one of air (+ 23.9). After 2 days, the 18OO2
4.1.2. Fluid phase
The medium that was added to the asks at the beginning of the
experiment had a dissolved oxygen concentration of 0.22 mmol L 1
and an oxygen saturation of 83% with a 18ODO value of 23.5 vs.
VSMOW. This isotope value is near atmospheric equilibrium (Fig. 1).
After two days (the 18ODO of the medium of the blank uid was about
1.5 lower and the DO concentration dropped to a saturation of 0.26%
(Table 2). As in the development of the headspace gas concentration,
oxygen concentrations of blank sample and sample no. 1 were also si-
milar in their uid phases with 0.06 and 0.07 mmol L 1. Also the
oxygen isotope composition between the blank and sample no. 1 de-
creased only slightly to a value of + 21.1 vs. VSMOW. Further de-
creases of the isotope composition of dissolved oxygen 18ODO in the
uid samples were also observed, however they did not drop as rapidly
as in the gas phase. They decreased continuously with a rate of about
0.6 per day to a value of +4.6 after 29 days (Sample no. 5).
The dissolved oxygen concentrations showed similar trends when
compared to those of the headspace, but at much lower concentrations.
They increased from 0.074 mmol L 1 in sample no. 1 to a value of
0.452 mmol L 1 in sample no. 5. The oxygen isotope composition of
water 18OH2O) remained stable with an average value of (9.16 1)
over the entire experiment as shown by repeated analyses from all
experimental asks.

297
M. Mader et al. Algal Research 26 (2017) 294301

Table 2
Measured temperature, stable isotope ratios of water, oxygen and oxygen concentrations of the uid phase during the experiment.

Days T 18OH2O 18ODO [O2](aq)

[C] s.d. [] VSMOW s.d. [] VSMOW s.d. [mmol L1] s.d. 1

Medium 24.6 0.1 9.19 0.1 23.5 0.0 0.22 0.030

Blank uid 0/2 28.0 0.1 9.19 0.1 21.9 0.4 0.06 0.001
Sample 1 2 27.2 0.1 9.18 0.1 21.1 0.2 0.07 0.003
Sample 2 7 26.4 0.1 9.21 0.1 16.6 0.2 0.09 0.006
Sample 3 11 26.2 0.1 9.18 0.1 11.4 0.2 0.11 0.004
Sample 4 18 25.3 0.1 9.16 0.1 8.0 0.2 0.16 0.004
Sample 5 29 25.3 0.1 9.08 0.1 4.6 0.9 0.45 0.075

VSMOW = Vienna Standard Mean Ocean Water.

Table 3
Photosynthesis-respiration ratios in comparison with optical density, algal biomass, CO2 and DIC concentration.

Days P/R ratio Optical density Calculated algal biomass Dry biomass CO2 DIC

1 1
[] s.d. [] s.d. [gL ] s.d. [gL ] [%] s.d. [mmolL 1] s.d.

Medium 0 0.1
Blank uid 0/2 0.1
Sample 1 2 0.7 0.16 0.001 0.15 0.07 85 3 16.9 0.03
Sample 2 7 5.3 0.1 0.28 0.001 0.20 0.07 0.27 77 3 15.8 0.03
Sample 3 11 7.7 0.3 0.43 0.008 0.27 0.08 0.34 72 3 13.9 0.03
Sample 4 18 2.6 0.1 1.25 0.015 0.66 0.08 0.83 64 3 12.6 0.03
Sample 5 29 0.4 4.67 0.028 2.27 0.09 2.25 46 3 8.8 0.03

Standard deviations below 0.07 for P/R ratio are not considered in this table.

4.2. Photosynthesis to respiration ratios 4.3. Optical density and algal biomass

The P/R ratio was calculated for the uid phases and started at
about 0.1 in the medium (Table 3/Fig. 2). It turned to a negative value
of 0.7 after two days (sample no. 1). Note that these rst values may
have had too low oxygen concentrations to compute reliable P/R ratios.
Seven days later the P/R ratio had turned to a value of 5.3 until a peak
of 7.7 was reached after 11 days. From then onwards, the P/R ratio
became smaller again and turned towards a value of 0.4 after 29 days.
The measured optical density was 0.16 after two days (Sample no.
1) and increased slowly to a value of 0.43 after 11 days (Sample no. 3).
It further increased to a value of 4.67 at the end of the experiment
(Fig. 2). The algal biomass showed the same trend as it was calculated
by a linear formula of the optical density. Sample no. 1 showed a bio-
mass of 0.072 g L 1 and increased to 0.292 after 29 days (Table 3).
4.4. CO2 and DIC concentrations

CO2 and DIC concentration decreased during the experiment and

5 showed an opposing trend to DO concentrations. The CO2 saturation in
8 the headspace was 85% after two days and declined to 46% after
P/R Ratio
29 days. In a similar pattern DIC concentrations decreased from 16.9 to
8.8 mmol L 1 from day 2 to da 29 (Table 3).
Optical density
4
6 5. Discussion
Optical density
P/R ratio

3
5.1. Inuences by outside air and possible elevated pressures in the asks
4
Negligible amounts of atmospheric oxygen were present in the ex-
2 perimental asks at the start of the experiment. Most of the oxygen that
was introduced was in dissolved form in the growth medium. Its dis-
2 solved oxygen stable isotope composition of + 23.5 (Table 2) in-
dominance of photosynthesis dicated equilibration with atmospheric O2 [39]. The decrease of the
1 dissolved oxygen concentration in the uid and the slight increase of
0 oxygen in the headspace indicated mixing of the introduced oxygen
with the large oxygen free headspace at the beginning of the experi-
0 ment.
0 10 20 30 Dissolved oxygen concentrations that were calculated from the gas
days phase with Henry's law constants were similar to the measured ones.
This means that altered dissolution behaviour of O2 by elevated pres-
Fig. 2. Photosynthesis versus respiration (P/R) ratio according to Quay et al. [45]
(squares and left axis) in comparison with the optical density (n = 3) of the algal biomass
sures within the ask could be neglected.
during the experiment (dotted line and right axis). Standard deviations of triplicate
measurements of optical density were smaller than symbol size. The light grey area shows 5.2. Photosynthesis
the time period in which photosynthesis dominates while the dark grey area indicates the
variance of P/R ratio as calculated from maximum and minimum measured values.
Photosynthesis depends on the availability of light, CO2, water, and

298
M. Mader et al.
the density of the photosynthetic organisms that is usually measured by
OD according to Lambert-Beers law. In our experiment, all these
parameters were suciently available. Because of the almost oxygen-
free conditions at the start of the experiment, photosynthesis can be
assumed to be the only process that increased oxygen concentrations
within the asks. This was also conrmed by almost oxygen free blank
and control samples. Photosynthetic activity started instantly after
lling the experimental asks and its light-dependent reaction caused
splitting of water molecules to produce molecular oxygen. According to
Henry's law, some of this newly generated O2 dissolved in the uid
phase. However, the larger part of it migrated into the headspace.
During the splitting of the water molecule 16O-enriched oxygen with
its average value of 9.2 versus VSMOW was transferred to the
molecular oxygen in both, the dissolved and headspace phases. This
process has been observed in many other studies, for instance by Guy
et al. [19]. This implies that the longer photosynthesis has been active,
the more 16O-enriched oxygen becomes introduced into the dissolved
and gaseous molecular oxygen phases. In closed systems one would
expect that the stable isotope ratio of the molecular oxygen approaches
the same 18O/16O ratio as the one measured in the water used in these
experiments. The underlying reaction can be formulated as:
CO2 + 12H2 O C6 H12 O6 + 6H2 O + 6O2 (5)
It can be further divided into the light-dependent reaction with:
12H2 O 24H+ + 6O2 Oxidation (6)
and the dark reaction with:
6CO2 + 24H+ C6 H12 O6 + CO2 Reduction (7)
Even though we need to assume that the production of O2 continued
after the experimental time of 29 days, the stagnating 18OO2 ratios in
the headspace gas indicated on-going addition of 16O-enriched oxygen
that were counterbalanced by eects of respiration. Further investiga-
tions of the steady state operation of such closed systems would be
necessary to show developments of oxygen dynamics together with its
stable isotope ratios.
5.3. Respiration
During the entire experiment neither the headspace gas nor the
dissolved oxygen reached the 16O-enriched isotope value of the water
used in the experiment. This means that a counterbalancing enrichment
in 18O of the molecular oxygen must have taken place. Such enrichment
of 18O can be caused by oxygen consuming processes that were hap-
pening at the same time. Consumption of oxygen is isotope selective
and preferably removes 16O from the dissolved oxygen pool. This pro-
cess, thus, enriches the remaining molecular oxygen in 18O [15]. This
was described by Eisenstadt et al. [48]. They analysed specic marine
and freshwater phytoplankton that consumed some of the produced
oxygen and found an enrichment of +4.5 to + 7 versus VSMOW at
low O2 levels of below 3 mol L 1. At these low O2 levels, major
consuming reactions, such as photorespiration, can be neglected since
the oxygenase activity of RuBisCO forming glycolate is only present at
high extracellular oxygen concentration or low carbon dioxide con-
centrations [49,50]. In their experiments, the enrichment of 18O in-
creased with rising O2 concentrations and may have been caused during
cyclic electron transfer in Photosystem II [48].
In our experiments, the dierence between the value of 18OO2 in
headspace gas and average of 18OH2O of substrate water ( 9.2) at
the end of the experiment amounted to 5.6 and shows consumption
of O2 also during the growth of for Galdieria sulphuraria. When con-
sidering the dissolved oxygen, this dierence was 13.8 between the
18ODO and the average 18OH2O isotope values at the end of the ex-
periment. This gap between substrate water and 18OO2 and 18ODO
may be specic for this type of algae. Future studies should, therefore,
also test if other species have similar eects and if systematic
299
Algal Research 26 (2017) 294301
dierences can be established.
Respiration seems to consume oxygen primarily in the uid phase, if
enough electron donor molecules are available, for instance in the form
of organic matter. Here aerobic respiration is the most prominent
oxygen consuming process that likely enriched the residual DO in 18O.
Therefore, it counteracts stable isotope shift towards 16O-enriched
isotope ratios that are caused by photosynthesis. These respiration
processes could have been caused by bacteria or by respiratory activity
of the algae themselves. Note that other methods that do not rely on
stable isotopes can also investigate respiration rates in submerged xed
bioreactors [51,52]. However, they require turning the oxygen supply
on and o, a method that is not possible in closed systems.
The dierence in oxygen isotope composition between headspace
oxygen and DO concentration can best be explained by preferable mi-
gration of the produced O2 into the headspace. This is because oxygen
has a poor solubility of, for instance, 0.284 mmol L 1 at 20 C [53].
Once the produced molecular oxygen reached the headspace, it was
much less aected by respiration because the biomass was in the liquid
phase. This was obvious by the more 16O-enriched isotope ratio of the
gas phase, when compared to the dissolved oxygen that was more in-
tensively aected by respiration. The stronger inuence by respiration
in the uid phase seems plausible because it also caused DO con-
centrations to increase at a lower rate of 0.5 mmol L 1 per day, when
compared to the concentration changes in the headspace. This is also
conrmed by the isotope values of the dissolved and gaseous oxygen
with more 18O-enriched values in the uid phase.
Both key processes that inuence stable isotope compositions and
concentration of oxygen were quantied by calculating the P/R ratio of
each sample. This ratio is inuenced by oxygen-producing and by
oxygen-consuming processes. At the beginning of the experiment, a low
algal density in the medium likely resulted in a P/R ratio < 1 and
indicated a dominance of respiration. Note that the oxygen con-
centrations were very low at this time and possible oxygen production
or consumption occurred at small rates, thus, causing larger un-
certainties. As expected, the production of oxygen increased after
2 days and reached a peak value after 11 days. At this time, oxygen
production by photosynthesis exceeded oxygen consumption by a factor
of 7.7. However, after this peak point, further increasing oxygen pro-
duction likely caused a rise in aerobic respiration. Note that it is not
possible to describe this turning point by a limitation of CO2 and DIC as
both parameters were suciently available to sustain photosynthesis
until the end of the experiment. As expected both parameters decreased
as a result of on-going photosynthesis.
In comparison, algal densities permanently increased during the
growth phase together with oxygen concentrations. These increasing
rates did not point out the time when photosynthesis started to become
limited again. This critical point was also not shown by oxygen con-
centrations alone. However, this changeover may be important in de-
termining the optimal functioning of a closed system algae reactor.
While it may be worth to collect algae products after this point, the
eciency of the system starts to decrease again until it reaches a status
in which respiration dominates the system.
6. Conclusions
This study employed a new stable isotope method of molecular
oxygen to determine the P/R ratios in a closed bioreactor experiment
with algae. This stable isotope approach can be seen as a new tracer for
photosynthetic eciency. Our study therefore presents a rst technical
approach for a new tool in bioengineering that enables to balance
oxygen-consuming and producing activities during algal growth. While
approved parameters, such as optical density, dry biomass and
oxygen concentration measurements, only focus on primary produc-
tion, the P/R ratios introduced here can also outline counteracting
aerobic respiration.
Including stable isotope measurements has a good potential to
M. Mader et al.
dene the critical point when a closed-system bioreactor starts to lose
its productivity. Changes of P/R ratios may serve as a useful early
warning system to either adapt parameters or to initiate a new algal
growth period for engineered bioreactors. The method, thus, employs a
new tool to decide, after which processing time respiration dominates
over photosynthesis and when the system becomes inecient in terms
of exploiting photosynthetic products.
In future studies, our method has the potential to provide better
insights into photosynthesis that produces ATP and NADPH for the
xation of CO2. Both enzymes are important for bioreactors because
they can be subsequently metabolised to biomass building blocks [54].
Moreover, the presented method could be helpful to control algal
growth and associated primary production in industrial bioreactors. It
may help to optimize specic light conditions and spectra, if stable
isotope ratios of molecular oxygen are responsive to their changes [55].
The method should also be applied and tested in open bioreactors in
order to weigh inuences of atmospheric and photosynthetic oxygen, as
they are expected to have strongly dierent isotope values.
More testing of this method would be necessary on other algal
strains in order to explore specic isotope changes and to nd out if
they develop individual optimal P/R ratios. More frequent measure-
ments could also provide more exact points of change. In addition, such
work should be complimented by carbon concentration and isotope
analyses because they are also inuenced by photosynthesis and re-
spiration. Overall, this study showed that natural abundance oxygen
stable isotope measurements may serve as novel and useful tools in
algae research and the optimization of their industrial production
strategies.
Acknowledgements
Funding was provided by the German Research Foundation (DFG
grant BA 2207/13-1) and a grant for major instrumentation to the chair
of Applied Geology (INST 90/678-1 FUGG). We also extend our thanks
to Chris Sillman from Thermo Fisher for advice and to Christian Hanke
for help with analyses.
Author contributions
Mader, Schwerna, Barth and Buchholz designed the study. Mader,
Schwerna and van Geldern performed laboratory sampling and ana-
lysis. Mader, Schwerna and Barth contributed to data analysis and in-
terpretation. Mader wrote the initial draft. All authors read and com-
mented on the drafts of this manuscript.
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