Materials and Method

Biologic material: Chlamydomonas reinhardtii

A sclareol/MO overexpressed producer Chlamydomonas reinhardtii (named as B4) was
constructed and provided by Bielefied University in Tris-Acetate-Phosphate agar plates.
Cultures were prepared in a sterile laminar airflow cabinet in 250 mL flasks using 100
mL of sterile TAP medium (Error: Reference source not found). For cultivation, an
orbital shaker incubator was used at 85 RPM, 25°C and in a 16:8 day/night regime with
a light intensity of 130 µmol m-2 s-1 during light hours. In this incubator, the level of
CO2 was 3% (v/v). Before inoculation in every photobioreactor, cultures grew until cell
concentration was enough to reach an optical density of at least 0.25 in the final
bioreactor. Materials, flasks, and bioreactors were sterilized utilizing autoclave
(Laboklav ECO 195-B, Steriltechnik AG) at 121°C for at least 15 min.

Determining biomass concentration

The biomass concentration was determined by measuring the optical density
(absorbance) of 1 mL sample using a spectrophotometer at 750 nm (DR 5000™ UV-
Vis, Hach Lange). Each culture sample was diluted with demineralized water so the
value of absorbance was between 0.2 and 0.5. Absorbances are correlated to the
biomass concentration by dry weight determinations (Chioccioli, Hankamer, and Ross
2014). These values were then used to calculate the biomass concentration with
equation 1 (Elarbab 2021). Moreover, the dried weight of 2 mL samples was also
determined by filtering and washing the pellet with 0.5 M ammonium formate
(Vejrazka et al. 2011). These samples were then dried in an oven at 100°C overnight.
Dried biomass was weighted and data was expressed in g L -1. These results were
compared with biomass concentration obtained with optical density to verify the
equation.

C x =0.4392∗OD750 nm +0.0599 (1)

Quantum yield determination

Quantum yield was measured, which is linked to the health of the culture since it
determines the photosynthetic efficiency of the culture (Posten and Feng Chen 2016). A
sample of 1.5 mL was placed in a cuvette (Sarstedt) in a dark place and after 10 min, the
hand-held fluorometer (AquaPen-C AP-C 100, Photon System Instruments) was used to
measure the quantum yield. Data obtained were expressed as a ratio with values
between 0 and 1.

Diterpenoid quantification
Samples of dodecane obtained from each experiment were analysed according to what
protocol by gas chromatography coupled with a flame ionization detector (GC-FID)

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(7890A, Agilent technologies). A stock solution of sclareol (Sigma-Aldrich 49944) of
150 mg/L was prepared. Dilutions in dodecane of 100, 50, 25, 10, 5 and 1 mg L -1 were
prepared to elaborate the calibration curve (Error: Reference source not found). The
same procedure was followed for the elaboration of the calibration curve for manoyl
oxide (Sigma-Aldrich 532665) (Error: Reference source not found). Each dodecane
sample was clarified by centrifugation at 3000 rpm for 10 min prior to analysis. A
volume of 200 µL of was placed in a glass vial that was later placed in the autosampler
of the chromatographer. The column used was an HP-5 30m x 0.320mm ID coated with
0.25µm of (5%-phenyl)-methylpolysiloxane (Agilent Technologies). The temperature of
the injector was 250°C and 1 µL was injected in splitless mode. A constant flow of 1
mL min-1 of hydrogen at 18.139 kPA was used as a carrier gas. The oven temperature
was held at 50°C for 0.45 min, then raised to 120°C at 22.04°C min-1, followed by
6.61°C min-1 to 160°C, later to 270°C at 22.04°C min -1 which was held for 1 min.
Finally, temperature was raised to 320°C at 22.04°C min-1 and held for 5 min. The
detector temperature was placed at 325°C. Areas of peaks with retention time related to
sclareol and manoyl oxide were then converted into mg L-1 of dodecane using the
calibration curve for sclareol and manoyl oxide respectively.

Calculations
Based on the concentration diterpenes obtained from GC, Biomass productivity,
diterpene productivities and yields were calculated (see equations 2 to 7). Parameters,
symbols and units are shown in table 1.

Table 1. Parameters and units used for calculations.

Parameter Symbol Unit

Diterpene concentration in dodecane a
C pd mg L-1
Diterpene concentration in culturea b C pc mg L-1
Volume of dodecane Vd L
Volume of culture Vc L
Biomass concentration a
Cx g L-1
Biomass productivity Px g L-1 d-1
Dilution rate D d-1
Extraction time t d
Diterpene Productivity P mg L-1 d-1
Diterpene yield Y %
Maximum daily yield Y max %
a
i subindex is used to specify a particular day
b
Only numerically since diterpenes do not accumulate in the culture

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 C pd∗V d
C pc= (2)
Vc

In batch:

C pc
P= (3)
t

C pc
Y= ∗100 (4)
C x∗1000

C pc . i
Y max = ∗100 (5)
C x. i∗1000

In photochemostat:

P x =C x∗D (6)

C pc
Y= ∗100 (7)
t∗P x∗1000

C pc .i−C pc . i−1
Y max = ∗100 (8)
P x∗1000

Flask cultivation
Under the flaks culture conditions previously described, extraction of sclareol and
manoyl oxide from flaks cultivation was performed using 5% dodecane overlay over
100 mL of culture starting at least in 0.25 of optical density (corresponding to a biomass
concentration of 0.17 g L-1). Samples of 1 mL of culture and 250 µL of overlay mixture
were taken regularly. Overlay sample was then clarified by centrifugation at 5000 rpm
for 10 min (Microstar 17R, VWR) and 200 µL of supernatant was sampled for sclareol
quantification. Culture growth was assessed by measuring optical density. Experiments
were done in biological replicates.

Algaemist
Algaemist are flat panels photobioreactor of two compartments (Figure 1) (de Mooij et
al. 2016). One compartment contains the culture and the other one contains the water
jacket for temperature control. The system is automated controlled. The culture
compartment faces the led-light source which is set in percentage which is calibrated (

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Error: Reference source not found). Medium is pumped into the culture with a
peristaltic pump set in percentage as well and reactor volume is maintained via gravity
due an overflow exit. Light and chemostat regimes can be controlled by an inner timer.
Filtered air (0.2 µm PTFE filter Acro 50, Pall corporation) is sparged to maintain the
culture well mixed and excess of air is released through an upper pipe to avoid
overpressure.

Figure 1. Algaemist photobioreactor (de Mooij et al. 2016)

Ex situ extraction
An ex-situ extraction experiment was performed using the Algaemist bioreactor which
was set at 825 µmol m-2 s-1 in a 12:12 day/night regime, and at a dilution rate of 0.5 d -1.
The daily collected overflow (harvest) was placed with 5% (v/v) of dodecane in a 500
mL flask covered with aluminium foil and agitated during 20 h at 500 rpm (Figure 8).
After this mixing step, broth and formed emulsion was placed in a decantation funnel.
Aqueous phase was separated 10 min later and emulsion and dodecane layer were
centrifugated (ScanSpeed 1580R, Labogene) at 3000 rpm for 15 min at 15°C. A sample
of dodecane was taken daily. New dodecane was used for every extraction day. Biomass
concentration was assessed by optical density and the quantum yield quantified.

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 Figure 2. Extraction process in the semi-batch approach.

In-situ extraction
In this first approach, the bioreactor was set at a light intensity of 825 µmol m-2 s-1 in a
12:12 day/night regime. The air flow was set at 250 mL min -1 and CO2 on demand to
control the pH. The temperature and pH were automatically controlled and kept at 25°C
and 7.5, respectively. The bioreactor was run at batch conditions to reach the maximal
growth (µ), moment when the photo-chemostat was started at a dilution rate of 0.5 d -1.
the formation of emulsion is deliberately induced inside the bioreactor via air sparging
(Figure 3). An experiment with a volume of dodecane equivalent to 20% of the culture
was pumped from the flask through a pipe with holes into the bioreactor in a rate of
1.17 mL min-1. Small solvent bubbles travelled along the bioreactor to the surface of the
culture where they replace part of the volume of the culture (about 36 mL). When this
volume was reached a mixture of dodecane and broth started to leave the bioreactor
through the overflow pipe and then collected in the harvest vessel. Attached to the
bioreactor, a funnel was used as a decantation vessel for separating the dodecane from
the aqueous phase by difference in densities. Two experiments were performed with 5%
of dodecane and with lower light intensities: 419 and 256 µmol m-2 s-1

A special dodecane-resistant hose made of platinum-cured perfluororelastomer and

expanded polytetrafluoroethylene (ePTFE) (STA-PURE series PFL, GORE) was used
as it has been previously found that silicon hoses were dramatically degraded by the
solvent and shear force caused by the action of the pump (Elarbab 2021; Kovács 2020).
After the whole volume of solvent had passed, it was then removed from the bioreactor
and from the overflow collector vessel (harvest). Collected solvent plus formed
emulsion was placed in the decantation funnel overnight covered with aluminium film.
Aqueous phase was then discarded, and solvent reintroduced into the bioreactor the next
day to continue with the extraction process. Volumes of solvent were measured every
day and samples of dodecane were taken for sclareol determination. No extraction was

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performed at night periods and during light ones, the bioreactor was at least 1 h without
contact with the solvent just before and after dark period. Biomass concentration was
determined by optical density and quantum yield was quantified. Pumps for TAP
medium and for dodecane were calibrated (Error: Reference source not found and

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Error: Reference source not found). pH meter was calibrated using 4.0 and 7.0 buffer
solutions. All connections were made using silicon hoses and polypropylene and
polyamide connectors.

Figure 3. Scheme of the extraction performed in Algaemist reactors. 1: Dodecane vessel; 2:

Bioreactor; 3: Decantation Vessel.

Infors-HT
The second strategy was tested in an Infors-HT, a fermenter-like photobioreactor of 3.6
L coupled with a light source set in 1835 µmol m-2 s-1 (calibration in Error: Reference
source not found) run with 12:12 day/night regime (Figure 4). A dodecane overlay
equivalent to 5% of the culture was placed in the bioreactor (Figure 5). A first
experiment was performed in batch culture for 8 days, when a complete removal of
dodecane took place. A second experiment was run in photo-chemostat. After maximal
growth was reached, dilution rate set at 0.25 day-1 and new dodecane was used (Figure
6). Temperature and pH were set at 25°C and 7.5 respectively and controlled
automatically. Samples of dodecane were taken daily for sclareol determination.
Biomass growth was measured by optical density and quantum yield was determined. A
double purpose pump was used for TAP medium and overflow for the overflow which
was calibrated (

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Error: Reference source not found). pH meter was calibrated using 4.0 and 7.0 buffer
solutions. Connections were completed using silicon hoses and polypropylene and
polyamide connectors.

A B

Figure 4. A. INFORS-HT fermenter (Infors HT 2019). B. Led-Light source.

Figure 5. Overlay of dodecane in the Infors-HT bioreactor.

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Figure 6. Schematical overview of the second strategy.

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