J. Algal Biomass Utln. 2014, 5 (2): 66 - 73
Disposable algae cultivation
ISSN: 2229- 6905
Disposable algae cultivation for high-value products using all around LED-illumination
directly on the bags
Tobias Hahne1, Bernhard Schwarze1, Michael Kramer2, Björn Frahm1,
1
Biotechnology & Bioprocess Engineering, Faculty of Life Science Technologies, Ostwestfalen-Lippe University of Applied
Sciences, Liebigstr. 87, 32657 Lemgo, Germany,
2
LED Linear, Pascalstr. 9, 47506 Neukirchen-Vluyn, Germany.
Corresponding Author: Björn Frahm, bjoern.frahm@hs-owl.de
Abstract
This paper investigated if a reusable LED-illumination directly on all sides of disposable bags is possible. Besides the traditional
cultivation of algae in sunlight illuminated vessels, the interest in cultivation using indoor-bioreactors grows. This development can
be explained by the potential of producing high-value substances such as food additives, cosmetics and pharmaceuticals via algae.
However, the requirements for the corresponding production are more complex with regard to sterility, process control and
reproducibility. These requirements can be fulfilled in illuminated indoor photobioreactors. There are non-disposable commercial
indoor photobioreactor systems available made of steel or glass to cultivate phototrophic organisms. For high-value products out of
(mammalian) cell culture, disposable systems are more and more applied. This trend is also interesting for photobiotechnology. The
illumination for disposable bags consists of reusable flexible LED-lines placed directly on all surfaces of the bag. This placement
reduces light losses and illuminates the bag from all sides. Moreover, it is simple, space-saving and flexible. Cultivations of the
organisms Chlorella vulgaris, applied as a well-known alga, and Euglena gracilis for the production of the substance paramylon
demonstrated the suitability of such an illumination applied to a disposable Wave-system.
Keywords: algae, disposables, high-value products, LED-illumination, photobiotechnology
Introduction
Cultivating algae opens a field of different possible products. These products can be split in two major groups. The
first group is produced outdoor in vessels such as open ponds in order to obtain large amounts of algae biomass
(Borowitzka 1999, Blanco et al. 2007). Natural sunlight and CO2 or, if appropriate, exhaust gases containing CO2 are
used for cultivation (Benemann et al. 1987, Chen 1996, Lee 2001, Pulz 2001). These unspecific sources of light and
CO2 lead to variable process parameters but also enable low production costs. The second group contains high-value
products like cosmetics, food additives and pharmaceuticals (Baddiley et al. 2008). Their role in industry increased
within the last years. Different products like antimicrobials, antivirals, antifungals, neuroprotective products,
therapeutic proteins, drugs, carotinoids etc. are derived from algae (Borowitzka 1992, Bhakuni and Rawat 2005,
Sreevatsan 2010). Usually artificial light and CO2 are applied due to more complex requirements for the
corresponding production with regard to sterility, process control and reproducibility. Therefore these requirements
are generally met in illuminated indoor photobioreactors (Pulz and Scheibenbogen 1998).
There are several concepts for state of the art photobioreactors such as the BIOSTAT PBR (Sartorius Stedim Biotech
GmbH, Göttingen, Germany) using transparent tubes made of glass that are lit from neon tubes as outside light
sources. The Labfors 5 Lux LED Flat Panel (Infors AG, Bottmingen, Switzerland) is illuminated by outside LEDs.
The DASGIP photobioreactor system (DASGIP Information and Process Technology GmbH, Jülich, Germany) uses
fixed LED-bars installed inside the stirred bioreactor. Another example is the dual sparging laboratory-scale
photobioreactor for continuous production consisting of a glass cylinder that is lit from outside light tubes and
agitates the algae suspension using air bubbles (Eriksen et al. 1998). Beside these reusable reactors, disposable
bioreactors are an alternative. Since Dr. Vijay Singh developed the first Wave-bioreactor in the end-1990s, disposable
systems became a suitable alternative to stainless steel reactors for corresponding applications (Rao et al. 2009).
Plastic bags partly filled with medium serve as disposable cultivation chambers. Power input is for example achieved
by a rocking motion. Their gentle cultivation conditions are attractive for cultivating shear-sensitive cells like
mammalian cells or hybridoma. Without need for cleaning and sterilization, disposables usually offer time- and costsavings for high-value products. Furthermore, they are flexible and simple in use and can be faster installed then
conventionally bioreactors. These advantages can also be used in photobiotechnology. Like in non-disposable
photobioreactor systems, providing enough light to the phototrophic cells is the main challenge.
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J. Algal Biomass Utln. 2014, 5 (2): 66 - 73
Disposable algae cultivation
ISSN: 2229- 6905
Disposable systems illuminated by bottom-mounted LEDs (non-commercial system, Lehmann et al. 2013), topmounted LEDs in distance to the bag (Applikon, Schiedam, Netherlands), fluorescent tubes installed in distance
above the bag (non-commercial systems) and, for outdoor application, flat panel airlift photobioreactors lit by natural
sunlight (Bergmann et al. 2013) are developed and tested so far.
The concept realized in this paper is an illumination directly on all surfaces of the disposable bag. This arrangement
minimizes light losses between light source and bag surface by distance and to the surrounding. Special attention has
to be paid to the selection of light sources since heating up of the bag material and the corresponding risks of damage
have to be avoided. The concept allows adjusting the illumination for each bag individually without necessity for
housing of each bag, e.g. concerning wavelengths or intensity depending on the cultivated organism or cell density
for example. The system has reusable flexible LED-lines placed directly on the bags surfaces. A corresponding
scheme and a photo are presented in figures 1 and 2.
Fig. 1. Scheme of the illumination consisting of reusable flexible LED-lines on all sides of a disposable bag.
Fig. 2. Photograph of example set-up consisting of reusable flexible LED-lines on all sides of a disposable bag.
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ISSN: 2229- 6905
The disposable bag also acts as a cooling system for the cultivation broth and LED-lines. The radiated heat is
absorbed by the medium and the head space. Some disposable systems do not feature a cooling device but only
heating possibilities for temperature control of the medium, e.g. via a heating mat. In such cases an additional cooling
device has to be added. An example is the set-up described in the Materials and methods-section. In the presented setup, the bag is partly in direct contact with the cooled bag holder between the LED-lines. A negative effect of the
slightly different bag bottom shape on flow patterns or other parameters has not been observed.
This study presents design and application of an illumination directly on all sides of standard disposable bags in order
to generate a simple method to grow phototrophic organisms in standard lab-scale disposable bioreactors. As a
presented example a Wave-system is used to cultivate the algae Chlorella vulgaris and Euglena gracilis. Chlorella
vulgaris has been selected since a lot of data is available for comparison to literature data. Euglena gracilis produces
paramylon, a substance known for properties such as anti-inflammatory effect, stimulation of immune system, growth
inhibition of some tumor types and positive effect on blood sugar level, insulin regulation and cholesterol level.
Materials and methods
Algae
As a first example, the green algae Chlorella vulgaris SAG 221-11b from the culture collection of algae (SAG)
Göttingen, Germany, was used in the experiments. It belongs to the division of the Chlorophyta and the class
Trebouxiophyceae. The spherical single-cell with a diameter about 2 to 10 µm was first isolated 1889 in a sweet
water lake by M. W. Beijerinck in the Netherlands. Chlorella vulgaris serves as a model organism in the experiments
because of the well-known growth characteristics (Rodrigo et al. 2007, Liang et al. 2009, Yeh et al. 2010).
As a second example, cultivations of the algae Euglena gracilis (G. A. Klebs, 1883) have been performed for the
production of paramylon, a reserve polysaccharide consisting of ß-1,3-glycosidic linked D-glucose molecules.
Euglena gracilis SAG 1224-5/25 has also been obtained from the culture collection of algae (SAG) Göttingen,
Germany.
Medium and shake flask cultivation
Chlorella vulgaris was grown in a medium composed by E. Kessler (Kessler and Czygan 1970). It is rich in salts with
the lack of an organic carbon source in order to inhibit the growth of bacteria or other microorganisms which could
contaminate the cultivation of the algae. Table 1 shows the medium composition.
Table 1: Composition of the applied Kessler medium for Chlorella vulgaris cultivation
Substance
KNO3
NaCl
MgSO4 * 7H2O
CaCl2 * 2H2O
FeSO4 * 7H2O
MnCl2 * 4H2O
H3BO3
ZnSO4 * 7H2O
(NH4)6Mo7O24 * 4H2O
EDTA (Triplex III)
NaH2P04 * 2H2O
Na2HP04 * 12H2O
distilled water
Mass [g]
0.81
0.47
0.25
0.014
0.006
0.0005
0.0005
0.0002
0.00002
0.008
0.47
0.6
1L
The components without the phosphates were mixed in 0.9 L and autoclaved for 20 minutes at 121 °C. Afterwards
the two phosphates were dissolved in 0.1 L distilled water, also autoclaved and added under sterile conditions at
temperatures below 70 °C.
Euglena gracilis has been cultivated in a medium consisting of non-sparkling Volvic mineral water (996 mL per
Liter) and 4 mL plant fertilizer (called SERAMIS Vitalnahrung für Grünpflanzen, produced by Mars GmbH,
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J. Algal Biomass Utln. 2014, 5 (2): 66 - 73
Disposable algae cultivation
ISSN: 2229- 6905
Mogendorf, Germany). The pH-value has been set to 3.4 by sulfuric acid, 0.2 molar. After autoclaving, vitamins B1
and B12 have been added: 6 mL/L of vitamin B1 stock solution and 0.5 mL/L B12 stock solution (each stock solution
10 mg per 100 mL) via a 0.2 µm sterile filter. The Chlorella vulgaris cultures were first grown in 25 mL unbaffled
flasks on a shaker (Certomat R, Sartorius AG, Göttingen, Germany) at room temperature (~ 24 °C). They were filled
with 12.5 mL algae suspension and shaken at a frequency of 120 rpm and a shake diameter of 25 mm. The shake
flasks were lit by a fluorescent lamp Fluora T8, 18 W (Osram, Munich, Germany) which led to a photon flux in the
range of 32 µmol photons (m2s)-1 photosynthetically active radiation (PAR) measured in the center of a shake flask
filled with demineralized water at the given filling volume. PAR was measured with a spherical microquantumsensor
US-SQS/L (Walz, Effeltrich, Germany). After four weeks the cultures were each passaged into baffled 500 mL shake
flasks containing 250 mL Kessler medium. There they have been cultivated until needed to inoculate the
photobioreactor (usually for one to three months).
The Euglena gracilis cultures were grown at 25 °C in 50 mL medium and as a second seed train step 70 mL medium,
each in 500 mL unbaffled shake flasks. Lighting conditions have been the same as described above, but shaking took
place at a frequency of 75 rpm. These conditions have been applied for the first four days of each seed train step to
initiate growth. Subsequently, the shaking flasks have been placed without power input and additional lighting at
standard lab conditions.
Bioreactor cultivation
The used disposable bioreactor was a Wave-system C 20 SPS-F made for disposable bags with filling volumes up to
20 L which is already a significant test scale for high-value products. 2 L disposable bags (GE Healthcare, Freiburg,
Germany) filled with 1 L Kessler medium were moved at 30 rocks per minute and an angle of 4.6° (Chlorella
vulgaris) or 25 rocks per minute and an angle of 4.7° respectively (Euglena gracilis).
The flexible LED-lines VarioLED Flex VENUS IP68 (LED Linear, Neukirchen-Vluyn, Germany) served as a
reusable light-source. 16 LED-lines with a light emitting surface of 16 x 375 mm were placed around the disposable
bag (see Fig. 1 and 2), 8 LED-lines in case of Euglena gracilis. The LED-lines stay in place due to their weight but
have been additionally fixed by transparent tape. For potential commercial use, bags featuring pockets/ears for
insertion of the lines are conceivable. The effective length for illumination due to the bag geometry was 310 mm.
8 LED-lines emitted red light, the other 8 blue light, each at 7.7 W m-1 LED-line. For Euglena gracilis, 4 red and
4 blue LED-lines have been sufficient to create adequate illumination. The spectral radiance was analyzed using a
Specbos 1211 USB VIS/NIR Spectroradiometer (Jeti Technische Instrumente GmbH, Jena, Germany). Plotting
measurements of spectral radiance [W (m2 sr nm)-1] over wave length showed a maximum of the distribution at
454 nm for the blue LED-lines and a second maximum of 638 nm for the red LED-lines (graph not presented).
Applying 8 blue and 8 red LED-lines to the 2 L disposable bag (filling volume 1 L) resulted in lighting conditions of
228 W m-2 equal to 38 W dm-3 for the one liter filling volume which each corresponds to 633-1294 µmol
photons (m2s)-1 photosynthetically active radiation (PAR). Measurement took place in a water filled bag 0.5 cm away
from inner wall (see Fig. 1). The variation in PAR of 633-1294 µmol photons (m2s)-1 results from the actual
measurement position (sensor 0.5 cm away from inner wall in front of LED-lines or 0.5 cm away from inner wall
between two pairs of LED-lines). The disposable bag has been illuminated in a 16 h : 8 h day/night-rhythm.
The heating mat of the bag holder and a cryostat cooling device (D8-G, Haake, Germany) controlled medium
temperature inside the bag (described in the chapter Results and discussion). The culture suspension was surface
aerated with 4 % CO2 in 50 L h-1 air flow for Chlorella vulgaris and 3 % CO2 in 20 L h-1 air flow for Euglena gracilis
via a MX4/4 gas mixing station (DASGIP Information and Process Technology GmbH, Jülich, Germany). Exhaust
gas flow passed a cooled device to settle out water content from the gas flow and is analyzed with a Hartmann &
Braun Advance Optima analyzer (ABB Analytical, Germany). The exhaust gas analyzer used for the Chlorella
vulgaris cultivations required a minimum gas flow of 50 L h-1. Dissolved carbon dioxide and dissolved oxygen
concentration as well as pH-value and temperature were determined when sampling using the CO2-Sensor (MettlerToledo, Greifensee, Switzerland), pO2-Sensor Oxi 520, the precision-pH-mV-meter pH 521 (both manufactured by
Wissenschaftlich-Technische Werkstätten GmbH, Weilheim, Germany) and a Pt100 temperature sensor (Sartorius
AG, Göttingen, Germany).
Biomass concentration was measured by analyzing samples with a photometer (spectralphotometer CADAS 100,
Hachlange GmbH, Düsseldorf, Germany) at 550 nm. Afterwards the extinction was inserted into the following
equation (1) as x to calculate the biomass concentration for Chlorella vulgaris (y).
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y  0.2942  x 2  0.2212  x  0.0488
(1)
In case of Euglena gracilis, turbidity has been measured using a Lovibond TurbiCheck (Tintometer group). Biomass
concentration has been determined by centrifugation of the sample at 3170 g for 30 minutes (corresponds to
4500 min-1 using the centrifuge Universal 320, Hettich), washing, second centrifugation at the same parameters,
drying at 105-107 °C for 16 hours (+/- 2 hours) and weighing.
Results and discussion
Both for Chlorella vulgaris as well as Euglena gracilis producing paramylon, an example cultivation in a disposable
system illuminated directly on all sides of the bag via reusable flexible LED-lines is presented. As an example for a
disposable bag bioreactor, a Wave-system has been used. At first the concepts of illumination and temperature
control of the bag (if not yet provided via the disposable bag bioreactor) are described followed by the cultivation
results.
Illumination and temperature control
The concept of illuminating the algae suspension in the bag is to install flexible LED-lines directly on all surfaces. So
the suspension can be lit from the bottom side, the sides and above. In order to achieve this a blue and red LED-line
are attached to each other, placed around the bag as shown in figure 1 and fixed to the bag and to the bag holder. In
the presented case red and blue LED-lines have been applied in order to directly supply specific wavelengths (see
Materials and methods). Of course different wavelengths or white LEDs emitting a wider spectrum can be used. The
16 applied LED-lines in the current set-up for Chlorella vulgaris lead to an illumination of up to 633-1294 µmol
photons (m2s)-1 photosynthetically active radiation (PAR). As described in Wilhelm et al. (Wilhelm and Jakob 2012)
such an illumination range is within the optimal range in order to maximize the photosynthesis rate of chlorophyll a.
Furthermore, optimal illumination conditions of the corresponding organism have to be considered. Therefore, 8
instead of 16 LED-lines have been applied for the cultivation of Euglena gracilis. Each LED-line can be individually
turned on or off, so that the amount of photosynthetically active radiation can be increased or decreased. This allows,
for example, the adjustment of light intensity depending on the organism or the supply of standard light intensities for
algae growth and the supply of high light intensities to create a stress phase in order to trigger the production of
corresponding substances.
When placing LEDs directly on the bag, the algae suspension is also heated by the LED-lines. The long-term
operation temperature of the chosen LED-lines was 44 °C even without additional cooling (measured between bag
surface above the headspace and LED-lines, which is the most crucial area and gave the maximum temperature).
Therefore the LED-lines are suitable for this purpose and direct contact of the LED-lines to the bag surface does not
represent the risk of bag damage. However, in order to control cultivation temperature, a cooling system was needed
for the bag which is often not included for such disposable bioreactor systems (used for mammalian cell cultures).
For this purpose a meander silicone tube (doutside = 11 mm, dinside = 8 mm) was installed inside the hollow bag holder.
It was connected with two polypropylene tubes to a cryostat that pumps distilled water with a temperature of 4 °C. In
order to control the temperature, the heating mat of the disposable system was placed under a part of the bag (see
Fig. 1). The system’s Pt100 measured the temperature of the bag. For a certain temperature set point the system can
heat against the cryostat and hold the temperature.
Cultivation results
After inoculating the bag with Chlorella vulgaris, a biomass concentration of 0.1 g L-1 was measured. In the
following figure 3 the cultivation courses of biomass concentration, CO2-concentration in exhaust gas and day-nightrhythm are presented.
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Fig. 3. Biomass concentration, CO2-concentration in exhaust gas and day-night-rhythm (16 h : 8 h) for the cultivation of
Chlorella vulgaris in the illuminated disposable system.
Figure 3 shows a satisfying growth of Chlorella vulgaris up to around 5 g L-1 biomass concentration, which is a range
also reported in literature (Mandalam and Palsson 1998) and proved the successful operation of the illumination
concept and application. The growth rate in the range of 0.33 g (Ld)-1 also fitted with other Chlorella vulgaris
cultivations. The two sudden decreases in biomass concentration were caused by addition of fresh medium to the bag
in response to evaporating medium during the cultivation. Evaporation is among others influenced by the gas flow of
50 L h-1 through the bag’s headspace. Decreasing the flow rate allows reducing evaporation. On the other hand,
evaporation can be advantageous with regard to downstream processing. The zigzag courses of exhaust-CO2
concentration reflect the changes caused by the metabolism of the algae due to day/night-rhythm. Fluctuations in the
exhaust gas O2-concentration were also visible by off-gas measurement (results not presented). The cultivation was
run for 40 days. The temperature fluctuated mainly between 26 °C without illumination and roughly 28 °C with
illumination (results not presented). During the night when the LED-lines have been automatically switched off the
temperature was constantly at 26 °C. When the LED-lines started to illuminate the bag, its temperature rose above the
set point. This behavior indicated that the heating during the night works properly. However the cooling system did
not yet completely remove the heat radiated by the LED-lines while they were illuminating the bag.
Furthermore, the algae Euglena gracilis has been cultivated in the same system at corresponding cultivation
parameters. Figure 4 presents the cultivation courses of biomass concentration, turbidity, pH-value and day-nightrhythm.
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Fig. 4. Biomass concentration, turbidity, pH-value and day-night-rhythm (16 h : 8 h) for the cultivation of Euglena gracilis in
the illuminated disposable system.
In this case, the three sudden changes in biomass concentration, turbidity and pH-value indicate the three points in
time during the cultivation where algae suspension has been harvested (800 mL) and new medium has been added
(800 mL). Medium evaporation has been minimized at the reduced gas flow rate. The target temperature of 25 °C has
been obtained at temperature deviations below 1 °C. The cultivation successfully resulted in 1.1 g/L cell dry weight
which has been analyzed and gave a mass percentage of 65 % paramylon. The paramylon has been further processed
and used for testing a cosmetic application (results not presented).
Conclusions
The concept of installing reusable flexible LED-lines directly on all surfaces of the bag of such a disposable system
has been presented and offers certain advantages. Cultivation results of Chlorella vulgaris as an alga well known in
literature as well as cultivation results of Euglena gracilis for the production of paramylon demonstrated the
feasibility. Temperature control can be further refined. Applying additional phototrophic organisms producing
interesting high-value products as well as other disposable scales are topic of further research.
Acknowledgements
We would like to thank Caroline Folz, Giorgina Platz, Gila Drews and Florian Drews for their work with Euglena
gracilis.
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