Algal Research 8 (2015) 3744

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

High light and carbon dioxide optimize surface productivity in a

Twin-Layer biolm photobioreactor
Larissa K.P. Schultze, Marie-Victoria Simon, Tong Li, Dorothee Langenbach, Bjrn Podola , Michael Melkonian
Universitt zu Kln, Botanisches Institut, Lehrstuhl 1, Biozentrum Kln, Zlpicher Str. 47b, 50674 Kln, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Immobilized cultivation of microalgae recently received increasing attention avoiding some of the major drawbacks
Received 1 September 2014 of suspension cultivation. Twin-Layer photobioreactors (TL-PBRs), a type of porous substrate bioreactor (PSBR),
Received in revised form 2 December 2014 have proven to be an efcient cultivation system for microalgal biolms. However, the TL-PBR had not yet been op-
Accepted 10 January 2015
timized with respect to light and CO2. The effect of light intensity up to 1486 mol m2 s1 as well as supplementary
Available online xxxx
CO2 (0.125%) was investigated using the green alga Halochlorella rubescens as a model. With a combination of
Keywords:
1023 mol photons m2 s1 and a CO2 level of 3% a surface productivity of 31.2 g dry matter m2 d1 was obtained.
Photobioreactor Along with a standing crop of 205 g m2 growth area (1011 mol photons m2 s1 at atmospheric CO2 level) these
Biolm values represent the highest biomass productivity and total biomass yield yet reported for a PSBR. This work, fur-
Twin-Layer thermore, demonstrates the successful transfer of laboratory scale data to a small prototype-scale greenhouse
Microalgae photobioreactor. With respect to an optimal Twin-Layer photobioreactor design, results suggest that the highest
Halochlorella footprint productivities (~50 g m2 d1 without supplementary CO2) can be obtained at high light dilution rates.
Productivity In contrast, a scenario using high irradiances and supplementary CO2 might be favourable from an economic
point of view.
2015 Elsevier B.V. All rights reserved.

1. Introduction biomass. Their sheet-like construction generates large surfaces for

Recently, the limitations of suspension cultivation of microalgae be-
came a focus of discussion, especially with respect to the requirements
for the production of low-cost biomass as a feedstock for biofuel produc-
tion [11]. In consequence, biolm-based cultivation systems are
regaining attention after having been largely neglected for decades:
Among the biolm photobioreactors, the porous substrate bioreactors
(PSBRs) constitute a new development that is subject to a new eld of
research and engineering [23]. PSBRs were apparently rst introduced
in a conguration termed Twin-Layer (TL) system, in which microalgae
are immobilized by self-adhesion on a hydrophilic, sheet-like porous
substrate, which effectively separates algal biomass from the bulk of
the culture medium [20,25,31]. Within the last two years, this type of
photobioreactor (PBR) has shown potential to solve some of the major
problems in suspension cultivation of microalgae (e.g. [18,22,24]):
PSBRs require low energy input for mass transfer and harvesting of
Corresponding author.
E-mail address: bpodola@uni-koeln.de (B. Podola).
URL's: http://www.melkonian.uni-koeln.de (L.K.P. Schultze),
http://www.melkonian.uni-koeln.de (M.-V. Simon), http://www.melkonian.uni-koeln.de
(T. Li), http://www.melkonian.uni-koeln.de (D. Langenbach),
http://www.melkonian.uni-koeln.de (B. Podola), http://www.melkonian.uni-koeln.de
(M. Melkonian).
favourable light dilution rates. Cell immobilization in a biolm exposed
to ambient air optimizes gas exchange by keeping photosynthetically
active cells directly at the liquid/gas interphase. Furthermore, biolm
immobilization signicantly reduces shear forces and spread of contam-
ination within a culture. Biotechnologically relevant species such as
Botryococcus braunii [10], Haematococcus pluvialis [37,41] or the
peridinin-producing dinoagellate Symbiodinium voratum [1], as well
as diverse microalgal species, used as live feeds for hatcheries in aqua-
culture [24], were effectively grown in PSBR biolms. Recently, the scal-
ability of the Twin-Layer technology was demonstrated at prototype-
scale for applications in biotechnology [24] and environmental biotech-
nology, e.g., wastewater purication [32].
Even though some of these studies show promising growth rates of
the unialgal biolms, knowledge of the optimization of growth condi-
tions is still fragmentary; however, it is essential for technical applica-
tions. Several recent studies provided insight on the effect of light
intensity on microalgal growth in a PSBR [1,10,16,18,41] and revealed
that, with supplementary CO2 (12%) and, in most instances, under
continuous (24 h) articial illumination, productivities of 6 to 15 g dry
biomass (DW) m2 growth area d1 can be observed, depending on
light intensity and species chosen. In these publications, however, max-
imal light intensities on the growth surfaces were restricted to 150-
400 mol m2 s 1 by the experimental setup. In contrast to these
ndings, a recent model developed by Murphy and Berberoglu [23] pre-
dicts that an increase of photosynthetic productivity to almost

http://dx.doi.org/10.1016/j.algal.2015.01.007
2211-9264/ 2015 Elsevier B.V. All rights reserved.
38 L.K.P. Schultze et al. / Algal Research 8 (2015) 3744
30 g DW m 2 d 1 may be attainable at light intensities above
1000 mol m2 s1 for the cyanobacterium Anabaena variabilis under
comparable conditions. This indicates that maximal productivities of
PSBRs may have not yet been achieved.
In the present study, we investigated the effect of light intensities up
to 1486 mol m2 s1 and of different concentrations of supplementary
CO2 (0.125%) on biomass productivity and total biolm dry weight of
the green alga Halochlorella rubescens grown in a Twin-Layer PBR. To ac-
count for more realistic application scenarios, we performed the exper-
iments in a long-term laboratory setup and under a discontinuous light
regime (14/10 h L/D photoperiod). The laboratory-scale results obtain-
ed were evaluated with respect to their applicability to a scaled-up
greenhouse prototype photobioreactor. Furthermore, we combined
our growth data with an analysis of light distribution in an outdoor
PBR model to estimate the PBRs areal productivity at different PBR con-
gurations (light dilution rates).
2. Materials and methods
2.1. Microalgal strain and suspension cultivation
A strain of the unicellular green alga Halochlorella rubescens
CCAC 0126 was provided by the Culture Collection of Algae at the
University of Cologne (CCAC; www.ccac.uni-koeln.de), Germany.
Stock cultures were grown in Bold's Basal Medium ([9]; www.
ccap.ac.uk) in 100 mL Erlenmeyer asks at 23 2 C and uores-
cent white light of 2040 mol m 2 s 1 using a light/dark cycle
of 14/10 h in a temperature-controlled walk-in growth chamber.
For the inoculation of the Twin-Layer PBRs, larger volumes were
grown in aerated Erlenmeyer asks at light intensities of about
100 mol m 2 s 1 under the same conditions.
2.2. Experimental setup
2.2.1. Laboratory-scale Twin-Layer photobioreactors
The laboratory-scale Twin-Layer photobioreactor (Fig. 1A) was con-
structed from a 50 cm long transparent PMMA tube based on the design
by Shi et al. [31]: a glass bre mat located in the PBR tube is continuously
supplied with 800 mL culture medium applied on top by means of a
A Laboratory-scale B Model of a Twin-Layer PBR
Twin-Layer test tubes for determination of light distribution
Distance between panels
E N adjustable to
20 / 30 / 50 / 70 / 100 cm
S W
2.0 m
alg
pcm
gf
p and high CO2 concentrations (25%) was tested in a roof top greenhouse
1.5 m
air
cm
Fig. 1. A: Laboratory-scale Twin-Layer test tubes. alg immobilized microalgae, pcm poly-
carbonate membrane as a carrier for microalgae, gf glass bre mat, air membrane pump
for air supply, cm culture medium. B: Model of a 1:1 Twin-Layer photobioreactor for the
determination of the diurnal light distribution on the reactor surfaces (for details see
2.3.1).
peristaltic pump at a ow rate of 35 mL min1, circulating back into
the medium ask. To avoid nutrient limitation for algal growth, the me-
dium was replaced every six days. Microalgae were immobilized at a
density of 25 g m 2 by ltration onto polycarbonate membranes
(PC40, 0.4 m pore size, 25 mm diameter, Whatman, Dassel,
Germany) on a circular area of 2.54 cm2 as described by Naumann
et al. [24]. Inoculated membranes were then placed onto the vertical
glass bre sheet in the PBR.
To investigate the effect of light intensity on algal growth, the TL-
PBRs were placed in front of a eld of 2 3 vertically orientated sodium
discharge lamps (SON-T AGRO 400W, Philips, Hamburg, Germany).
Light intensities were adjusted to 1011, 794, 630, 528, 434, 323, 197,
86, 49, and 22 mol m2 s1 by varying the distance between lamps
and PBRs and/or covering tubes with a 50% neutral grey lter (type
209 0.3ND, LEE Filters, Hampshire, UK) to reduce the light intensity.
To adapt freshly inoculated cultures to high irradiances, discs were
kept in the PBRs for 24 h at 50% of the nal light intensity before taking
the rst biomass samples. The light/dark cycle was set to 14/10 h and
experiments were conducted in a growth chamber at a temperature of
26 2.5 C measured inside the TL-PBR tubes. During the experimental
period of 31 days, the TL-PBR tubes were continuously aerated by mem-
brane pumps with 0.75 L min1 ambient air.
With respect to the applicability of the biomass productivity de-
termined in the laboratory scale experiments to a production situa-
tion using natural sunlight, the effect of (solar-induced) diurnal
changes in light intensity during the day was investigated. The ex-
perimental setup consisted of one set of TL-PBR tubes subjected to
three different variable light intensities for 14 h. Light intensities
were manually modied every 2 h between 06:00 and 20:00 (Sup-
plementary Table S1). Here, light intensities where adjusted to rep-
resent irradiances as they were recorded at the top (ranging from
107 to 1020 mol m 2 s 1), central (47406 mol m 2 s 1), and
bottom (55206 mol m 2 s 1) area of the Western surface of a TL
sheet at 50 cm panel distance as measured using an outdoor TL
panel model over one day (Section 2.3.1). Furthermore, a second
set of TL-PBRs was set up illuminated for 14 h with three xed light
intensities (445, 208, 118 mol m 2 s 1 ), corresponding to the
weighted daily averages of the three PBR positions mentioned
above. Irrespective of the two different modes of application, both
setups received the same total amount of photons per day (5.9,
10.5 and 22.4 mol photons m 2, respectively). Experiments were
performed in the growth chamber at 25.0 3.5 C as described
above.
The effect of supplementary carbon dioxide supply was studied at a
light intensity of 200 10 mol m2 s1 using uorescent tubes and a
temperature of at 23 2 C in a growth chamber over a period of 9 days
(samples were taken on day 0,3,5,7, and 9, respectively). CO2 was ap-
plied in concentrations between 0.012 and 3.0% (v/v) by mixing pure
CO2 with compressed air, resulting in a constant gas ow through the
TL-PBR of 1.253 L 1 min. A combination of high light intensities
(1023 and 1486 mol m2 s1 provided by sodium discharge lamps)
(temp. 28.031.5 C inside the TL-PBR due to high irradiances) in a sim-
ilar experimental setup.
2.2.2. Cultivation in a prototype Twin-Layer photobioreactor
To monitor growth of H. rubescens under near-to-application condi-
tions, a Twin-Layer sheet of 1.0 m2 (80 90 cm inoculated area) was set
up in a prototype TL-PBR using printing paper as a substrate as de-
scribed by Naumann et al. [24]. Unlike the previous PBR setup, the
drip irrigation system was replaced by a capillary mat (80 g m2,
ISOLA AS, Eidanger, Norway) to supply culture medium over the total
module width more evenly onto the Twin-Layer from a reservoir con-
taining 7 L medium on the top of the module, constantly relled from
the main reservoir. To allow long-term cultivation of microalgae using
paper as substrate without fungal contamination, 25 L of a brackish
 L.K.P. Schultze et al. / Algal Research 8 (2015) 3744
water culture medium was employed using initially a 1:2 mixture (v/v)
of ASP12 [19] and Bold's Basal Medium [9]; after day 20 of cultivation
ASP12 was replaced by a Large Scale Brackish Medium (LSBM)
based on Bold's Basal Medium and prepared by dissolving 21.1 g L1
Tropic Marine sea salt (Dr. Biener GmbH, Wartenberg/Angersbach,
Germany) in demineralized water and addition of 2.94 10 3 M
NaNO3, 4.31 10 4 M K2HPO4, 1.29 103 M KH2PO4,
1.71 104 M EDTA (Titriplex), 5.53 104 M KOH, 0.15 109 M vi-
tamin B12, 4.10 10 9 M biotin, 0.30 106 M thiamine-HCl, and
0.80 109 M niacinamide. To avoid nutrient limitation, media were
exchanged every 7 days.
Light intensity on the growth surface was adjusted to an average of
52 (3663) mol m2 s1 through a combination of ambient illumina-
tion and sodium discharge lamps (L/D 14/10 h) in the roof top green-
house at temperatures of 22 1.5 C and a relative humidity of 30
10%.
2.2.3. Sampling and determination of dry biomass
Laboratory-scale TL-PBRs were sampled by removing lter discs
from the reactor. To determine biomass on the originally inoculated
area only, a cylindrical template of 18 mm diameter was used to remove
cells spreading beyond the edges of the inoculated area. Filters were
dried at 105 C to constant weight and biomass was measured gravi-
metrically. From the prototype-scale PBR, biomass growth was deter-
mined according to Naumann et al. [24].
2.3. Spatial and diurnal light distribution on the growth surfaces of a Twin-
Layer photobioreactor and modelling of PBR productivity
2.3.1. Measurement of light distribution
Under optimal irradiation conditions, the distribution of sunlight in a
model of a Twin-Layer photobioreactor was monitored on a cloudless
mid-summer day (July 6th, Cologne, Germany) from 07:30 to 19:30
CEST at two hour intervals. To mimic a prototype-scaled TL-PBR at dif-
ferent setups, two mesh-print plywood panels coated with pale green
paperboard of 2.00 1.50 m were vertically mounted in parallel on a
steel rack in the northsouth axis (Fig. 1B). The rack allowed a rapid ad-
justment of the inter-panel distance to 20, 30, 50, 70, and 100 cm, re-
spectively. Each panel was equipped with 5 5 equally spaced holes
of 27 mm diameter each. Photosynthetic active radiation (PAR) was
measured on the vertical surface of the panels with a quantum sensor
(LI-190SA, LI-COR Biosciences GmbH, Bad Homburg, Germany) by
pointing the sensor through the holes in the in-plane level of the
board towards the opposing panel. At each of the seven points in time,
a total of 50 sampling points on the Eastern and Western panel surfaces
were recorded for each of the ve panel distances (see above), and, the
irradiances on the horizontal area above the panels were determined
(Fig. 2).
Irradiance [m ol photons m -2 s-1 ]
2000
1750
1500
1250
1000
750
500
1305 Local
250
mol m-2 s -1 noontime
0 the experimental period were tted with a quality of R2 N 0.98 (ex-
8 10 12 14 16 18 20
Daytim e (CEST)
Fig. 2. Solar irradiation (mol photons m2 s1 SD, n = 410) on horizontal level above
the outdoor model photobioreactor. Values were recorded on a cloudless mid-summer
day in Cologne, Germany.
39
2.3.2. Estimation of areal productivity
Light distribution data between PBR panels are available for seven
time points during the day at 50 positions on the model PBR surface
(see Section 2.3.1). Each of the 50 values for the respective sampling
time points was dened to represent the light intensity for the 2 h inter-
val (sampling time 1 h) at the area surrounding the measuring posi-
tion (40 30 cm). The 7 time intervals thus comprise a period of 14 h
from 06:30 to 20:30 which is here dened as the period of photosyn-
thetic activity. Based on growth rates determined for different light in-
tensities (Fig. 4), the daily biomass growth on the reactor surface can
be calculated per 2 h-interval for each of the 25 areas on the east and
west panel surfaces and summed up to the overall daily productivity.
With respect to the different panel distances and over a period of
30 days (for the sampling points used for biomass determination, see
Fig. 3), the areal productivity was estimated in terms of growth rate
and total accumulated biomass (by integration of the nonlinear function
of the growth rates).
2.4. Data analyses
All regressions of growth kinetics were calculated with GraphPad
Prism v5.04 statistical software (GraphPad Software Inc., La Jolla, USA).
To determine individual growth rates at any sampling point in
time during the long-term experiment (31 days) at different light in-
tensities (Fig. 3), the rst order derivate was calculated from best-t
functions relying on three replicates at each of the 11 sampling
points. Here, pre-dened functions were used to t growth kinetics
for the different light intensities, respectively (saturation function
one site total for growth kinetics measured at light intensities
197 mol m 2 s 1 and third order polynomial for light intensi-
ties 86 mol m 2 s 1).
For all data sets, linear growth phases were veried by GraphPad
Prism's linear regression model and growth rates within this period
were derived from the slope of the linear function. To compare growth
rates of algae at different experimental conditions statistically, the built-
in statistical test based on the analysis of covariance (ANCOVA) was
employed to compare slopes of linear growth phases. Plots of
irradiance-dependent growth rates were mathematically tted by ap-
plying the model of Platt et al. [26].
3. Results
3.1. Immobilized growth and light
Total biomass on the bioreactor surface and daily growth
rates were monitored at ten light intensities from 23 to
1011 mol m 2 s 1 over a period of 31 days. Within the experimen-
tal period with a light/dark cycle of 14/10 h, the maximal biomass
yield reached was 205 g DW m 2 at the highest light intensity of
1011 mol m 2 s 1 (Fig. 3), whereas the lowest light intensity of
22 mol m 2 s 1 resulted in the lowest biomass yield (approx.
55 g m 2 ). In general, the nal standing crop increased with in-
creasing light intensity (Fig. 3). Under continuous (24 h) illumina-
tion, which has been applied in many of the recent studies
employing Twin-Layer type PSBRs, for 10 days at 607 to 1019 mol
photons m 2 s 1, the biomass yield was 3545% higher compared
to the 14/10 h light/dark regime, with the higher yields (in % in-
crease compared to 14/10 h light/dark regime) obtained at the
lower light intensities (Fig. 3, open circles). Growth functions within
cept for the values at 434 mol photons m 2 s1, which could not
be tted to the models) and daily growth rates were calculated
based on the rst order derivate (Fig. 4). On day 2, the growth rate
increased from 1.5 g m 2 d 1 at 22 mol photons m 2 s 1 to
12 g m 2 d 1 at 1011 mol photons m 2 s 1 (which was the
highest growth rate observed under these conditions, Fig. 4). In the
40 L.K.P. Schultze et al. / Algal Research 8 (2015) 3744

200 528 434

170
140
110
Total biom ass [g m -2 ]

80 1011 794 630

50
1019 826 607
20

200 323 197 86 49 22

170
140
110
80
50
20

2 6 10 14 18 22 26 30 2 6 10 14 18 22 26 30 2 6 10 14 18 22 26 30 2 6 10 14 18 22 26 30 2 6 10 14 18 22 26 30
Cultivation tim e [days]

Fig. 3. Total dry biomass of Halochlorella rubescens on Twin-Layer surfaces (g m2 SD, n = 3) recorded over a growth period of 31 days for a 14 h (black/white circles) light cycle and 24 h
continuous illumination (open circles) for different light intensities ranging from 22 to 1019 mol m2 s1 and without any supplementary CO2.

rst 13 days of the experiment no full light saturation in terms of bio-
mass productivity occurred up to the maximal irradiance applied in
the experiment. From day 16 onwards, light saturation was observed
at light intensities N200 mol m2 s 1 (Fig. 4). In general, growth
rates at the respective light intensities decreased with time (and in-
creasing standing crop), the decrease being more pronounced at the
higher light intensities (up to 70%; Fig. 4). The efciency of light use in
terms of biomass (g) produced with 1 mol of photons observed on
day 2 ranged from 1.3 at 22 mol photons m 2 s 1 to about 0.25 at
1011 mol photons m2 s 1 (Fig. 4). However, a sharp decrease of
the efciency of light use was observed at light intensities from 22 to
500 mol m2 s1, whereas a tendency for saturation occurred above
500 mol m 2 s 1. In the course of the experiment of 31 days, the
light use efciency, on average, dropped by about 50%.
In contrast to a laboratory situation in which microalgae are subject-
ed to light of a xed intensity during the light period, an outdoor PBR is
exposed to an oscillating photon ux density due to the diurnal solar
cycle. To investigate whether biomass growth depends on these two
different modes of light application, two light regimes (illumination
with a xed light intensity and with a simulated diurnal solar cycle,
see Section 2.2.1) were applied during a 14 h photoperiod, supplying
the same amount of total photons within this time.
Fig. 5 shows biomass productivity of these two modes of application
for three different daily photon ux densities of 5.9, 10.5 and
22.4 mol m2 d1, corresponding to the different positions on the reac-
tor surface (see 2.3.1). Biomass growth within 14 days could be tted to
a linear model (r2 0.98) with growth rates ranging from 4.7 to
6.2 g m2 d1 for increasing light intensities, whereas no signicant dif-
ference between the two modes of light application (xed intensity or
simulated diurnal solar cycle) was observed.
3.2. Effect of addition of carbon dioxide
The effect of elevated carbon dioxide concentrations on microalgal
growth was investigated at 200 mol photons m2 s1 over a period
of 9 days. The growth rate was determined by tting to a linear model
with r2 0.95. Addition of CO2 in the range of 0.12 to 3.0% (v/v) stimu-
lated biomass growth in a concentration-dependent manner (Fig. 6A).

Light use efficiency [g biom ass (m ol photons)-1]

12 1.50
2d 4d 6d 8d 10 d
10 1.25
8 1.00
Grow th rate [g m -2 d-1 ]

6 0.75
4 0.50
2 0.25

12 1.50
13 d 16 d 20 d 25 d 31 d
10 1.25
8 1.00
6 0.75
4 0.50
2 0.25

100 300 500 700 900 100 300 500 700 900 100 300 500 700 900 100 300 500 700 900 100 300 500 700 900
-2 -1
Light intensity [m ol m s ]

Fig. 4. Biolm age-dependent productivity of Halochlorella rubescens on Twin-Layer surfaces (growth rate in g DW m2 d1 SD, n = 3) at different light intensities ranging from 22 to
1011 mol m2 s1 and a 14 h light cycle without supplementary CO2. The photosynthetic efciency (PE) in terms of g DW mol photons1 is indicated by the broken line.
 L.K.P. Schultze et al. / Algal Research 8 (2015) 3744 41

120 140
5.9 mol m -2 d -1 10.5 m ol m -2 d-1 22.4 m ol m -2 d-1 2.0% 3% 5%
100 constant 120 1023 mol
]
-2

]
illumination 1486

-2
Total Biomass [g m

100

Total Biomass [g m
80 simulated
diurnal
60 cycle 80

40 60

20
4.7 g m-2 d-1 5.2 g m-2 d-1
0 20
2 6 10 14 2 6 10 14
Tim e [days]
Fig. 5. Total dry biomass of Halochlorella rubescens on Twin-Layer surfaces (g m2 SD,
n = 4) depending on the mode of light application (constant light intensity or simulated
diurnal solar cycle, both for 14 h d1, each) at identical total daily photon ux density over
a period of 14 days. The three different photon ux densities (5.9, 10.5 or
22.4 mol m2 d1) refer to total daily light intensities measured on a model TL-PBR at bot-
tom, central or top positions, respectively (see 2.3).
Even at the lowest CO2 concentration applied (0.12%), a signicant in-
crease of the growth rate by 27% was observed (Fig. 6B). At 3% CO2 the
productivity increased by more than 50% compared to ambient air. To
investigate the effect of the addition of CO2 on growth at the maximal
productivity at high light intensity, CO2 concentrations of 2, 3 or 5%
were applied in combination with high light intensities of 1023 and
1486 mol m2 s1 respectively (Fig. 7). The linear growth phase was
used to compare results of different setups. At 2 or 3% CO2, the biomass
productivity reached 30 g m 2 d 1 or 31.2 g m 2 d 1 respectively
(corresponding to a 250% increase compared to cultivation at atmo-
spheric CO2 concentration) and did not differ signicantly between
these two concentrations. However, at 3% CO2, the linear growth rate
was maintained one day longer than at 2% CO2, leading to a higher
nal standing crop of more than 120 g m2 after 5 days (Fig. 7). At a
CO2 level of 5%, the growth rate decreased by 20% compared to the
growth rate observed at 2% or 3% CO2, however, growth still resulted
in a nal total biomass of 110 g m2 at 5% CO2 due to a longer phase
of linear growth (4 days instead of 3 days (at 3% CO2) or 2 days (at 2%
CO2; Fig. 7)).
3.3. Long-term productivity in a prototype photobioreactor
To investigate whether the results obtained in the laboratory-
scale setup as described above can be transferred to a semi-real
12.0
11.5 A
40
6.2 g m-2 d-1
30.0 g m-2 d-1 31.2 g m-2 d-1 24.0 g m-2 d-1
2 6 10 14 0
0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5
Tim e [days]
Fig. 7. Total dry biomass of Halochlorella rubescens on Twin-Layer surfaces (g m2 SD,
n = 4) at 1023 and 1486 mol photons m2 s1 at different CO2 concentrations in the
gas phase (v/v in %).
production situation, one module (sheet) of 1 m 2 of a prototype-
scale Twin-Layer photobioreactor with paper as a substrate for
algal immobilization and growth was inoculated with H. rubescens.
At a light intensity of about 50 mol m 2 s 1 (corresponding to a rel-
atively narrow distance between TL sheets in a reactor), a linear
growth with a growth rate of 3.0 g m 2 d 1 was maintained for
42 days (Fig. 8A). This result shows no signicant difference in
terms of growth rate compared to the laboratory-scale TL-PBR intro-
duced above (3.0 g m 2 d 1 over 31 days). After day 42 the growth
rate decreased and after day 63 the stationary phase with a nal
standing crop of about 160 g m 2 was reached. At the maximal bio-
mass density, the biolm reached a thickness of approximately 1 mm
(Fig. 8B). Contaminations by bacteria or eukaryotes (other
microalgae, protists or fungi) were rarely observed by light micros-
copy and did not impair growth or development of the densely
packed layers of H. rubescens during the experimental period of
77 days.
4. Discussion
4.1. Optimizing surface productivity: effect of light and carbon dioxide
Our results demonstrated that light intensity and addition of car-
bon dioxide have signicant effects on the surface productivity of a
Twin-Layer PSBR over an order of magnitude. At ambient CO2 con-
centration and light intensities b 250 mol m 2 s 1 the surface pro-
60 ductivities shown here for H. rubescens are comparable to [1,4,5,14,
B 39] or even considerably higher than productivities reported from
com pared to am bient air [%]

11.0 50 other types of laboratory-scale conventional biolm or PSBR systems

Increase of grow th rate
Grow th rate [g m -2 d-1 ]

10.5 [27]. However, the effect of much higher irradiances on microalgal

10.0 40 growth has to our knowledge not yet been studied in biolm-based
9.5 r=0.98 cultivation platforms. This, presumably, may be attributed to the
9.0 30 fact that light saturation of photosynthesis or photoinhibition is
8.5 commonly observed at light intensities ranging from 100 to a few
8.0 20 hundred mol m 2 s 1 (e.g., [8]) for most microalgal species studied
7.5 in suspension cultures. Our results demonstrated that in early
CO2 concentration
7.0 10 growth phases of a biolm, biomass productivity can increase by
in ambient air
6.5 more than 50% to about 30 g m 2 d 1 if irradiances increased from
6.0 0 197 to 1023 mol photons m 2 s 1, leading to the highest standing
crop of more than 200 g DW m 2 reported from a PSBR to date. Dur-
0.12
0.21
0.35
0.50
1.0
3.0

0.01 0.1 1 10
ing the experiment, photoinhibition was not apparent and full satu-
Concentration of CO2 [%]
ration of photosynthesis was not observed even at the maximal light
intensity used. Liu et al. [18] provided data suggesting that full satu-
Fig. 6. A: Biomass productivity of Halochlorella rubescens on Twin-Layer surfaces (g DW
m2 d1 SD, n = 48) determined over 9 days at 200 mol photons m2 s1 at differ-
ration of photosynthesis does not occur below 300 mol m 2 s 1
ent CO2 concentrations in the gas phase (v/v in %). B: CO2-dependent increase of growth using a Twin-Layer type PBR with Acutodesmus obliquus, whereas
rate (%) compared to an atmospheric CO2 level. other publications show full light saturation at lower irradiances of
42
200
Lab-scale TL tube
175 49 mol m-2 s -1
GR 0-31d: 3.0 g m-2 d-1
Total Biom ass [g m -2 ]
150
125
100
75
50
25
0
0 10 20
Fig. 8. A: Total biomass of Halochlorella rubescens on Twin-Layer surfaces (g m2 SD) at laboratory scale (n = 4) and during a long-term growth experiment with a greenhouse prototype
PBR (n = 3) at light intensities of about 50 mol m2 s1. GR growth rate. B: The biolm of Halochlorella rubescens on the greenhouse prototype PBR using printing paper as a substrate
(with harvested area on the left) at a nal density of about 160 g m2 dry matter.
about 150 mol m 2 s 1 [10,41] in Twin-Layer PBRs cultivating
other microalgal species. This might indicate that, probably, the abil-
ity to cope with high light intensities might arise from the adaptation
of microalgal strains to these conditions in combination with a dense
algal layer, e.g., as reported for suspensions of Arthrospira platensis in
a at plate PBR at light intensities of a few thousand mol m 2 s 1
[29].
In microalgal biotechnology, most cultivation platforms rely on
supplementary carbon dioxide to stimulate growth. For PSBRs,
several studies report growth rates of 6 g m 2 d 1
(Botryococcus braunii, 100 mol photons m 2 s 1 , 1% CO 2 ) to
15 g m 2 d 1 (Acutodesmus obliquus, 300 mol photons m 2 s 1,
2% CO 2 ) for different microalgae grown with supplementary CO 2
(12%) and irradiances of 100300 mol photons m 2 s 1 [10,
1618,41]. In a rotating biological contactor-based biolm
photobioreactor (non-PSBR type) at 422 mol photons m 2 s 1 ,
the effect of CO 2 addition into the culture medium led to a 10-
fold increase of surface productivity of Chlorella sorokiniana up to
20 g m 2 d 1 on the reactor surface [3]. With H. rubescens, we ob-
tained growth rates of 10 or 10.5 g m 2 d 1 at 200 mol m 2 s 1
at 1 or 3% supplementary CO2 , respectively. This corresponds to a
CO 2 -induced increase of growth rates by 4045%. In a modied
Twin-Layer type PBR the effect of CO 2 compared to cultivation
with ambient air was shown by Ji et al. [16] for Pseudochlorococcum
sp. growing at 100 mol photons m 2 s 1 , demonstrating a 35
80% (depending on biolm age) increase of productivity at a CO 2
level of 2%.
It is well known that the stimulating effect of supplementary CO2 on
microalgal growth is further enhanced at higher light intensities (e.g.
[15]). Consequently, with respect to surface productivity, a combination
of high light intensities and supplementary CO2 led to the highest sur-
face productivity of 31.2 g m2 d1 (this study). Neither by increasing
light from 1023 to 1486 mol m2 s 1 nor by raising the CO2-level
from 3 to 5%, could biomass productivity be further increased (this
study). This may indicate that, with respect to light and CO2, near to op-
timal growth parameters for H. rubescens might have been reached
under the given experimental conditions.
To assess the performance of a photobioreactor, i.e., its ability to con-
vert sunlight into biomass, the photosynthetic light use efciency (PE) is
of importance. Under optimal (theoretical) conditions, plants can utilise
about 1012% of the available sunlight for CO2 xation [12,36], which is
comparable to a theoretical maximal biomass productivity of about
1.5 g mol PAR photons 1 [2]. In our Twin-Layer biolm under the
lowest irradiance, an ambient CO2 level, and in an early growth stage
of the biolm, the PE was 1.3 g mol photons 1. In contrast,
the PE did not exceed 0.3 g mol photons1 at light levels of
L.K.P. Schultze et al. / Algal Research 8 (2015) 3744
B
Single-module TL-PBR 1 m surface
39-63 (52) mol m-2 s -1
GR 0-42d: 3.0 g m-2 d-1
30 40 50 60 70 80
tim e [d]
300 mol m2 s 1 at ambient CO2 levels, which is signicantly
lower than values of 0.50.9 g mol photons1 reported from several op-
timized suspension-type or biolm PBRs at N 400 mol m2 s1 [3,42].
However, our results show that the application of 23% CO2 at
1023 mol m2 s1 increased the PE to 0.6 g mol photons1, suggesting
that the low PEs observed (Fig. 4) arise from CO2 limitation under high
light conditions.
Scope for optimization of productivity can be found in the con-
stant and signicant decrease of growth rates over the 31 days of bio-
lm cultivation. Similar observations were made in other PSBRs [10,
16]. This phenomenon was most prominent at light intensities of
1011 mol m 2 s 1 (see Results). Here, the nal total biomass
yield could have been about 80% higher if the initial productivity of
12 g m 2 d 1 could have been maintained over the whole growth
period of 31 days. A reason for this observation might be found in
dark respiration in microalgal cultures, often affecting the net pro-
ductivity of microalgal production systems: in a growing biolm of
a certain thickness increasing dark zones may lead to increasing
total biomass losses by dark respiration if oxygen is present, whereas
illuminated zones and, accordingly, daily biomass formation by pho-
tosynthesis remain stable over time. However, to identify possible
limiting factors for maximal biomass productivity as a starting
point for further optimization, insight into unialgal phototrophic
biolms (e. g. , by microsensor studies) with respect to internal gra-
dients (light, gases, and nutrients), physiological processes, and
physical properties is required.
4.2. Implications for PBR design
The design of a Twin-Layer PBR and other types of PSBRs often in-
volves light dilution by arranging sheet-like surfaces in a vertical, paral-
lel orientation [18,22,24], with dilution factors ranging from 4 to 20
(growth surface per footprint area). In the present study we have inves-
tigated a direct impact of light and CO2 on biomass productivity on the
growth surface without considering the footprint performance of the
reactor. Consequently, the determination of this important measure
for PBR assessment under outdoor (greenhouse) conditions in a long-
term setup under real operation conditions would be a next step for
technology development and, in general, cannot be replaced by the ex-
trapolation of laboratory data. Nevertheless, to give a gure of the po-
tential performance of a TL-PBR, we estimated footprint productivity
by using light distribution data measured between parallel TL-sheets.
In this context, we have successfully shown that laboratory-scale
growth rates can be transferred to a small prototype greenhouse system
with supplementary articial light at moderate light intensities (equiv-
alent to a high light dilution factor and thus high photosynthetic
 L.K.P. Schultze et al. / Algal Research 8 (2015) 3744 43

Distance between PBR-sheets [cm] (coverage vert./hor.) leads to more cost-effective cultivation (i.e., material savings in terms
20 (15) 30 (10) 50 (6) of surface-related costs compensate for biomass losses at the footprint
70 (4.3) 100 (3)
level as well as higher costs for footprint-related investments). In this
scenario, an appropriate TL-PBR would rely on a reduced surface area
Grow th rate PBR footprint [g m -2 d-1 ]

60 1.2

Total aereal biomass PBR footprint

A B and a low light dilution rate at high irradiances and biomass density
50 1.0 as proposed by Richmond [30] for suspension PBRs. In particular, the ap-
plication of supplementary CO2, which proved to stimulate growth ef-
40 0.8 fectively under these conditions (by up to 250%), would be highly

[kg m -2 ]
advantageous to overcome CO2 limitation in the biolm.
30 0.6

20 0.4 5. Conclusions

10 0.2 High surface productivities of up to 31 g dry microalgal biomass m2

growth area d1 were achieved in a Twin-Layer type PSBR by increasing
0 0.0 light and carbon dioxide supply. Even though the highest footprint pro-
5 10 15 20 25 30 5 10 15 20 25 30 ductivities were obtained at high light dilution rates (i.e., at low to mod-
Tim e [days] erate light intensities), lower footprint productivities allowing exposure
to high light intensities may be preferable from an economical point of
Fig. 9. Potential PBR productivity at different spacings of Twin-Layer modules (light dilu- view, considering footprint requirement and costs for PBR infrastruc-
tion rates) over a period of 31 days. Values were calculated based on light distribution in ture. Preliminary data show that results of this study are applicable to
model PBR and light-dependent growth rates determined without supplementary CO2.
a scaled-up production setting and suggest a long-term pilot assess-
A: PBR productivity (growth rate) per reactor footprint (g DW m2 d1). B: Total aerial
biomass yield per reactor footprint (g DW m2) as a sum of daily productivities over ment of a TL-PBR under greenhouse conditions. Until now, research
the growth period of 31 days. on PSBRs has revealed signicant benet of this reactor type to the
eld of technical microalgal cultivation. In contrast, understanding of
efciency). Furthermore, we have demonstrated that the mode of light basic processes in articial unialgal biolms, such as growth or distribu-
application (xed light intensity vs. illumination in a diurnal cycle) does tion of resources and products (light, gas, nutrients), is still insufcient
not affect productivity if the total daily photon availability is equal, compared to suspension cultures. We propose the development of a
which is in accordance with observations by Meseck et al. [21] and biolm growth model to gain essential knowledge for further reactor
Toro [35]. By using the light-dependent productivities determined in design.
this study in combination with light data recorded with a 1:1 scale TL Supplementary data to this article can be found online at http://dx.
model during a Central European summer day with optimal sunlight doi.org/10.1016/j.algal.2015.01.007.
availability (see Section 2.3.1), we have calculated footprint productiv-
ities for different panel distances (light dilution rates). With a TL-PBR, Acknowledgements
aerial productivities of 8 to 52 g m2 d1 can be expected without sup-
plementary CO2, depending on panel distance and biolm age (Fig. 9). The authors thank Franka Hofmann and Sonja Darschnik for their
At the lowest sheet distance (20 cm), about 1.1 kg of dry biomass can dedicated support in experimental work. This study was supported by
theoretically be generated within 30 days on 1 m2, whereas a 100 cm the University of Cologne (KST 158901001).
distance between modules yielded a standing crop of only 300 g dry bio-
mass on the same ground area and within the same time. With an aver-
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