Protist, Vol.

156, 239251, August 2005

http://www.elsevier.de/protis
Published online date 11 July 2005
ORIGINAL PAPER
The 96-Well Twin-Layer System: A Novel Approach
in the Cultivation of Microalgae
Eva C.M. Nowack
1
, Bjo rn Podola, and Michael Melkonian
Botanisches Institut, Universita t zu Ko ln, Lehrstuhl 1, Gyrhofstr. 15, 50931 Ko ln, Germany
Submitted March 10, 2005; Accepted April 19, 2005
Monitoring Editor: Robert A. Andersen
A novel system for the growth and maintenance of microalgae has been developed that allows the
cultivation of a large number of strains with little manual effort. The system is based on a 96-well
microtiter plate in which a membrane lter constitutes the bottom of each well. Algal strains are
immobilised on the membranes and provided with culture medium through contact with layers of
glass bre located beneath the membranes in a special cultivation chamber. The conguration
effectively separates culture medium from algal cells which allows the simultaneous exchange of the
culture medium for 96 strains within a few minutes without the need to transfer the algae. If necessary,
algal strains can be transferred using multi-channel pipettes. We demonstrate that a large variety of
microalgal strains including delicate agellates can be reliably grown in the system under axenic
conditions and without cross-contamination. As an array system, the 96-well twin-layer system using
immobilised algae is also amenable to high-throughput and massively parallel applications
increasingly sought after in algal bio- and environmental technology.
& 2005 Elsevier GmbH. All rights reserved.
Key words: cultivation; culture collections; cell immobilisation; microalgae; microtiter plates; lter plates.
Introduction
Algae occupy a prominent position in the living
world in terms of their ecological importance and
genetic diversity. It has been estimated that their
species numbers may exceed several million, of
which microalgae constitute the major part (An-
dersen 1992). To describe this diversity as well as
to make it available for research and application, it
is imperative to rst establish cultures. Although
considerable progress has been made in recent
years in the cryopreservation of microalgae (e.g.
Day 2004), to maintain many of their strains, algal
culture collections still rely on the serial transfer of
individual strains from suspension or agar cul-
tures. This approach, established as a standard
method for a great variety of microalgae by Ernst
Georg Pringsheim in the 1920s (Mollenhauer
2003), is both labour and cost intensive and limits
the holding capacities of culture collections. In
consequence, only a small percentage of the
global microalgal biodiversity is currently repre-
sented in culture.
ARTICLE IN PRESS
1
Corresponding author;
fax +49 221 4705181
e-mail eva.nowack@uni-koeln.de (E.C.M. Nowack)
& 2005 Elsevier GmbH. All rights reserved.
doi:10.1016/j.protis.2005.04.003
To reduce the time interval between serial
transfers, algae are usually maintained under
suboptimal growth conditions, i.e. low tempera-
tures (o20 1C) and low light intensities
(o50 mEm
2
s
1
), often in combination with an
abbreviated photoperiod of 12h or less (e.g. Day
et al. 1999). Some authors recommended long-
term maintenance of microalgae by encapsulation
of cells in alginate gels. Using this method, a
variety of strains could be preserved over periods
of 1236 months without transfer (Hertzberg and
Jensen 1989; Lukavsky 1988). However, the
preparation of encapsulated cultures as well as
their transfer is very labour intensive, and thus this
method seems to by unsuitable for the cultivation
of a large number of strains.
The twin-layer system, a novel cultivation
technique for microalgae, was recently introduced
by Podola and Melkonian (2003) in the context of
the construction of an algal biosensor. Compared
with traditional cell immobilisation techniques, which
rely on encapsulation of cells in polymeric matrices
(Robinson et al. 1986), in the twin-layer system,
immobilisation is achieved by the ltration of cells on
a membrane lter. The functional unit of the system,
the vertically orientated twin-layer, is composed of
(1) the source-layer, a brous tissue to which a
continuous, pump-driven ow of culture medium is
connected and (2) the substrate-layer, a porous
membrane lter, on which the algae are immobi-
lised. Source- and substrate-layers self-adhere to
each other. Because aqueous solutes can pass
through the substrate-layer, the immobilised cells
are continuously supplied with culture medium
through the source-layer, whereas the immobilised
cells are retained on the light-exposed surface of the
substrate-layer due to its small pore size.
The aim of the present work was to develop a
low-maintenance system for the cultivation of
microalgae based on the twin-layer technology.
To allow half-automatic processing with multi-
channel pipettes, the device was based on the
standard 96-well microtiter plate format. Using
these benchmarks, we developed a cultivation
system for microalgae that may help to overcome
some current limitations of suspension cultures.
Results
Construction and Functionality of the 96-
Well Twin-Layer System
A detailed schematic illustration of the 96-well
twin-layer system is presented in Figure 1. The
functional unit of the system consists of a
horizontally orientated twin layer. A stack of eight
layers of glass bre sheets forming a reservoir for
70ml of culture medium, represents the source-
layer of the system. As substrate-layer, a 96-well
MultiScreen lter plate is used. The bottom of
each well consists of a separate membrane lter,
ARTICLE IN PRESS
Figure 1. Schematic illustration of the 96-well twin-layer system. (A): cultivation chamber (open), (B):
cultivation chamber (closed), (C): detail of A depicting the contact zone between the source- and substrate-
layer.
240 E.C.M. Nowack et al.
with a pore size of 0.22 mm. This membrane lter
constitutes the substrate for the cultivation of the
microalgae which are immobilised on it by ltra-
tion. This functional unit is enclosed in the
cultivation chamber. The chamber is composed
of a base, frame, and cover (Fig. 1). There are
three boreholes in the base [one near the end of its
long side (Fig. 1A) and two at the opposite short
side (not shown in Fig. 1)] that are plugged with
septa of rubber through which an exchange of
culture medium can be performed. Six plugs of
Teon foam in the frame enable gas exchange
between the environment and the small head
space which is formed by the frame above the
lter plate. A glass cover closes the chamber and
allows illumination of the cultures from above. The
chamber is held together by the pressure of four
clamps (Fig. 1B). This pressure guarantees a close
contact between source- and substrate-layers
(Fig. 1C) which is necessary for the functionality
of the system. Sealings in the base and in the
frame ensure air-tight closure of the chamber.
To increase the interval between two exchanges
of culture medium, we enhanced the volume of
culture medium in the chamber by the use of a
larger number of glass bre sheets. To investigate
whether nutrients contained in the culture medium
in lower layers of glass bre can diffuse to the
cultures, a test system was constructed. This
system consisted of four quadratic sections of
lter plates, each containing 16 wells, inoculated
alternately with cultures of Haematococccus
pluvialis or Scenedesmus rubescens at a density
of 2.5mg chlorophyll a cm
2
. These strains are
capable of synthesising secondary carotenoids
upon nutrient limitation. The test plates were
placed in four Petri dishes containing 1, 2, 4, or
8 layers of glass bre respectively, saturated with
culture medium. The area of the glass bres
corresponded to that of the test plates. Figure 2
shows a photograph of the test plates taken 40 d
after inoculation of the cultures (cultures were
grown in a water-saturated atmosphere at a
temperature of 2472 1C). It became clear that
nutrients from the lower layers of glass bre
contribute to the growth of the two algal strains
as the test system with eight layers of glass bre
(Fig. 2D) was the greenest of the four test systems.
In consequence, in all further experiments eight
layers of glass bre were used.
Periodical Exchange of Culture Medium
In the 96-well twin-layer system, an exchange of
culture medium for all 96 strains can be performed
simultaneously without opening the chamber. For
this purpose, the chamber is brought to an upright
position and the septa at the top and bottom of
the chamber are pierced by syringes. Using the
upper syringes, fresh culture medium can be
injected. The fresh medium displaces the ex-
hausted medium from the glass bre sheets, and
the latter is collected in the reservoir (Fig. 1A). This
reservoir holds approximately 20 ml that can be
taken up in steps by the lower syringe.
To examine whether this procedure accom-
plishes the complete exchange of culture medium,
the following test was performed: The source-
layer of a chamber was saturated with 70ml of a
dye solution [0.44% (w/v) Uranin (sodium uor-
escin, Merck, Darmstadt, Germany)]. In the next
step, the chamber was washed with H
2
O (Milli-Q)
in steps of 20ml. The solute leaking from the glass
bre sheets was collected in steps by withdrawing
20ml in each step and the concentration of the
solution was determined spectrophotometrically.
Figure 3 depicts the decrease of Uranin concen-
tration in successive 20ml fractions. After 60ml
was withdrawn, the Uranin concentration started
to decrease and after withdrawal of 100ml, the
Uranin was almost completely removed (r4.5%
of the initial concentration), demonstrating that the
replacement of the culture medium using syringes
is highly efcient.
ARTICLE IN PRESS
Figure 2. Cultures of Scenedesmus rubescens (S)
and Haematococcus pluvialis (H) were inoculated
alternately in 16-well horizontal test systems and
grown for 40 d at a temperature of 241C with source-
layers composed of (A): 1, (B): 2, (C): 4, and (D): 8
layers of glass-bre sheets.
96-Well Twin-Layer Algal Culture System 241
To determine the interval between two ex-
changes of culture medium that is necessary to
maintain healthy cultures, the following experi-
ment was performed: three cultivation chambers
were inoculated in parallel with nine different algal
strains (Cosmarium elegantissimum, Cystodinium
sp., Eudorina elegans, Haematococcus pluvialis,
Klebsormidium nitens, Klebsormidium sp., Lyng-
bya halophila, Nostoc muscorum, Staurodesmus
convergens) at a density of 2.5mg chlorophyll a
cm
2
. Over a time period of 77 d, the cultures
were kept at a temperature of 24721C and a light
intensity of 25mEm
2
s
1
. During this time, the
culture medium was exchanged every 14, 21, or
35d. The physiological status of the cultures was
estimated by chlorophyll uorometry. As an
example, Figure 4 shows the development of
quantum efciency of electron transport (DF/F
m
)
over time for cultures of Klebsormidium nitens.
DF/F
m
remained stable over time irrespective of
the time interval between exchanges of culture
medium. After a short adaptation time of up to 4d
only minor changes of the quantum efciency
were observed during the cultivation period.
Similar results were obtained for the other eight
strains investigated: using a Kruskal-Wallis test,
the collected data of the differences in quantum
efciencies at the start and end of each experi-
ment from all nine algal strains were compared for
the different intervals between exchanges of
culture medium (14, 21, 35 d), and no signicant
differences between the data sets were observed
p 0:05.
Transfer of Algae
Growth of cultures in the 96-well twin-layer system
appeared to saturate after some time even when
nutrients were continuously provided by periodical
exchange of the culture medium. The thickness of
the algal layer rarely exceeded 2mm, but even
after prolonged culturing (up to 230 d), suspension
cultures could be re-established from 50 immobi-
lised strains tested. Dead cells were not encoun-
tered in signicant numbers in older immobilised
cultures. Algae transferred with a multi-channel
pipette from an old to a new 96-well twin-layer
system readily continued growth. However, over
the maximum period of time tested (i.e. 230 d),
transfer of algae did not appear to be necessary
for maintaining cultures.
ARTICLE IN PRESS
Figure 4. Development of quantum efciency of electron transport (DF/Fm) in immobilised cultures of
Klebsormidium nitens over a time period of 75 d. The culture medium was exchanged every 14 d (A), 21d (B),
or 35 d (C) (indicated by the dotted vertical lines). Each value represents the average from six different
cultures.
Figure 3. Spectrophotometrically (l 487 nm) de-
termined Uranin concentration of the solution that
leaks out of the 96-well twin-layer system during the
wash-out procedure of the source-layer with H
2
O
(Milli-Q), saturated with a 0.44% (w/v) Uranin
solution.
242 E.C.M. Nowack et al.
Cross-Contamination
To evaluate the likelihood of cross-contamination
between neighbouring wells, the following ap-
proach was chosen: axenic cultures of Eudorina
elegans and non-axenic cultures of Klebsormi-
dium sp. (M1939) were cultivated side by side in a
cultivation chamber at a temperature of 24 1C.
After 32d (with two exchanges of culture medium),
the sterility of the cultures was controlled in ve
parallels each. The sterility status of both cultures
remained unchanged in all wells. Additionally, we
observed that axenic cultures of E. elegans
remained in this status over a period of 32 d,
when non-sterile culture medium was delivered to
the source-layer.
Suitability of Algal Strains for Cultivation in
the 96-Well Twin-Layer System
In the course of this study, 90 different algal strains
from most major algal classes were analysed for
their suitability to be cultivated in the 96-well twin-
layer system by means of chlorophyll uorometry,
visual characterisation and re-establishment of
suspension cultures (Table 1).
In several strains, the stability of the quantum
efciency of electron transport and the results
from the visual characterisation and re-establish-
ment of suspension cultures were incongruent
(Table 1). Figure 5 shows the development of
quantum efciency of electron transport (DF/F
m
)
in two immobilised Tetraselmis strains [Tetraselmis
sp. (M1325) and Tetraselmis tetrathele] over a
cultivation time of 150d. In the rst 98d, a
signicant decrease of quantum efciencies was
observed down to 38 and 59% of the starting
values, respectively. In contrast, the visual char-
acterisation of the cultures revealed thick, dark-
green layers of algae, which after resuspension in
culture medium yielded motile cells (even after an
extended cultivation time of 245 d). To evaluate
whether the observed lower quantum efciency
values resulted from a stratication of the culture,
with the top layer of cells differing physiologically
from the lower layers, after 98 d cultivation time,
the cultures in each well were resuspended in
100ml culture medium, thoroughly mixed and
subsequently re-immobilised (indicated by the
vertical dotted lines in Fig. 5). This procedure
resulted in recovery of the quantum efciency
values when measured 2d later (Fig. 5).
Of the 90 strains tested during this study, 83%
were judged to be suitable for growth and
maintenance in the 96-well twin-layer system,
and 17% appeared to be unsuitable under the
conditions chosen. By changing the culture con-
ditions, however, some strains which failed to
grow under standard conditions, were capable of
growth. For example, a strain of Cryptomonas
(M1488), which did not grow under standard
conditions, exhibited growth upon reduction of
the light intensity to 3mEm
2
s
1
. Using Cyano-
phora paradoxa, we determined the effect of a
combination of different temperatures, light in-
tensities, and inoculum densities on growth
(Fig. 6). The growth density of the cultures
measured as the amount of chlorophyll a per
cm
2
after 45d of cultivation differs considerably.
At 241C, no growth was detected; in contrast,
signicant growth was observed at 141C. The
effects of temperature and light intensity on
growth were determined as highly signicant by
variance analysis (ANOVA, po0.001). Whereas
temperature explained 95.7% of the total variation
(po0.001), only 0.7% could be attributed to light
intensity (p 0.054). The combined effect of
temperature and light intensity on growth of C.
paradoxa was non-signicant. The inoculum den-
sity was not integrated in the variance analysis
because growth density is expressed by the same
parameter (chlorophyll a per area). When the
inoculum density (2.5mg chlorophyll a per cm
2
)
was subtracted from the nal growth density, the
observed differences in the growth density at the
two inoculum densities were non-signicant
(p40.05; U-test, two directional).
Discussion
Construction and Materials
In this contribution, we demonstrated that micro-
algal cultures can be maintained in a horizontal
twin-layer system without continuous ow of
culture medium (as previously described by
Podola and Melkonian 2003). The varying colora-
tion of the immobilised cultures of Haematococ-
cus pluvialis and Scenedesmus rubescens after a
cultivation time of 40d revealed that the different
nutrient status of the cultures was dependent on
the number of glass bre sheets used as the
source-layer. Therefore, it was apparent that
immobilised cells can take up nutrients from
the lower layers of the glass bre. By using
eight sheets of glass bre to support the
growth of cultures, we were able to extend the
interval between exchanges of culture medium
considerably.
ARTICLE IN PRESS
96-Well Twin-Layer Algal Culture System 243
ARTICLE IN PRESS
Table 1. List of algal strains tested for their suitability to be cultivated in the 96-well twin-layer system. Origin
of the strains: ASW: Culture Collection of Algae at Vienna University (Vienna, Austria); CCAC: Culture
Collection of Algae at the University of Cologne (Cologne, Germany); CCAP: Culture Collection of Algae and
Protozoa, Dunstaffnage Marine Laboratory (Oban, Scotland); CCMP: Provasoli-Guillard National Center for
Culture of Marine Phytoplankton (Bigelow Laboratory for Ocean Sciences, West Boothbay Harbor, Maine,
USA); M: Algal research culture collection Melkonian (Cologne, Germany); NIES: Microbial Culture Collection
(Tsukuba, Japan); PLY: Plymouth Culture Collection (Plymouth, UK); SAG: Sammlung von Algenkulturen at
the University of Go ttingen (Go ttingen, Germany); UTEX: Culture Collection of Algae at the University of
Texas (Austin, Texas). The suitability + or - is judged by three criteria: (1) stability of the quantum
efciency (%DF/F
m
) through 70 100 d (X stands for cases in which the steady state uorescence (F
t
) did
not reach the minimum level of 0.15); (2) visual characterisation: (VC) classied as growth +, stagnation
(+), and bleaching , and (3) re-establishment of suspension cultures from immobilised cells cultivated
for at least 150 d with the 96-well twin-layer system (R150 d). nd no data recorded. Culture conditions (CC)
are indicated as follows: abbreviation of culture medium cultivation temperature [721C]. The
abbreviations used are: ASP: ASP-12, H: HSM, W: Waris-H, WSi: Waris-H+Si, W3V: Waris-H+3V (for details
see Methods).
Strain no. Species %DF/F
m
VC R150 d Suitable CC
M2266 Amphidinium sp. 98.7 + + + ASP-14
M2209 Ankistrodesmus sp. 127.9 + + + W-14
SAG B 2.84 Asterionella formosa Hassall 94.2 + nd + WSi-14
CCAC 0049 Asteromonas gracilis Artari X nd ASP-14
M0401/1 Carteria sp. 83.8 + nd + W-14
M2015 Carteria sp. X nd W3V-14
CCAC 0010 Chlamydomonas coccoides
Butcher
78.6 + + + ASP-14
SAG 83.81 Chlamydomonas reinhardtii CW-15
Dangeard nd + + + H-14
M1963 Chlamydomonas sp. 80.9 + + + W3V-14
M1977 Chlamydomonas sp. nd + nd + W3V-14
M1982 Chlamydomonas sp. nd + nd + W-14
M1990 Chlamydomonas sp. 77.3 + + + W3V-14
SAG 211-11b Chlorella vulgaris Beijerinck 89.8 + + + W-14
SAG 211-12 Chlorella vulgaris Beijerinck 84.5 + nd + W-14
M1995 Chlorogonium sp. 66.3 + + + W3V-14
CCAP 909/9 Chromulina chionophila Stein nd + nd + W-14
M1625 Chroomonas sp. nd + nd + W-24
CCAC 0117 Cosmarium elegantissimum Lundell 100.8 + + + W-24
M1488 Cryptomonas curvata Ehrenberg 170.9 (+) nd W-24
M1490 Cryptomonas curvata Ehrenberg X (+) + + W-14
M2201 Cryptomonas sp. X (+) nd W3V-14
M2286 Cryptomonas sp. X (+) nd W3V-14
M2287 Cryptomonas sp. X (+) + + W3V-14
CCAC 0074 Cyanophora paradoxa Korshikov nd + nd + W-14
M1153 Cylindrotheca fusiformis Reimann
et Lewin
84.3 + nd + ASP-14
SAG B 59.87 Cystodinium sp. 143.4 + + + W-14
M2116 Dunaliella lateralis Pascher and
Jahoda
X (+) nd W-14
PLY 430 Dunaliella minuta Lerche 13.5 (+) + + ASP-14
CCAP 19/9 Dunaliella parva Lerche 62.8 + + + ASP-14
CCMP 362 Dunaliella parva Lerche 81.4 + + + ASP-14
SAG 183.80 Dunaliella primolecta Butcher 76.4 + + + ASP-14
CCMP 1320 Dunaliella tertiolecta Butcher 40.8 + + + ASP-14
CCAC 0011 Eudorina elegans Ehrenberg 62.4 + nd + W-24
CCAC 0081 Euglena archaeoplastidiata
Chadefaud
59.6 + nd + W-24
244 E.C.M. Nowack et al.
ARTICLE IN PRESS
Table 1. (continued)
Strain no. Species %DF/F
m
VC R150 d Suitable CC
SAG 1224-5/25 Euglena gracilis Klebs nd + + + W-24
M2022 Eunotia sp. X (+) nd WSi-14
SAG B 13.82 Glaucosphaera vacuolata
Korshikov
X nd W-14
M1849 Gymnodinium sp. X (+) + + W-24
CCAC 0055 Haematococcus pluvialis Flotow
em Wille
97.8 + + + W-24
M2243 Hydrodictyon reticulatum Bory 83.4 + + + W3V-14
M0939 Hymenomonas sp. 79.4 + + + ASP-14
M2068 Klebsormidium accidum (Ku tzing),
Silva, Mattox and Blackwell
nd + + + W-14
SAG 335-1a Klebsormidium nitens (Menegluni in
Ku tzing) Lokhorst
78.1 + nd + W-24
M1939 Klebsormidium sp. 107.8 + + + W-24
M1941 Klebsormidium sp. 109.1 + + + W-14
M2009 Klebsormidium sp. 92.3 + nd + W-24
CCAC 0119 Klebsormidium subtile (Ku tzing)
Tracanna ex Tell
103.3 + nd + W-14
M1895 Klebsormidium subtile (Ku tzing)
Tracanna ex Tell
84.9 + nd + W-14
M1164 Lyngbya halophila Hansgirg 88.0 + + + W-14
NIES 255 Monomastix minuta Skuja X nd W-14
M1772 Navicula sp. 90.7 + + + WSi-14
NIES 485 Nephroselmis olivacea Stein X nd W-14
CCAP 1960/4B Nephroselmis olivacea Stein X nd W-14
NIES 483 Nephroselmis olivacea Stein X nd W-14
M0931 Nephroselmis sp. X nd ASP-14
M1762 Nitzschia communis Rabenhorst 99.1 + + + WSi-14
M1771 Nitzschia sp. 76.2 + + + WSi-14
M1167 Nostoc muscorum C. Agardh 79.2 + nd + W-24
M1314 Ophiocytium sp. 73.8 + + + W3V-14
M2160 Ophiocytium sp. 76.1 + + + W3V-14
M2161 Ophiocytium sp. 70.5 + + + W3V-14
M2010 Oscillatoria sp. nd + nd + W3V-14
M1784 Pandorina sp. 73.5 + + + W3V-14
M2100 Pediastrum sp. nd + + + W3V-14
SAG 1965-3 Pedinomonas minor Korshikov 97.6 + nd + W-14
UTEX LB 2255 Peridinium inconspicuum Lemm. nd + + + W-24
M1917 Phacotus sp. 64.0 + nd + W3V-14
M2284 Phacus sp. nd W3V-14
M2017 Pinnularia sp. X (+) + + WSi-14
SAG 1380-1C Porphyridium purpureum (Bory)
Drew and Ross
46.3 + + + ASP-14
SAG 61.81 Pseudokirchneriella subcapitata
(Korshikov) Hinda k
86.5 + + + W-14
M2069 Scenedesmus rubescens
(Dangeard) Kessler
76.2 + + + W-24
M1398 Scherffelia dubia Pascher nd + nd + W-24
M2074 Sphaerellopsis sp. 91.8 + + + W3V-14
M1843 Spirogyra sp. nd + + + W-14
M2147 Staurastrum planktonicum Teiling 68.3 + + + WSi-14
CCAC 0120 Staurodesmus convergens
(Ehrenberg) Teiling
80.9 + nd + W-24
M1825 Stauroneis sp. X + + + WSi-14
96-Well Twin-Layer Algal Culture System 245
The cultivation chambers exclusively consist of
material that can be sterilised by autoclaving, a
prerequisite for the long-term maintenance of
algal cultures. The lter plates are commercially
available as sterile units. That the sterility of
cultures could be maintained, when these were
supplied with non-sterile culture medium, demon-
strated that the 0.22 mm pore size of the PVDF
membranes, effectively prevented contamination
of the immobilised algae. This is corroborated by
the fact that it was possible to cultivate axenic and
non-axenic strains in neighbouring wells without
cross-contamination. If necessary, the culture
medium may be sterilised by ltration when
injected into the chamber to preserve thermo-
labile constituents such as vitamins or chelators
(Huebert and Shay 1992) or prevent precipitation
of salts often encountered during autoclaving
(Harrison et al. 1980).
Handling
By dispensing with the pump-driven ow of
culture medium, the horizontal twin-layer system
is basically maintenance-free during the time
interval between exchanges of culture medium.
Cross-contamination or mix-up of cultures during
the exchange of culture medium is prevented
because the chamber remains closed during this
process. The 96-well standard format makes the
system also accessible to half-automatic proces-
sing using multi-channel pipettes which also
reduces the likelihood of a mix-up of strains. A
possible disadvantage of the system may be the
fact that to remove a single strain from the
chamber, the whole system has to be opened.
To minimise the risk of bacterial contamination
during removal of strains or transfer of algae,
sterile conditions are imperative and appropriate
measures such as the use of shielding devices
may be useful.
Nutrient Supply
The greatest advantage of the 96-well twin-layer
system compared to serial subculture of single
strains, in our estimation, is that the exchange of
culture medium is independent of the transfer of
algae and can be performed simultaneously for all
96 strains within a few minutes without opening
the system. This results in a signicant reduction
of handling time.
An upper limit for the time interval between
exchanges of culture medium, necessary to
maintain healthy cultures, was not determined.
However, at a cultivation temperature of 241C,
an exchange of culture medium every 35d
turned out to be sufcient. We anticipate that
at lower temperatures, which are usually used
to maintain stock cultures, the time interval
between exchanges of culture medium may be
signicantly extended. Indeed, re-establishment
of suspension cultures from six different Tetra-
selmis strains cultivated at 14 1C in the twin-layer
system, and not supplied with fresh culture
medium over a period of 105d was achieved
(results not shown).
ARTICLE IN PRESS
Table 1. (continued)
Strain no. Species %DF/F
m
VC R150 d Suitable CC
M1317 Synechocystis sp. nd + nd + W-24
M1826 Synedra sp. 106.5 + + + WSi-14
CCMP 880 Tetraselmis astigmatica Norris and
Hori
98.5 + + + ASP-14
SAG 1.96 Tetraselmis chui Butcher 58.6 + + + ASP-14
CCAP 66/9 Tetraselmis convolutae (Parke and
Manton) Norris et al.
90.1 + + + ASP-14
M1325 Tetraselmis sp. 86.7 + + + ASP-14
CCMP 947 Tetraselmis sp. 31.0 + + + ASP-14
M1739 Tetraselmis sp. 90.0 + + + ASP-14
M1828 Tetraselmis sp. 63.5 + + + ASP-14
PLY 272 Tetraselmis tetrathele (G.S. West)
Butcher
80.2 + + + ASP-14
M2382 Tribonema sp. nd + nd + W3V-14
M0950 Volvox aureus Ehrenberg nd (+) nd W3V-14
246 E.C.M. Nowack et al.
Suitability
For many strains, the quantum efciency of
electron transport (DF/F
m
) was an adequate
indicator of the physiological status of the im-
mobilised cultures, as previously described for
algae in suspensions (Schreiber et al. 1995). In
other cases, as shown for two marine Tetraselmis
strains, quantum efciency of electron transport
and the results from visual characterisation and
re-establishment of suspension cultures did not
correspond (see Results, Fig. 5). It is likely that the
observed reduction of quantum efciency is
caused by stress conditions. Maxwell and John-
son (2000) described that chlorophyll uorescence
in higher plants originates from just the upper few
cell layers of a leaf. We thus assume that only cells
from the upper cell layers of an algal culture
contribute to the uorescence signal. Such cells
are directly exposed to light radiation. Light
intensities that trigger photoinhibition are generally
described to be much higher than the light
intensities used in our study, i.e. between
250mEm
2
s
1
(Samuelsson et al. 1985) to full
sunlight (Ha der et al. 1998). For species that live
permanently submerged in their natural habitat
and especially for agellates, which in suspension
prot from the possibility of changing their loca-
tion due to phototaxis, a position in the upper cell
layers of an immobilised culture may be consid-
ered as light-stressed. Cells in lower cell layers,
however, are shaded by the upper layers, and
thereby protected against direct radiation. It is
likely that these cells contribute signicantly to the
optical appearance of the cultures and their
capability to establish suspension cultures. This
assumption is supported by the results of the re-
immobilisation experiment performed with the
marine Tetraselmis cultures (see Results).
We used a combination of chlorophyll uoro-
metry, visual characterisation and re-establish-
ment of suspension cultures, to determine the
suitability of a given test strain to grow in the
96-well twin-layer system. Applying these
criteria, 83% of the algal strains tested could be
ARTICLE IN PRESS
Figure 5. Development of quantum efciency of
electron transport (DF/F
m
) in immobilised cultures of
Tetraselmis sp. (M1325) and Tetraselmis tetrathele
over a cultivation time of 150 d. After a cultivation
time of 98 d, the cultures were resuspended and re-
immobilised (dotted vertical line). Displayed are the
average values of DF/F
m
and its standard deviation
from 6 different cultures. The cultivation conditions
were (1) temperature: 14 1C; (2) light intensity:
3575mEm
2
s
1
; and (3) exchange of culture
medium: every 21d.
Figure 6. Growth density (in terms of mg chlorophyll
a cm
2
) of immobilised cultures of Cyanophora
paradoxa over a period of 45d under different
combinations of culture conditions as indicated at
the bottom of the graph: temperature (T in 1C), light
intensity (LI in mEm
2
s
1
) and inoculation density
(ID in mg chlorophyll a cm
2
). The bars represent the
average growth density with standard deviation of
six different cultures for each set of culture condi-
tions.
96-Well Twin-Layer Algal Culture System 247
maintained in the 96-well twin-layer system. This
result is comparable to data presented by Lu-
kavsky (1988) for the long-term maintenance of 31
microalgal strains immobilised in Ca-alginate:
81% of the cultures survived over a period of 32
months. As explained above, we think that this
method is technically too complex to be used
routinely on a large scale. Furthermore, there is
evidence that immobilisation of cells on a twin-
layer is better suited for growing sensitive organ-
isms than encapsulation of cells in gels. In
Lukavsky s study, only 10% of the strains tested
were agellates. Flagellates are arguably more
sensitive to cell immobilisation than other micro-
algae. In our study, 57% of the 90 strains
examined were agellates.
For most of the algal strains tested in the 96-well
twin-layer system, the culture conditions were
identical. The results obtained for C. paradoxa
under variable culture conditions demonstrated
that changes in the culture conditions may have a
profound effect on the growth of an immobilised
culture. This observation suggests to us that a
growth optimisation strategy would lead to suc-
cessful maintenance of those algal strains which
failed to grow in the 96-well twin-layer system
under standard growth conditions.
Further Applications
In addition to its potential for growth and main-
tenance of a large number of algal strains, the 96-
well twin-layer system may also be of interest for
specic applications. There are a numerous
studies describing the physiological reaction of a
single or a few algal species to a specic stimulus.
The 96-well twin-layer system offers the possibility
for massively parallel applications by exposing
many algal strains simultaneously to specic
culture conditions, bioactive compounds, patho-
gens, etc. and to record the physiological reaction
of each strain in parallel. The advantages of the
twin-layer system compared to the standard 96-
well microtiter plate are the long-term stability of
the cultures, which have access to a much larger
volume of culture medium through the source-
layer, and the possibility to manipulate the
composition of the culture medium without affect-
ing the cultures. The twin-layer system should also
be particularly useful for genetic manipulation of
sensitive cells (such as cell wall-less mutants of
Chlamydomonas) or selection of mutants. Diffu-
sion of compounds secreted by one strain to
neighbouring strains should greatly facilitate the
study of allelopathic interactions among algae
with the 96-well twin-layer system. During our
studies, however, no such allelopathic effects
were observed.
We also anticipate that the isolation of new
strains from natural samples and their establish-
ment as axenic cultures may be facilitated by the
96-well twin-layer system. Sensen et al. (1993)
described the use of uorescence-activated cell
sorting (FACS) for the production of axenic clonal/
single cell-derived algal cultures. Meanwhile, cell
sorting is widely used in algal ecology and to
establish clonal/single cell-derived algal cultures
directly from natural samples (e.g. Surek and
Melkonian 2004). In our laboratory, the growth of
single algal cells immobilised on membranes has
already been demonstrated for a few taxa
(Naumann, unpubl. observations). Therefore, it
should be possible to sort single algal cells from
natural samples directly onto the membranes of
the 96-well twin-layer system.
Methods
Microalgal stock cultures: Microalgal strains
used in this study are listed in Table 1. All strains
were cultivated as suspension cultures without
aeration in Erlenmeyer asks containing 50ml
culture medium. The culture media used for the
cultivation of stock cultures were WARIS-H
(McFadden and Melkonian 1986), variations of
this medium [WARIS-H-3 V (WARIS-H with a 3-
fold vitamin concentration) and WARIS-H+Si
(WARIS-H containing 0.5mM Na
2
SiO
3
9 H
2
O)]
as well as HSM (Sueoka 1960). Marine species
were grown in articial seawater medium ASP-12
(Provasoli 1963) with replacement of TRIS buffer
by 3 mM HEPES. The light intensity varied
between 20 and 40mEm
2
s
1
at a light/dark-
cycle of 14/10h, the cultivation temperature was
either 24721C or 14721C (Table 1).
Assembly of the chamber: To assemble the
cultivation chamber, the following steps were
performed: eight layers of glass-bre sheets were
soaked for 10min in 500ml H
2
O (Milli-Q) and
subsequently rinsed thoroughly to remove nish or
other contaminations. A chamber with rinsed,
dried glass-bre sheets was accurately closed
by means of four clamps and autoclaved for
20min at 1211C. Under a laminar ow cabinet,
150ml of culture medium was injected step-
wise through the upper septa [which were
surface-sterilised with 70% (v/v) ethanol] into the
chamber. The excess 80ml of culture medium was
withdrawn.
ARTICLE IN PRESS
248 E.C.M. Nowack et al.
Inoculation and maintenance of cultures in
the 96-well twin-layer system: For the inoculation
of the lter plates with microalgae, chlorophyll a
was used as surrogate parameter for algal
biomass. To determine the chlorophyll a concen-
tration of suspension cultures, chlorophyll a was
extracted with DMSO using the method of Hiscox
and Israelstam (1979) and its concentration
determined spectrophotometrically according to
Jeffrey and Humphrey (1975). For algal taxa not
dealt with in Jeffrey and Humphrey, equations
were adjusted accordingly. As shown by Hiscox
and Israelstam (1979), the equations originally
published by Jeffrey and Humphrey for pigment
solutions in acetone can be applied without
correction also to DMSO solutions.
For the immobilisation of algal cells, the cham-
ber, assembled and sterilised as described above,
was opened under a laminar ow hood and a lter
plate placed on top of the stack of wet glass bre
sheets. Each well was inoculated with 60ml of a
dense algal suspension. Thin, homogenous algal
layers of a density of 2.5 or 5mg chlorophyll a
cm
2
were obtained by the slow permeation of
excess culture medium from the substrate- into
the source-layer. Depending on the experiment,
the immobilised algae were exposed to a light
intensity of 340 mEm
2
s
1
, a light/dark-cycle of
14/10h and a temperature of either 24721C or
14721C.
To transfer the cultures from an old to a new 96-
well twin-layer system, the immobilised cultures
were resuspended in approximately 200ml of
culture medium by means of an 8-channel multi-
pipette (Pipetman Ultra Multichannel, Gilson) and
then an aliquot transferred. For some mucilage-
forming algae, this required thorough mixing of the
suspension.
Examination of the suitability of immobilised
cultures for cultivation in the 96-well twin-layer
system: The physiological status of algae immo-
bilised in the 96-well twin-layer system was
estimated by a combination of (1) chlorophyll
uorometry, (2) visual characterisation, and (3) re-
establishment of suspension cultures.
(1) Chlorophyll uorometry was performed by
means of a PAM-2000 Chlorophyll Fluorometer
(WALZ, Effeltrich, Germany). Chlorophyll a uor-
escence (l4700nm) was induced by pulse-
modulated excitation light of a frequency of
20kHz and a wavelength lo 670nm. F
t
, the
steady-state uorescence of light-adapted immo-
bilised algae was measured after a 10min
illumination period with white light (Krypton
q668 100W, 45mEm
2
s
1
) during continuous
illumination. F
m
, the maximal uorescence of
light-adapted immobilised algae, was induced by
applying a saturation pulse of white light
(23004500mEm
2
s
1
) for 400 ms. The mea-
surements were performed by covering the lter
plate with a 2mm thick transparent plastic plate
and positioning the end of the glass-bre optic of
the uorometer directly on this plate over the top
of the well to be examined. The effective quantum
efciency of electron transport DF/F
m
was calcu-
lated according to Genty et al. (1989) and used to
estimate the physiological status of the immobi-
lised cultures. Algal cultures were regarded as
stable +, if their DF/F
m
did not decrease by
more than 25% within a period of 70100d.
Otherwise they were regarded as non-stable .
(2) By visual inspection of immobilised cultures
using a stereomicroscope, three different states of
immobilised cultures could be distinguished,
namely growth, stagnation, and death. In Table
1, these stages are referred to as +, (+), and
respectively. A culture was classied as
growing if the development of a homogenous
algal layer of increasing thickness was observed
within four weeks after immobilisation of the cells.
It was classied as dying if bleaching was
observed within 4 weeks. The third stage, stagna-
tion, was characterised by no discernable growth
but also no bleaching within 4 weeks, and in some
strains (e.g. Cryptomonas), correlated with co-
pious mucilage production.
(3) Re-establishment of suspension cultures
from immobilised cultures was achieved by the
transfer of cells from the 96-well twin-layer system
into a Petri dish containing 7ml of culture medium.
The growth of a dense suspension culture after
14d was referred to with +, and the failure of
growth with . A positive result in one of the
three criteria (see above) was regarded as
sufcient to classify a strain as suitable (+ in
Table 1). When all three criteria failed, the strain
was regarded as unsuitable for growth in the 96-
well twin-layer system ( in Table 1).
Sterility tests: To estimate the sterility of
immobilised cultures, sterility tests were per-
formed. For this purpose, the surface of an algal
layer was rinsed with 200ml of sterile culture
medium without resuspending the immobilised
algae. Approximately 150ml of the medium
was then transferred to 7ml of a sterile standard
growth medium for bacteria. This medium
contained (per L) 8.0g Bactopeptone (DIFCO
Laboratories, MI, USA), 1.0g glucose, 1.0g beef
extract (Merck, Darmstadt, Germany), and 1.0g
yeast extract (ICN Biomedicals, CA, USA).
ARTICLE IN PRESS
96-Well Twin-Layer Algal Culture System 249
To conrm results from sterility tests, probes
were analysed by phase-contrast light micro-
scopy.
Materials used in the 96-well twin-layer
system: 96-well MultiScreen lter plates contain-
ing PVDF membranes with a pore size of 0.22 mm
were purchased from Millipore, Schwalbach,
Germany (no. MAGVS2210). Glass bre sheets
(Isola 26091K, 80gm
2
) were obtained from Isola
AS, Eidanger, Norway. The cultivation chamber
itself consists of Delrin (polyacetal, POM), a highly
heat-resistant and thereby autoclavable plastic.
The bore holes in the base were plugged with
autoclavable injection septa of 13mm diameter
made of rubber (DIN 58366). The Delrin frame
contained six inlays of teon foam (Berghof,
Eningen, Germany). The cover of the chambers
was a 3mm thick glass plate. For the exchange of
culture medium, sterile disposable 50 ml syringes
with 2-gauge needles were used.
Statistical analysis: The KolmogorovSmir-
nov test was applied to analyse the Gaussian
quality of a data set. To calculate signicant
differences between average values, the Man-
nWhitney U-test or, if there were more than two
sets of data, the KruskalWallis-test was used.
Regression curves and the calculation of the area
under a graph were performed by means of
GraphPad PRISM Version 4.01. The inuence of
a single or a set of factors on a tested value was
examined by variance analysis (ANOVA). To
achieve a Gaussian distribution of the data, the
data were subjected to a log (x+1) transformation.
For statistical analysis, SPSS 11.0 statistic soft-
ware (SPSS Inc.) and GraphPad PRISM Version
4.01 (GraphPad Inc.) were used.
Acknowledgements
This work was carried out in close collaboration
with the members of the experimental workshop
of the Department of Botany, University of
Cologne, whose uncomplicated and professional
cooperation we thankfully acknowledge.
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