Hydrurus foetidus (Chromista, Chrysophyceae): A large
freshwater chromophyte alga in laboratory culturepre_606 105..112
Dag Klaveness*1
and Eli-Anne Lindstrøm2
1
University of Oslo, Department of Biology, Microbial Evolution Research Group (MERG), and 2
Norwegian Institute for
Water Research (NIVA), Gaustadalléen 21, Oslo, Norway
SUMMARY
The psychrophilic freshwater alga Hydrurus foetidus
(Villars) Trevisan has previously resisted laboratory culturing
in liquid media. We investigated the use of
microbiological techniques adapted to specific requirement
for low temperature and high turbulence of the
alga. We found that successful culturing required
cleaning steps, where pieces of the alga were dissected
out and plated on agar under cold conditions. From
here, unialgal colonies could be isolated and inoculated
into liquid media under appropriate conditions. Turbulence,
created by a laboratory shaker, is critical. Sufficient
light (30–100 mmol m–2
s–1
) and low temperature
(here 2–3°C) are necessary. This rather common but
highly under-investigated freshwater alga has potential
for basic and applied research when available as laboratory
cultures.
Key words: algal culture, freshwater alga, Hydrurus,
periglacial, psychrophile.
INTRODUCTION
The large (up to 10–30 cm), branched, freshwater
golden alga Hydrurus foetidus (Villars) Trevisan is distributed
worldwide (Wehr & Sheath 2003), but
restricted to cold streams, particularly in alpine and
periglacial environments (French 2007). In climates
with defined seasons, it is mainly found in the spring
during snowmelt, when the light conditions are improving.
However, the alga may also be found throughout
the summer and autumn in streams with low water
temperatures; for example, in rivers that drain glaciated
landscapes (Ward 1994; Rott et al. 2006a) or in the
cold springs of the Alps (Cantonati et al. 2006). Hydrurus
has previously resisted laboratory culturing in liquid
media (e.g. Rostafinski 1882a; Esser 2000) and is not
available from any culture collection. Pioneers like
Klebs (1896) and Geitler (1927) kept Hydrurus alive
for some time under natural or laboratory conditions,
but never succeeded in establishing laboratory cultures
in liquid media. It is extremely sensitive to temperature
changes, transport and careless laboratory conditions,
which may lead to cellular changes, sometimes sporulation,
bacterial attacks, deterioration and lysis. In
order to fix samples for electron microscopy, scientists
brought their fixatives to the river banks to perform the
initial processing (Vesk et al. 1984; Hoffman et al.
1986). Very few studies have been done on gene
sequences from Hydrurus – only three sequences from
natural material are available via the National Centre for
Biotechnology Information (NCBI) GenBank database –
one (5S) from Japan (Lim et al. 1986) and two (18S
and 28S rRNA) from Norway. Today, cultures of unicellular
(e.g. Ochromonas, Mallomonas) and colonial (e.g.
Dinobryon, Synura) golden algae (the classes Chrysophyceae
and Synurophyceae) are maintained to some
extent in culture collections and laboratories; but
golden algae may be more demanding and require
closer attention than members of many other algal
classes. Among the more critical studies of golden
algae under exacting laboratory conditions are those
investigating their physiological requirements (e.g.
Hutner et al. 1953; Klaveness & Guillard 1975;
Lehman 1976; Maberly et al. 2009).
The Kingdom Chromista, in which the golden algae
are affiliated, also includes the large brown algae (the
class Phaeophyceae), which provide shelter and food to
fauna of the near-shore marine hydrosphere. Large
brown algae contain protective carotenoids and lifesupporting
unsaturated lipids. Hydrurus is the only
large freshwater representative of the Kingdom Chromista;
it has a wide inland distribution (cf. Wehr &
Sheath 2003) and represents an interesting candidate
as a source of nutrients comparable to its marine counterparts.
Hydrurus is a valuable food source for fungi
and protists (Bursa 1934; Rott et al. 2006a,b) and for
larvae and adult insects in cold streams (Ward 1994;
Milner et al. 2001, 2009)
A comprehensive database on the distribution and
locations of Hydrurus in Norway is available to us
*To whom correspondence should be addressed.
Email: dag.klaveness@bio.uio.no
Communicating editor: I. Mine.
Received 30 October 2010; accepted 2 November 2010.
doi: 10.1111/j.1440-1835.2010.00606.x
Phycological Research 2011; 59: 105–112
© 2011 Japanese Society of Phycology
(Lindstrøm & Klaveness, manuscript in preparation) for
primary information on the environmental requirements
of Hydrurus in Norway. A large amount of environmental
information from locations where Hydrurus grows in
abundance is available from the international literature
(e.g. Ström 1926; Geitler 1927; Bursa 1934; Wehr &
Sheath 2003; Rott et al. 2006b). This kind of information
is helpful for culture initiation. But reliable clonal
cultures are necessary to sort out important and critical
factors for growth and reproduction, and to test environmental
factors individually and in combination. For
culturing, we note that a large alga like Hydrurus with
its pronounced mucopolysaccharide sheath might have
a limited capacity for gas exchange in water, unless
turbulence can keep every branch of the thallus
exposed. Furthermore, very cold water has a large carrying
capacity for dissolved gases, and may also provide
a continuous supply of nutrients under natural conditions.
In the laboratory, the agar plating technique,
pioneered in the case of Hydrurus by Geitler (1927, p.
796 and footnote), provides direct access to air as well
as nutrients. The early works of the scientists Klebs
(1893, 1896), Geitler (1927) and Bursa (1934) therefore
provided inspiration to continue the work towards
achieving consistent Hydrurus growth in liquid media.
The major purpose of this work was therefore to develop
a method for growing reproducible cultures of H. foetidus
in liquid medium. Our results, materials, and cooperation
are offered to improve the knowledge of this
widespread and interesting organism.
MATERIALS AND METHODS
Easy access to the Alpine Research Center at Finse
(www.finse.uio.no located at 60°36′ N, 7°30′ E) in
southern Norway, and to the torrent river that drains
lake Finsevatn (map at Fig. 1) close to the center,
provided us with fresh material throughout the spring
months. The research center has a cold incubator
(Sanyo MIR-153 with a door window) with external light
sources and a timer to supply a 14:10 h LD (light
: dark) cycle. The prominent spring populations of
Hydrurus in this river were first identified by Ström
(1926) as H. foetidus. It is still present in abundance
seasonally, and we fully confirmed Ström’s identification
based on the most precise and detailed morphological
descriptions available: Rostafinski (1882b),
Klebs (1893), Pascher (1913), Geitler (1927), Bursa
(1934), Mack (1953), Hovasse and Joyon (1960),
Fukushima (1962), Joyon (1963), Canter-Lund and
Lund (1995), Nicholls and Wujek (2003). The taxonomic
history of this genus is complicated, and the
morphological separations between species proposed
earlier are uncertain due to a pronounced phenotypic
plasticity. For more information, we suggest the overviews
by Rostafinski (1882a) and Silva (1950). Until
investigations have been done by modern methods on
material and cultures from different areas, we follow
the recommendations by recognized authorities, from
Pascher (1913) to Kristiansen and Preisig (2001),
treating this genus as monotypic with H. foetidus as the
only species.
For sequencing and for the culturing efforts reported
here, material was secured from the river on 7 March
2007. The river was still close to a minimum of winter
flow, and the water was flowing with a depth of
2–15 cm at the site of collection. Snowmelt is still
minimal at this location in the beginning of March, so
the river temperature was close to that of the icecovered
lake near the surface (1–2°C). The gross morphology
of the thalli collected were typically arbuscular,
3–7 cm in length, with central axes and branches upon
which the minor branchlets were organized. The entire
thallus is covered by a characteristic polysaccharide
sheath. Information on the partial 18S and 28S rRNA
gene sequences from this material are now available at
the NCBI GenBank at accession numbers FM955256
and FM955257. Unialgal cultures from this material
are offered to culture collections, to generate interest
and faciliate comparison with local strains.
The methods chosen for isolation and culturing of
Hydrurus are modifications of standard classical
microbiological techniques; glasswares were used in
most cases, and when plastics were necessary we
avoided direct contact with the biological material (cf.
Blankley 1973; McDonald et al. 2008). We exposed
tiny fractions of the alga directly to moist air within
Petri dishes filled with an agar substrate that contained
dissolved nutrients (Fig. 2). The medium was
Guillard & Lorenzen’s WC (Guillard & Lorenzen 1972)
made with glass-distilled water, without organic buffer,
without added silicate, and no further pH-adjustments
(WC simplified; here = SWC, Table 1). The liquid
medium was autoclaved as 70-mL batches in 125-mL
Bellco flasks with stainless steel caps (Fig. 2). This
medium could be solidified with 1.25% Difco Bacto
Agar, this should be a three-vessel procedure to avoid
harmful interference between components (cf. Allen
1968) and loss of nutrient availability by precipitation.
First, 1 mL of the diPotassium-hydrogenphosphate
stock solution (= stock solution 5 in Table 1) was
diluted to 10 mL in a glass culture tube with a stainless
steel cap; then, 490 mL of glass-distilled water
was placed in a 1-L (or larger) culture flask with stainless
steel cap and the remaining six stock solutions
(Table 1) were added in appropriate amounts to
achieve a final volume of 1 L of medium; finally,
500 mL of glass-distilled water was mixed with 12.5 g
of agar in another flask. All three vessels were autoclaved
at 121°C for 15 min; then the three solutions
were mixed while hot and poured into sterile plastic
Petri dishes. After cooling, the condensed water on the
© 2011 Japanese Society of Phycology
106 D. Klaveness and E.-A. Lindstrøm
lids was carefully drained, and the plates were stacked
and stored upside-down in polyethylene bags at the
ambient temperature within the culture facility. The
liquid medium attained a pH within the range of 7.1–
7.4 upon equilibration with air; the addition of agar
did not change this substantially.
A glass Pasteur pipette was melted closed and
curved at the tip with a spiritus flame. Tiny pieces of
Figs 1–6. 1. Map of area and location from where Hydrurus foetidus was collected. The major watercourses and ponds are white,
landscape with topography are indicated in grey. Equidistance of terrestrial height curves are 5 m. Cottages and buildings are indicated
as dark grey structures, while the dirt road is shown by a double line. The Alpine Research Center, indicated by ARC, is at the center to
the right side of the map. Scale bar = 200 m. Lake Finsevatn (in the upper left corner) is dammed up to a water level of 1215 m above
sea level, by a concrete structure (indicated by black solid line, at DAM in figure). The river does not freeze during winter and is accessible
at the spot indicated by the large arrow – with some effort and technical gear. 2. Culturing vessels for growing H. foetidus. (lower left)
a 9-cm Petri dish with agar medium; (lower right) a 15-cm screwcapped culture tube with an agar slant; and (top) a 175-mL Bellco bottle
with 50 mL of medium. 3. Macrophoto of colonies on an agar plate, grown for 9 weeks. Scale bar = 2 mm. 4. Fringe of a colony on
an agar plate, photographed through a microscope (40¥ objective without a coverglass). Spreading out on the agar plate appeared to be
difficult; colonies remained quite compact. Scale bar = 10 mm. 5. Multicellular branch of a thallus in liquid culture. Scale
bar = 10 mm. 6. Larger magnification of the distal part of thallus grown in liquid culture. Note a broadly conical apical cell (middle right)
initiating a lateral branch, but still within the common polysaccharide sheath. The surface of the polysaccharide sheath at this stage of
isolation still appears rugulose due to epiphytic bacteria. Scale bar = 10 mm.
© 2011 Japanese Society of Phycology
107Culturing method for Hydrurus foetidus
fresh material from the river were carefully macerated
and then streaked with this fine tool in a criss-cross
pattern on the agar plates. The inoculated plates were
stored in zip-locked polyethylene bags and maintained
upside-down in the cold incubator at 3°C, with approximately
30 Ϯ 15 mmol m–2
s–1
(depending on the distance
from the light source) of fluorescent light,
provided on a 14 : 10 h LD cycle. The inoculated agar
plates were later moved to Oslo inside cold thermos
bottles, and then placed in a cold room maintained at
2.6°C with 14 : 10 h LD cycles and 30–100 mmol
m–2
s–1
of soft white fluorescent light during the L
phase. Light was measured at the positions of the
culture vessels in the directions of the light sources,
with a recently calibrated, cosinuscorrected Li-Cor
Quantum sensor, mounted on a LI-1000 DataLogger.
Temperatures in the culture chamber and room
were logged with calibrated Onset TidBit v1 and v2
dataloggers.
Observations, photography and measurements of
cell sizes were done with a Nikon Diaphot microscope
equipped with an eyepiece reticle and a Nikon D1
digital camera. Both the eyepiece reticle and the photographic
records were carefully calibrated by a stage
micrometer (Zeiss Oberkochen, Germany), at all magnifications
used.
Two methods were tested for growing Hydrurus in
liquid medium. The simplest was the inoculation of
single colonies from agar plates into flasks (Fig. 2). The
flasks were then mounted on a shaker (simple linear
movement, 20 mm amplitude at 40 rpm). The other
method was inoculation into 40 ¥ 300 mm tapered
glass tubes that had an air inlet at the bottom; this
method still requires more work for optimization. Here,
we report the most important experiences during the
work that resulted in successful flask culturing of
Hydrurus.
RESULTS
Periodic inspection of the agar plates at the Alpine
Research Center revealed that the survival of algal fragments
was quite successful, with visible growth along
the edges and a substantial volume increase (Figs 3,4).
There was, however, a heavy growth of bacteria on the
plate surface. To eliminate the bacteria, Hydrurus colonies
were dissected out, shaken, rinsed in the medium,
and restreaked/reisolated; this reduced the bacterial
problem and removed all foreign algal and cyanobacterial
epiphytes. We found that this ‘clean’ Hydrurus
could be kept alive on an agar surface for at least one
year without transfer when the Petri dishes were stored
in sealed polyethylene bags under moderate light in
cold room conditions. Agar slants in screwcapped tubes
(Fig. 2) were also useful for cold transport and mailing
samples.
When clean colonies from one agar plate, grown from
the macerated tissue of one single branch tip of material
from the river, were picked and inoculated into
flasks with medium, a certain degree of morphological
plasticity was expressed. In some bottles, aggregations
of cells proliferated as typically colored, slimy layers on
the glassware and loosely branched filaments in the
liquid. In other flasks, star-shaped or branched colonies
developed from the agar plate colony. These consisted
of fast-growing, weakly colored central axes (cf. Bursa
1934: ‘Gallertzylinder’ p. 77, ‘Zentralzylinder’ p. 115)
that developed branches of typical structure and color
(Figs 5,6). A complete plant of Hydrurus consisted of
three types of cells: (i) central axis cells that were
apparently able to produce firm polysaccharide for the
central axis structure (cf. Tab. II in Rostafinski
(1882b), or figs 13–26 in Graham & Wilcox 2000);
and: (ii) more or less isodiametric cells that formed
branches emanating from the axial structures (as in
Figs 5,6); these developed within soft to fluid polysaccharide
coats (cf. also fig. 297 on p.161 in CanterLund
& Lund 1995) – and 3) the meristematic top cell
of each branch.
To conserve both types of ‘tissue’ – the central axis
and the branches within soft polysaccharide coats – it
was initially considered important to propagate pieces
consisting of both types. However, we noticed that
colonies transferred from agar plates very soon after the
initial isolation led significantly to the formation of
firmer central axes. Under the conditions described
Table 1. List of stock solutions for liquid medium SWC (=Guillard
and Lorenzen’s WC-medium simplified).†
No. of
stock
solution
Components Amount of the component
dissolved in distilled
water to make a
100 mL stock solution
1 CaCl2·2H2O 3.68 g
2 MgSO4·7H2O 3.70 g
3 NaHCO3 1.26 g
4 NaNO3 8.50 g
5 K2HPO4 871 mg
6 Na2EDTA 436 mg
FeCl3·6H2O 315 mg
CuSO4·5H2O 1.0 mg
ZnSO4·7H2O 2.2 mg
CoCl2·6H2O 1.0 mg
MnCl2·4H2O 18 mg
Na2MoO4·2H2O 0.6 mg
H3BO3 100 mg
7 Thiamin·HCl 10 mg
Biotin 50 mg
Vitamin B12 50 mg
†To make a 1 L medium, add 1 mL of each stock solution.
Stock solutions 1–6 can be stored for some time in refrigerator.
Stock solution 7 should be split in small batches and kept frozen.
© 2011 Japanese Society of Phycology
108 D. Klaveness and E.-A. Lindstrøm
here, the continued transfer of excised pieces from
liquid cultures to new liquid cultures every 3 weeks (or
when a thallus in a flask reached approximately 30 mm
in size), favored the growth of branch tissue. These
colonies showed gradual rearrangements of the central
axes into loosely organized, but still discrete structures.
This organization of the thallus then remained stable
under the type of turbulence we applied here. The size
of the different cells in a thallus from culture (as shown
in Fig. 7) was measured under the microscope and are
given in Table 2.
Algal cultures with good growth rates often outgrow
the bacteria during exponential growth phases, probably
because the leachates of secondary metabolites
are kept under control. The foul smell characteristic of
H. foetidus was not apparent in these cultures, despite
the presence of bacteria. As long as the algae were
growing rapidly, only few bacteria were observed along
the edges of the polysaccharide sheaths or in the
medium. However, if the growth of the alga slowed,
within a short period of time (days or weeks) a decomposition
process commenced. Then, the characteristic
smell of H. foetidus became palpable.
DISCUSSION
We succeeded in establishing and propagating H. foetidus
in liquid cultures by using simple, wellestablished
techniques, ensuring that only clean
glassware (no plastics) was in direct contact with the
alga, and maintaining some degree of skill and
patience. The pioneer connoisseurs of the art of protist
and algal culturing (e.g. Ogata, Geitler, Lwoff, Droop,
Provasoli, Guillard) never renounced cleanliness and
quality – obligate requirements for success – and attention
and patience gave results of interest.
Our simplification of the medium described by Guillard
and Lorenzen (1972) avoided adding silicate; this
may be rational for media intended for isolation purposes,
given that glassware is involved in their preparation
and storage. Studies of silicate requirements in
cultures indicated early that silicate leaching from
glasswares, from silicate contamination of chemicals,
and from the use of glass stills could supply silicate
compounds (e.g. Werner 1977). A pH adjustment was
not required for the SWC medium, when time was
allowed after autoclaving for equilibration with air at the
ambient temperature. Chlorinity was kept low by avoiding
the addition of HCl, and other potentially inhibiting
ions for golden algae (e.g. potassium; cf. Lehman
1976) were well controlled by moderate concentrations.
The buffering capacity was provided by the bicarbonate
system; therefore, this required attention during
the cleaning and rinsing of glassware and preparation of
medium. Continuous equilibration of the bicarbonate
system was controlled by applying turbulence during
algal growth. The careful handling of bottles (e.g.
placing the bottles on melting ice in polystyrene containers
when outside the growth facilities) may have
prevented sporulation during periods without turbulence;
moreover, secondary establishment was also
avoided in the splash zones on the glass walls of the
bottles. The shaker speed was chosen by trial and error
and had to be reduced to avoid spilling medium. Thus,
the degree of turbulence was not optimized for algae
growth. This compromise may have influenced the
developmental morphology of the pieces inoculated
into our bottles. Bursa (1934, p. 77) concluded from
field studies that the water flow rate (‘Strömung’) had
an influence on the development of the central axis; low
flow rates appeared to have led to poor development.
The turbulence in our cultures was non-directional with
respect to the free-floating plants. However, in the river,
there is a substantial directional drag on the sessile
thallus; under those conditions, growth may require a
different central axis. The quality of the central axis
may be regulated by turbulence, laminar flow rate, and
shear stress; this should be investigated in future
studies under rigorous experimental conditions.
The cell sizes (particularly the categories 2., 3., and
combined in category 5., in Table 2) agree well with the
size ranges reported by other authors from wild material
(e.g. 8–16 ¥ 12–22 mm by Mack 1953; 7–20 mm by
Joyon 1963; 5–15 mm by Vesk et al. 1984) – but there
are no separate measurements of the different cell
categories in the literature, as reported here. It has
been shown that cells ‘aus dem Innern des Lagers’
(=central axis cells?) may attain extreme shapes and
Fig. 7. Recently excised branch of a larger (>30 mm), freefloating
H. foetidus colony. The colony was grown in a flask on a
shaker, this branch was excised and transferred into new medium
and grown for one week before this picture was taken. It displays
the typical shape of a free-floating colony grown through several
transfers (as explained) in liquid cultures on a shaker with nondirectional
turbulence at 2.6°C and a photosynthetically active
radiation quantum flux of up to 100 mmol m–2
s–1
. Scale
bar = 5 mm.
© 2011 Japanese Society of Phycology
109Culturing method for Hydrurus foetidus
AA-elongation (e.g. Berthold 1878, cf. also Mack
1953).
Answers to questions and speculations that have
arisen in the literature regarding the morphological plasticity
or nature of the highly polymorphous species H.
foetidus, will require more careful observations (e.g.
observations and biometry on different cell types, cf.
Table 2 here), well-designed experiments in the laboratory
on cultivated strains like this one (numbered G
070301), and new isolates from other locations. New
isolates may provide new sequences that reveal genetic
diversity behind the apparent morphological plasticity of
H. foetidus. Bacteria-free cultures are obligatory for the
study of physiological processes and certain nutrient
requirements (e.g. Mainx 1929; Hutner et al. 1953;
Klaveness & Guillard 1975; Vieira and Klaveness 1986;
Danger et al. 2007). When phagotrophic protists or
potential mixotrophs, like golden alga, are involved (e.g.
Hardin 1942; 1944; Bird & Kalff 1987), knowledge of
the bacterial flora is essential (cf. Jennings 1908: ‘To
make the conditions of existence the same, it is not
sufficient to attend merely to the basic fluid; the bacteria
must also be the same’). In our current state of knowledge,
it is a primary challenge to obtain bacteria-free
cultures of Hydrurus for different kinds of physiological
studies, without use of antibiotics that could be mutagenetic
or harmful to organelles. Because Hydrurus is a
large, freshwater, inland alga that is phylogenetically
affiliated with marine seaweeds, it may be of nutritional
and biotechnological interest in addition to serving as a
model system for the brown branch of plants (see Baweja
et al. 2009 for challenges).
We invite academic laboratories to share our agar
slants with ‘Palmellen’ in order to take part in improving
culturing conditions and to further study this strain
(G 070301). This study may also inspire the investigation
of new local strains of H. foetidus.
ACKNOWLEDGMENTS
Logistic and financial support from the Microbial Evolution
Research Group (MERG) at the University of
Oslo, and technical assistance from the staff at Finse
Alpine Research Center are gratefully acknowledged.
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Cell category Cell shape n Apical-antapical (AA)
distance and range (R),
mm
Lateral diameter (LL)
and range (R), mm
1. Apical cell of branches Half spheroid to conical
with flat or angular base
10 AA = 12.0 Ϯ 2.3 LL = 14.8 Ϯ 2.5
R = 9–16 R = 12–19
2. Intercalary cell of
dense branches
Angular or compressed 10 AA = 11.1 Ϯ 1.7 LL = 13.0 Ϯ 2.3
R = 9–13 R = 10–18
3. Free cells of loose
branches
Sphere to spheroid 10 AA = 13.5 Ϯ 1.7 LL = 12.6 Ϯ 1.1
R = 12–17.5 R = 11.5–14
4. Central axis cells Ellipsoid to ovoid 10 AA = 25.2 Ϯ 4.7 LL = 10.2 Ϯ 1.2
R = 20–32 R = 8–12
5. Branch cells (2. + 3.
above) combined
20 AA = 12.3 Ϯ 2.1 LL = 12.8 Ϯ 2.1
R = 9–17.5 R = 10–18
The first 10 cells (n) observed of each cell category was measured. Apical end of cells were towards the top of thallus or branches (=
direction of growth), close to where the chloroplast is located (cf. Fig. 6). Lateral diameter was taken as maximum diameter 90° on the
apical-antapical distance. Data given are means Ϯ standard deviations. R indicates ranges encountered among the cells of each category.
© 2011 Japanese Society of Phycology
110 D. Klaveness and E.-A. Lindstrøm
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