Annals of Botany 114: 1733, 2014

doi:10.1093/aob/mcu085, available online at www.aob.oxfordjournals.org

INVITED REVIEW

Ecophysiology of gelatinous Nostoc colonies: unprecedented slow growth

and survival in resource-poor and harsh environments
Kaj Sand-Jensen*
Freshwater Biological Laboratory, Biological Institute, University of Copenhagen, Universitetsparken 4,
2100 Copenhagen, Denmark
* E-mail ksandjensen@bio.ku.dk

Downloaded from http://aob.oxfordjournals.org/ at Library of Medical Center of Fudan University on May 12, 2015
Received: 17 February 2014 Returned for revision: 6 March 2014 Accepted: 1 April 2014

Background The cyanobacterial genus Nostoc includes several species forming centimetre-large gelatinous col-
onies in nutrient-poor freshwaters and harsh semi-terrestrial environments with extended drought or freezing. These
Nostoc species have filaments with normal photosynthetic cells and N2-fixing heterocysts embedded in an extensive
gelatinous matrix of polysaccharides and many other organic substances providing biological and environmental pro-
tection. Large colony size imposes constraints on the use of external resources and the gelatinous matrix represents
extra costs and reduced growth rates.
Scope The objective of this review is to evaluate the mechanisms behind the low rates of growth and mortality, pro-
tection against environmental hazards and the persistence and longevity of gelatinous Nostoc colonies, and their
ability to economize with highly limiting resources.
Conclusions Simple models predict the decline in uptake of dissolved inorganic carbon (DIC) and a decline in the
growth rate of spherical freshwater colonies of N. pruniforme and N. zetterstedtii and sheet-like colonies of
N. commune in response to a thicker diffusion boundary layer, lower external DIC concentration and higher
organic carbon mass per surface area (CMA) of the colony. Measured growth rates of N. commune and
N. pruniforme at high DIC availability comply with general empirical predictions of maximum growth rate (i.e. doub-
ling time 1014 d) as functions of CMA for marine macroalgae and as functions of tissue thickness for aquatic and
terrestrial plants, while extremely low growth rates of N. zetterstedtii (i.e. doubling time 23 years) are 10-fold
lower than model predictions, either because of very low ambient DIC and/or an extremely costly colony matrix.
DIC uptake is limited by diffusion at low concentrations for all species, although they exhibit efficient HCO3
uptake, accumulation of respiratory DIC within the colonies and very low CO2 compensation points. Long light
paths and light attenuation by structural substances in large Nostoc colonies cause lower quantum efficiency and as-
similation numberand higher light compensation points than in unicells and otheraquatic macrophytes. Extremely low
growth and mortality rates of N. zetterstedtii reflect stress-selected adaptation to nutrient- and DIC-poor temperate
lakes, while N. pruniforme exhibits a mixed ruderal- and stress-selected strategy with slow growth and year-long sur-
vival prevailing in sub-Arctic lakes and faster growth and shorter longevity in temperate lakes. Nostoc commune and its
close relative N. flagelliforme have a mixed stressdisturbance strategy not found among higher plants, with stress
selection to limiting water and nutrients and disturbance selection in quiescent dry or frozen stages. Despite profound
ecological differences between species, active growth of temperate specimens is mostly restricted to the same tempera-
ture range (035 8C; maximum at 25 8C). Future studies should aim to unravel the processes behind the extreme per-
sistence and low metabolism of Nostoc species under ambient resource supply on sediment and soil surfaces.

Key words: Gelatinous colonies, cyanobacteria, Nostoc commune, Nostoc flagelliforme, Nostoc pruniforme, Nostoc
zetterstedtii, carbon use, carbon concentrating mechanisms, photosynthesis, light use, growth, long-lived, survival,
desiccation tolerance, nutrient-poor.

IN T RO DU C T IO N structure in rice fields, freshwater lakes, ponds and streams and

The cyanobacterial genus Nostoc includes many species that are
highly diverse with respect to morphology, functional properties,
biotic relations and habitat distribution (Dodds et al., 1995).
Nostoc species have filaments with normal photosynthetic cells
and N2-fixing heterocysts and they periodically form resistant
akinetes for survival and short motile filaments (hormogonia)
for reproduction (Mollenhauer et al., 1994). Some species are
free-living and many species engage in loose or obligate cooper-
ation with land plants and fungi (e.g. lichens; Mollenhauer,
1988; Kaasalainen et al., 2012). A third, fascinating Nostoc
type forms large gelatinous colonies of variable shape and
# The Author 2014. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
on alternating wet and dry soils or rock surfaces (Dodds et al.,
1995; Gao et al., 2012). The large gelatinous species require
special adaptations to obtain sufficient light, nutrients and dis-
solved inorganic carbon (DIC) (Sand-Jensen et al., 2009b) in
water and to survive the extreme variations in temperature,
water supply and irradiance on naked soils and rock surfaces
(Tamaru et al., 2005; Li et al., 2011; Yu and Liu, 2013). While
phototrophs in resource-rich environments have been treated in
numerous original papers, reviews and books (Larcher, 2003;
Lambers et al., 2008), studies on the adaptations of phototrophs
to resource-poor and physically extreme environments are rare.
The objective of this review is to evaluate the mechanisms
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18 Sand-Jensen Ecophysiology of Nostoc colonies
behind the extremely low rates of growth and mortality and high
persistence, longevity and protection against environmental
hazards of gelatinous Nostoc colonies and their ability to econo-
mize with highly limiting resources.
The review focuses on four colonial Nostoc species: the sheet-
like Nostoc commune, the closely related N. flagelliforme and
two species that form spherical colonies, N. pruniforme and
N. zetterstedtii. All four species resemble each other by
forming centimetre-large gelatinous colonies, but they differ in
distribution, habitat preference, growth rate, environmental tol-
erance and appearance (Fig. 1). Nostoc commune is a globally
widespread terrestrial and semi-aquatic species forming inactive
crusts when dry, but rapidly changing to 1 5-mm thick sheet-
like gelatinous structures when wet (Scherer et al., 1984;
Marsh et al., 2006; Novis et al., 2007). Nostoc commune has un-
precedented tolerance of environmental extremes (Satoh et al.,
2002; Tamaru et al., 2005; Sand-Jensen and Jespersen, 2012).
It tolerates desiccation during dry seasons and can survive for
.100 years on a museum shelf (Cameron, 1962). It tolerates
freezing to 60 8C in Antarctic deserts (Davey, 1989; Novis
et al., 2007) and to 269 8C in liquid helium in the laboratory
(Sand-Jensen and Jespersen, 2012), but rapidly resumes metab-
olism when rewetted at physiologically suitable temperatures
between 0 and 30 8C (Scherer et al., 1984; Taranto et al., 1993;
Sand-Jensen and Jespersen, 2012; Mller et al., 2014). Nostoc
flagelliforme is also widely distributed in arid and semi-arid
areas throughout the world (Gao et al., 2012; Ye et al., 2012).
It has a thread-like appearance but resembles N. commune in
its genetics, growth habitat, colony composition and extreme tol-
erance to and rapid recovery from desiccation, freezing and salt
stress (Gao and Zou, 2001; Qiu et al., 2004; Huang et al., 2005;
Yong-Hong et al., 2005; Liu et al., 2010; Arima et al., 2011). We
have observed Nostoc with the typical morphology of both
N. commune and N. flagelliforme on the Great Alvar on Oland,
Sweden (Sand-Jensen et al., 2010). Nostoc flagelliforme has
been classified as a variety of N. commune in arid regions of
F I G . 1. Images of colonies of rehydrated Nostoc commune (upper left), Nostoc
zetterstedtii (upper right, usually more spherical) and N. pruniforme (lower
middle).
Spain (Aboal et al., 2010) and recent DNA studies group those
two species together with N. sphaeroides and N. punctiforme
in a common clade (Arima et al., 2011; Gao et al., 2011),
which makes species identity dubious. Nonetheless, we maintain
the distinction between N. commune and N. flagelliforme here in
accordance with most available literature.
Nostoc pruniforme forms a beautiful, dark green, spherical
colony with a smooth surface like a plume. It is more common
and widely distributed both geographically and ecologically in
oligo- and mesotrophic freshwaters in temperate and sub-
Arctic regions than its rare freshwater cousin, N. zetterstedtii
(Dodds et al., 1995; Raun et al., 2009). Danish alkaline lakes
housing N. pruniforme contain .0.7 mM DIC (Sand-Jensen
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et al., 2009b). In temperate lakes, N. pruniforme appears to
grow in diameter from 0.2 cm in late spring to 2 3 cm in mid-
summer and it can rapidly die off, supposedly because of attacks
by viruses or bacteria at high summer temperatures (K. Sand-
Jensen, unpubl. data). In contrast, in a cold (4 8C), nutrient-poor
spring in Oregon, USA, N. pruniforme reached a handball size of
15 17 cm in diameter and weighed 2.6 kg after sustained slow
growth for 9 14 years (Dodds and Castenholz, 1987). Very
large colonies are also found in cold transparent lakes in
Greenland (K. Sand-Jensen, unpubl. data). In addition to their
larger size and persistence, they differ from Danish temperate
specimens by having a more solid surface and a denser gel.
Two additional species, N. calcareous and N. sphaericus
(Mollenhauer et al., 1994) resemble N. pruniforme but are not
discussed further here.
The fourth species, N. zetterstedtii, is a rare, highly persistent
organism whose colonies resemble firm blackberries. It lives in
only 50 100 nutrient-poor soft-water lakes in Sweden and in a
few other lakes in neighbouring countries (Bengtsson, 1986,
1995; Mollenhauer et al., 1999). Soft-water lakes usually have
DIC concentrations of 0.02 0.3 mM and concentrations of
total phosphorus ,0.1 0.3 mM in open waters (Sand-Jensen
et al., 2009b). Among the freshwater phototrophic species
studied so far, Nostoc zetterstedtii has unprecedented low
growth and mortality rates (Sand-Jensen and Mller, 2011). In
oligotrophic Lake Varsjo, Sweden, it takes 3 years for
N. zetterstedtii to double its colony mass and 30 years to
reach the recorded maximum diameter of 7 cm (Sand-Jensen
and Mller, 2011). There is no apparent mortality of grazers
and pathogenic bacteria and viruses (Sand-Jensen and Mller,
2011). The persistence of N. zetterstedtii is confirmed by its
ability to survive for 14 months in the dark at 5 8C with intact
colony structure and unaltered fresh mass (K. Sand-Jensen,
unpubl. data).
All mentioned Nostoc species must have common structural
and functional properties linked to the large size and extensive
matrix of the colonies. However, the species differ with respect
to metabolism, growth, mortality, tolerance to environmental
stress and habitat preference. In this review, I first examine
some of the main structural properties of the three main
species (N. commune, N. pruniforme and N. zetterstedtii) with
supplementary data for N. flagelliforme related to the centimetre-
large size and gelatinous matrix of the colonies. Second, I evalu-
ate theoretically the diffusive flux of DIC to the colony surface
for a set of environmental constraints on boundary layer thick-
ness and external concentrations. Third, I examine the functional
consequences of the large colony size for the use of DIC and light
 Sand-Jensen Ecophysiology of Nostoc colonies 19

in photosynthesis and growth. Fourth, I compare the tolerance of TA B L E 1. Shape, range of linear dimensions and surface
Nostoc species to environmental stresses related to temperature, area:volume ratio (SA:V ) of three species of Nostoc colonies,
freezing and desiccation. Fifth, I evaluate the growth, mortality Nostoc trichomes, submerged leaves and single phototrophic
and longevity of the three Nostoc species and the ecological unicells
implications of these properties. The final section points out
that costs and benefits of colony formation should be quantified Species and Shape Diameter/ SA:V
phototrophic type thickness (cm) (cm 1)
in future studies. For this comparative analysis of the eco-
physiology of gelatinous Nostoc colonies, I use published data N. commune Flat structure 0.1 10 to 20
and include supplementary unpublished data to ensure a compre- 0.5 2 to 4
hensive comparison. N. pruniforme Sphere 0.2 30
17.0 0.36
Nostoc zetterstedtii Sphere, 0.2 30
S I Z E , S HA P E AN D CO M PO S I T IO N granulated surface 7.0 0.851.7

Centimetre-thick colonies with photosynthetic filaments embed-
ded in a gelatinous matrix exert large physical constrains on
functional properties.
Size and shape
Large size of phototrophs imposes constraints on the use of light
and uptake of inorganic carbon and nutrient ions from the sur-
rounding water (Nielsen and Sand-Jensen, 1990; Agusti et al.,
1994). For a body of a given shape, surface area (SA L 2)
scales to the second power of the linear dimension (L), volume
(V L3) to the third power, and surface area:volume ratio
(SA:V L 1) to the inverse of the linear dimension. These allo-
metric relationships reflect that the supply rates of light and
external resources are scaled to the outer surface while the
requirements for production and maintenance of the cells and
the gelatinous matrix are mainly scaled to the volume.
The spherical shape adopted by N. pruniforme and
N. zetterstedtii is a simple shape of low SA:V ratio (3 r 1,
where r is the radius of the sphere). Individual filaments of
Nostoc embedded in the gelatinous matrix and free-living fila-
ments of cyanobacteria and algae are cylindrical and maintain
a high SA:V ratio inversely scaled to the transverse radius of
the filament (2 r 1) but independent of filament length. The
SA:V ratio of the flat sheet-like structure of Nostoc commune,
multicellular algae and leaves of mosses and higher plants is in-
versely scaled to thickness (Lth) of the structure (SA:V 2 Lth1)
but is independent of its surface area, volume and total mass as
long as tissue thickness remains constant.
The gelatinous colonies of N. commune, N. pruniforme and
N. zetterstedtii all have relatively thick tissues (0.1 17 cm)
and very low SA:V ratios (0.4 20 cm 1) compared with the
thin Nostoc trichomes within the gel (SA:V 4000 8000 cm 1)
and the 0.01- to 0.3 cm-thick submerged leaves of mosses and
angiosperms (SA:V 702000 cm 1) (Table 1). Nostoc commune
sheets typically vary in thickness from 0.1 to 0.5 cm when wet
and have SA:V ratios of 2 20 cm 1. The smooth spherical col-
onies of N. pruniforme vary from a diameter of 0.2 cm and
an SA:V ratio of 30 cm 1 to an astonishing maximum diameter
of 17 cm and an exceedingly low SA:V ratio of only 0.36 cm 1.
Small colonies of N. zetterstedtii are smooth, have a diameter
of 0.2 cm, just like N. pruniforme, while large colonies, reach-
ing a recorded maximum diameter of 7 cm, have a granulated
surface that can be regarded as consisting of small hemispheres
(exposed area 2 pr2 and planform area pr2) distributed all over
a large spherical surface. The granulated surface area has a
2-fold larger surface area relative to a smooth surface and the
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N. trichomes Cylinders 5 10 4 8000
10 10 4 4000
Submerged leaves Flat structure 1 10 3 2000
30 10 3 70
Single-celled algae or Sphere 2 10 4 30 000
cyanobacteria 2 10 3 3000
SA:V ratio is only 1.7 cm 1 for the largest N. zetterstedtii col-
onies. The granulated surface could, in theory, double the
diffusion-limited uptake of external solutes for N. zetterstedtii
by doubling the surface area compared with smooth colonies
of N. pruniforme of the same diameter (see section Size
effects, diffusive supply and loss of solutes). The granulated
surface could also contribute to the formation of thinner diffu-
sion boundary layers (DBL) around the surfaces of
N. zetterstedtii when the granules increase micro-turbulence
(Boudreau and Jrgensen, 2001). The micro-topography of the
granulated surface can result in thinner DBLs at the protruding
tips of the granules and thicker DBLs in the crevices between
the granules (Carpenter and Williams, 1993; Jrgensen, 2001)
so the diffusion geometry is difficult to interpret. Although diffu-
sive fluxes remain experimentally unexplored, they are, nonethe-
less, highly relevant considering that N. zetterstedtii usually
grows in lakes with lower concentrations of DIC, N and P than
N. pruniforme (Sand-Jensen et al., 2009b).
Composition of colony matrix
A high proportion of Nostoc colonies is composed of the extra-
cellular matrix, primarily of polysaccharides of high viscosity
and molecular weight. The hydrolysed polysaccharides from
N. flagelliforme were mainly composed of glucose (43 %),
galactose (30 %), xylose (21 %), mannose (6 %) and small
amounts of glucuronic and uronic acids (Jia et al., 2007;
Pereira et al., 2009). Much the same main sugars were hydrolysed
from hot water extracts of polysaccharides from field samples of
N. commune, N. flagelliforme and N. sphaeroides, though small
amounts of arabinose appeared in N. flagelliforme and rhamnose
and fucose in N. sphaeroides (Huang et al., 1998). The compos-
ition of extracellular compared with intracellular polysacchar-
ides was suggested to be quite different in N. commune and
N. flagelliforme, perhaps due to a selective mechanism for excre-
tion of polysaccharides (Mehta and Vaidya, 1978). Different
studies of N. commune have shown the complex chemistry of
the hydrolysed matrix, which includes six to nine monosacchar-
ides, uronic acid, deoxy-sugars, pyruvate, acetate and peptides
(Pereira et al., 2009). Scanning electron microscopy has revealed
20 Sand-Jensen Ecophysiology of Nostoc colonies
a net-like matrix of polysaccharides around Nostoc filaments
(Cupac and Gantar, 1992), suggesting that their ultrastructure
could influence colony shape. The chemical structure, the 3-D
network and the presence of special peripheral groups, such as
nosturonic acid, are probably essential for the outstanding
moisture absorption and moisture retention capacity of Nostoc
polysaccharides. These properties are highly interesting in
biomedical applications involving living cells and tissue
(Yu et al., 2010; Li et al., 2011).
When the gelatinous matrix of N. commune, N. flagelliforme
and N. pruniforme is hydrated its water content is very high,
leading to a soft structure that is easy to cut with a finger nail,
as opposed to the very solid structure of N. zetterstedtii, which
can only be cut, with great force, using a scalpel. Summer col-
onies of N. pruniforme from mesotrophic Lake Esrum with a
fresh mass (FM) of 3.5 g have a mass specific density of 1.01 g
FM cm 3 while colonies of N. zetterstedtii from oligotrophic
Lake Varsjo have a specific density of 1.13 g FM cm 3
(Raun et al., 2009; Sand-Jensen et al., 2009b). Both species
have gradually decreasing mass specific density for larger col-
onies, which tend to be hollow and water-filled in the centre of
N. pruniforme and possess a massive, though less dense, struc-
ture without trichomes in N. zetterstedtii (Table 2). The carbon
mass per surface area [CMA, equivalent to leaf mass per
surface area for plants (LMA)] is closely scaled to structural
and functional properties of macroalgal thalli (Markager and
Sand-Jensen, 1994, 1996) and plant leaves (Nielsen and
Sand-Jensen, 1989, 1991; Lambers and Poorter, 1992, 2004;
Poorter et al., 2012). The CMA increased by a power of 0.25
relative to colony FM for N. zetterstedtii and only 0.05 for
N. pruniforme, because the first species maintains massive col-
onies and the second species tends to develop hollow colonies
with increasing size (Table 2). Thus, in two summer collections,
CMA was 8-fold higher for N. zetterstedtii than N. pruniforme
for colonies of 0.1 g FM, while the difference was 14-fold for
colonies of 3.5 g FM.
The typical differences in CMA values for 3.5 g FM colonies
of N. zetterstedtii (1400 mmol organic C cm 2) and
N. pruniforme (100 mmol organic C cm 2) have important
implications. Assuming the same net photosynthesis relative to
TA B L E 2. Organic carbon content per surface area (CMA, mmol
C cm 2) relative to colony fresh mass (FM,g) of three Nostoc
species
Species Relationship Source
N. commune CMA 184 + 47 K. Sand-Jensen, unpubl. data
(mean + s.e.)
N. pruniforme Log CMA 0.0474 log C. Mller and K. Sand-Jensen,
FM + 1.88 unpubl. data
CMA for 0.1 g
FM colony 69
CMA for 4.0 g
FM colony 81
N. zetterstedtii Log CMA 0.26 log Sand-Jensen et al. (2009)
FM + 3.13 Sand-Jensen and Mller (2011)
CMA for 0.1 g
FM colony 587
CMA for 4.0 g
FM colony 1531
colony surface area of both species and the same proportion of
net photosynthesis being incorporated into new biomass, the
turnover rate of the colony mass will be 14-fold lower for
N. zetterstedtii than for N. pruniforme. With the same assumption
of constant photosynthesis per surface area independent of
colony size, biomass turnover and relative growth rate should
decline with colony weight in proportion to the increase in
CMA; i.e. an 8- to 9-fold decline across the range of colony
weights of N. zetterstedtii from 0.1 to 4 g FM. We have recorded
a decline in the relative growth rate and the surplus of photosyn-
thesis minus respiration per colony mass with increasing colony
size in N. pruniforme, but did not find the same strong size de-
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pendence for N. zetterstedtii (Sand-Jensen and Mller, 2011).
We propose that allocation to extracellular products and their dif-
fusive loss to the environment may decline with colony size and
that these processes are relatively more important for the meta-
bolically less active N. zetterstedtii. Reduced loss of exudates
with higher colony size could counteract the expected decline
in growth rate in larger colonies (see section Size effects, diffu-
sive supply and loss of solutes).
As emphasized, a very high proportion of the colony volume
and fresh mass is made up of the water-rich colony matrix com-
pared with the denser Nostoc filaments. Still very high propor-
tions of the carbon content of the colony are bound in the
matrix. In N. commune, polysaccharides in the matrix constitute
.60 % of the colony dry mass (Hill et al., 1997). Among spher-
ical Nostoc colonies, filaments are confined to an outer shell,
which occupies a falling proportion of the volume with increas-
ing colony size, such that the contribution of the matrix to the
total organic content should also increase. If we assume that
the organic carbon:chlorophyll a mass quotient in Nostoc fila-
ments has a typical range of 30 50 at 15 8C and moderate irra-
diances (Geider, 1987; his Fig. 13), we estimate that the matrix
constitutes 18 45 % of the organic carbon mass in the smallest
colonies of N. pruniforme, while it is 37 62 % in large colonies
of 3.5 g FM. The proportion of organic carbon in the matrix is
much higher in N. zetterstedtii, being 50 70 % in small col-
onies weighing a few milligrams and  90 % in large colonies
weighing 3.5 g FM. The smaller proportion of living Nostoc fila-
ments in the colony mass in N. zetterstedtii than in N. pruniforme
will, as already mentioned, result in less photosynthetic produc-
tion of new organic material relative to colony mass and therefore
much lower growth rates for N. zetterstedtii than for
N. pruniforme.
Functional properties of colony matrix
The gelatinous matrix of Nostoc colonies is composed of a
complex mixture of polysaccharides (Bertocchi et al., 1990;
Yousimura et al., 2012), which is responsible for forming and
maintaining the colony shape and protecting the cells against en-
vironmental hazards and pathogens (Tamaru et al., 2005;
Knowles and Castenholz, 2008). It is fascinating, yet unexplored,
how the different colony shapes come about. A variety of disac-
charides, including sucrose and non-reducing trehalose, also par-
ticipate in the protection of cells and matrix (Caiola et al., 1996;
Hill et al., 1997; Potts, 1994, 1999, 2000). Poly- and disacchar-
ides protect membranes and macromolecules against freezing,
heating, desiccation and salt stress (Tamaru et al., 2005;
Sakamoto et al., 2009; Yoshida and Sakamoto, 2009).
 Sand-Jensen Ecophysiology of Nostoc colonies
The formation and accumulation of trehalose in
N. flagelliforme and N. punctiforme are under strict regulation
in response to water limitation (Yong-Hong et al., 2005;
Yoshida and Sakamoto, 2009), presumably because trehalose
is an essential, though costly, product that diverts organic
carbon away from growth processes. The functional importance
of the gel is supported by the observation that strains of
N. commune with no gel and strains where the gel has been phys-
ically removed from the filaments become susceptible to injury
(Tamaru et al., 2005). Addition of exogenous polysaccharides
to endolithic Nostoc sp. and Chlorella sp. increases their toler-
ance to desiccation (Knowles and Castenholz, 2008), probably
by retaining a water layer around the cells and preventing mem-
branes from cracking and macromolecules from undergoing de-
structive conformational changes. The main polysaccharides
observed in the colony gel of N. commune have a strong capacity
for in vitro moisture absorption and water retention, permitting a
.10-fold increase in mass when desiccated colonies become
rehydrated (Li et al., 2011; Sand-Jensen and Jespersen, 2012).
The gel has a strong adsorption capacity for heavy metal ions
that are toxic to cyanobacteria in free ionic form (Bender et al.,
1994; De Philippis et al., 2007; Pereira et al., 2009). Non-reducing
sugars, UV-screening pigments and water stress proteins also
assist in the environmental protection of Nostoc colonies (Hill
et al., 1997; Potts, 1999). In response to increasing intensity and
duration of UV exposure of N. flagelliforme, contents of protective
scytonemin and mycosporine-like amino acids increase (Budel
et al., 1997; Yu and Liu, 2013). A large number (573) of the
genesthat were probed (6903) in N. punctiforme responded signifi-
cantly to UVA exposure, 473 genes being up-regulated and
only 100 being down-regulated (Soule et al., 2013). As expected,
genes encoding antioxidant enzymes and the sunscreen scytone-
min were up-regulated, while many genes involved in the
synthesis of photosynthetic pigments were down-regulated.
Also, Nostoc polysaccharides are capable of reducing oxidative
damage by scavenging superoxide anions and hydroxyl radicals
(Li et al., 2011).
Antibiotic production has been recorded in many Nostoc
species (de Cano et al., 1986; Bloor and England, 1991;
Gromov et al., 1991), including gelatinous colonies (Hameed
et al., 2013). When N. zetterstedtii was homogenized in
water and mixed 1:10 v/v with natural lake water containing
bacteria, DNA synthesis stopped immediately (Sand-Jensen
et al., 2009a). The polysaccharide nostoflan, isolated from
N. flagelliforme, has in vitro and in vivo antiviral effects on a
variety of enveloped viruses, including influenza A virus
(Kanekiyo et al., 2005, 2007, 2008). Symbiotic Nostoc in a
great variety of lichens either contains toxic microcystins or
has the genes for producing them in 12 % of 803 lichen speci-
mens collected worldwide (Kaasalainen et al., 2012). Micro-
cystins occurred in 52 different variants in the 803 lichen
specimens (Kaasalainen et al., 2012); they are small cyclic pep-
tides and are known to be highly toxic to eukaryotes by inhibiting
protein phosphatases (MacKintosh et al., 1990). Microcystins
are active at multiple sites with gradually declining effects on
nitrogenase activity, respiration, photosynthesis and growth of
competing cyanobacteria. Three different classes of peptide
compounds (anabaenopeptin, cryptophycin and nostocyclopep-
tides) could protect the toxin-producing cyanobacteria against
bacteria, viruses, fungi, fish and ducks and could be deadly to
21
mice and dogs around paddy fields (Nowruzi et al., 2012).
Their most important ecological effects, however, involve the in-
hibition of pathogenic viruses and bacteria, potential grazers and
competing phototrophs, not mice and dogs.
S I Z E E F F E C T S , D I F F U S I V E S U P P LY A N D LOS S
OF SOLUTES
As organisms increase in size, diffusion gradually becomes a less
efficient means of supplying external dissolved substances.
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Size effects
Because resource uptake in phototrophic organisms scales to
surface area while resource demand scales to volume and mass,
a first principle dictates that relative growth rate scales to SA:V
and CMA (Nielsen and Sand-Jensen, 1990; Lambers and
Poorter, 1992, 2004). Studies on N. pruniforme and
N. zetterstedtii show that chlorophyll content and photosynthetic
capacity relative to surface area are approximately independent
of colony size because the Nostoc trichomes are mainly confined
to the 1 2 mm-thick outer periphery of the spherical colony,
while its dark central part is mostly devoid of trichomes and
photosynthetic activity (Raun et al., 2009; Sand-Jensen et al.,
2009b). If Nostoc colonies produce hollow spheres with an
outer shell of uniform thickness and a water-filled central
cavity, CMA would be independent of colony size, just as in
the uniformly thick thalli of many macroalgae, plant leaves
and the sheet-like structure of N. commune. This is also almost
the situation for N. pruniforme in summer collections in
Danish lakes, where CMA increases with colony mass to a
power of only 0.05, corresponding to a 1.3-fold increase in
CMA for a 100-fold increase in colony FM (Table 2).
Spherical colonies could in this case maintain an almost un-
altered growth rate with increasing size provided the extent of dif-
fusive supply of limiting DIC and nutrients remained constant
relative to surface area, or that this diffusive flux was not rate-
limiting. Because colonies of N. zetterstedtii are massive,
CMA is high and increases by a power of 0.75 with respect to
colony size and thereby should contribute to a low and declining
growth rate in larger colonies.
For growth performance, it is therefore crucial how resource
fluxes and mass density respond to changes in environmental
conditions and colony size and how photosynthates are allocated
between new cells, colony matrix and protective organic
compounds.
Resource supply by diffusion and turbulence
The diffusive flux (Fa) of a dissolved substance to a unit
surface of a spherical colony is determined by colony size (e.g.
radius r), thickness of the DBL surrounding the surface (d),
the concentration gradient across the boundary layer and the dif-
fusion coefficient (D), according to eqn 1 (Table 3). The flux
is highly dependent on size for small colonies, whereas for
large colonies (r d) the flux gradually approaches the two-
dimensional situation for diffusion to a flat plate where the flux
relative to surface area is independent of colony radius (eqn 2
in Table 3).
22 Sand-Jensen Ecophysiology of Nostoc colonies

The flux of HCO3 was calculated to a unit surface of smooth
spherical colonies like those of N. pruniforme (eqn 1) of variable
size for realistic thicknesses (0.3, 1 and 3 mm) of the DBL in
moving and quasi-stagnant water and differences in concentra-
tions of 0.1, 0.5 and 2.0 mM (mM mmol L 1 mmol cm 3)
between the external water and the colony surface representative
of what would be expected in lakes of low, intermediate and high
HCO3 concentrations, respectively (Fig. 2). Profiles of dissolved
oxygen measured with microelectrodes above the top surface of a
Nostoc parmelioides colony as a function of water velocity mea-
sured 1 mm above the colony suggested DBL thicknesses of
0.1 mm at 6 10 cm s 1, 0.2 0.3 mm at 1 cm s 1 and
.0.4 mm in quasi-stagnant conditions (Fig. 3 in Dodds et al.,
1995). The lower part of the colony resting on the surface has
greater DBL thicknesses. Nostoc zetterstedtii could be expected
to mostly experience concentration differences of 0.1 0.3 mM
HCO3 and N. pruniforme 0.7 2 mM. When N. commune (and
N. flagelliforme) is supplied with rainwater the DIC concentra-
tion is very low. However, when water with dissolved HCO3 is
received from the carbonate-rich soils of the typical growth habi-
tats, the expected DIC range is 0.4 2 mM (Sand-Jensen et al.,
2010; Christensen et al., 2013). Because of the granulated
surface of N. zetterstedtii, the flux is perhaps doubled relative

TA B L E 3. Equations 15 describe the diffusive flux relative to
surface area (Fa), volume (Fv), shell volume (Fvs) and entire colony
(Fcol) of smooth spherical colonies with a given radius (r) of a solute
with diffusion coefficient D across a DBL (d) with the concentration
gradient Cm Co, where Cm and Co are concentrations in the bulk
medium and at the colony surface, respectively. Equation 6 is for a
hollow sphere with an outer shell of thickness Ls. Equations 7 and 8
describe the concentration of a solute at the colony surface (Cr)
produced at a rate of Pv per unit volume of a massive (eqn 7) or
hollow colony (eqn 8) and lost by diffusion to stagnant external
water of zero concentration
No. Equation
1 Fa r 2 (r(r + d) d 1) D (Cm Co) r 1 (1 + r/d) D (Cm Co)
2 Fa d 1 D (Cm Co); (r d)
3 Fa r 1 D Cm; (Co 0)
4 Fcol 4p r D Cm; (Co 0)
5 Fv 3 r 2 D Cm; (Co 0)
6 Fvs r 1 Ls 1 D Cm; (Co 0)
7 C (r) 1/3 Pv D 1 r 2; (Cm 0)
8 C (r) Pv Ls r D 1; (Cm 0)
Equations are derived from Nobel (1983) and Denny (1993).
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to the smooth surface of N. pruniforme, while the flux to the
sheet-like colonies of N. commune is much lower than the flux
to small spherical colonies, but only slightly lower than the
values for large colonies of N. pruniforme, because they physic-
ally approach the two-dimensional diffusion geometry. Only the
diffusive flux of HCO3 has been calculated, because HCO3 con-
stitutes .95 % of the DIC pool in alkaline water (.0.5 mM) and
85 % in soft water (0.1 mM) at air saturation with 0.015 mM
CO2. All tested Nostoc colonies can use HCO3 actively (see
later).
With an HCO3 gradient of 2 mM through a 1-mm-thick DBL,
the potential flux is 1440 nmol C cm 2 h 1 for colonies with a
radius of 1 mm (5 mg FM) and about half that value (790
nmol C cm 2 h 1) for colonies with a 10-fold higher radius of
10 mm (4.5 g FM) (Fig. 2). The potential flux for the two-
dimensional diffusion geometry to a sheet-like structure like
that of N. commune is 720 nmol C cm 2 h 1 and is independent
of size. Fluxes decline 20-fold when the HCO3 gradient is
20-fold smaller (i.e. 0.1 mM across the DBL). In contrast, a
thinner DBL of 0.3 mm increases the flux most for large colonies
because they are more restricted by diffusion geometry than
small colonies. Thus, for a gradient of 2 mM, the calculated
flux increases to 3120 nmol C cm 2 h 1 for a small spherical
colony (radius 1 mm), to 2470 nmol C cm 2 h 1 for a large

10 000
20, 03
20, 10
Carbon flux (nmol DIC cm2 h1)

20, 30
05, 03
1000 05, 10
05, 30
01, 03
01, 10
01, 30
100

10
01 1 10
Colony radius (cm)

F I G . 2. Calculated diffusive flux of HCO3 [J r 2 [r r (r r) D (Cm Co)]; Nobel, 1983] as a function of radius (r) for spherical colonies exposed to three ambient
concentration levels (Cm: 0.1, 0.5 and 2.0 mM HCO3 ) and three levels of boundary layer thickness (DBL: d r r; 0.3, 1.0 and 3.0 mm). The combinations are indi-
cated in the key in the figure, with Cm first and d second. The molecular diffusion coefficient (D) is set at 1.0 10 9 m2 s 1 (Zeebe, 2011) and the HCO3 concentration
at the colony surface is assumed to be zero.
 Sand-Jensen Ecophysiology of Nostoc colonies
DIC uptake (mol g1 colony h1) 25
20
15
10
05
0
05
0 05 10 15
Oxygen release (mol g1 colony h1)
F I G . 3. DIC exchange of N. zetterstedtii with water (open symbols) and with
both water and the colony volume (filled symbols) as a function of oxygen ex-
change with water and colony volume during 18 h of photosynthesis in the
light. The dotted line represents the 1:1 molar exchange of DIC and oxygen.
Colonies were incubated at three DIC levels containing initially 0.99 mM
(circles), 0.29 mM (triangles) and 0.10 mM (squares). After Sand-Jensen et al.
(2009b); reproduced with permission of the Association for the Sciences of
Limnology and Oceanography, Inc.
spherical colony (radius 10 mm) and to 2400 nmol C cm 2 h 1
for a sheet-like structure.
What do these fluxes imply in terms of potential growth rate
and biomass turnover? The answer is different for
N. pruniforme, with carbon mass relative to surface area of
100 mmol C cm 2 for large colonies (radius 10 mm) compared
with 1400 mmol C cm 2 for N. zetterstedtii of similar size.
Assuming an inward flux during 12 daytime hours, a large
HCO3 gradient of 2 mM across a DBL thickness of 1 mm, the po-
tential turnover time of the biomass would be 11 d for
N. pruniforme and 147 d for N. zetterstedtii, not accounting
for night-time respiration, while a small gradient of 0.1 mM
could only permit a potential turnover time of 210 d for
N. pruniforme and 2940 d for N. zetterstedtii emphasizing the
profound influence on carbon turnover of higher CMA for
N. zetterstedtii compared with N. pruniforme. This difference
should be enhanced by N. pruniformes preference for hard-
water lakes, ensuring a higher flux, and N. zetterstedtiis predom-
inant existence in soft-water lakes. Thus, typical turnover times
of N. pruniforme could be of the order of 11 d under suitable con-
ditions in hard-water lakes containing 2 mM DIC for colonies
measuring 10 mm in radius as opposed to 2940 d (8 years) for
N. zetterstedtii of the same size growing in soft-water lakes
with only 0.1 mM DIC available. If we account for the possibility
of a 2-fold higher inward HCO3 flux for N. zetterstedtii because
of the granulated colony surface, the turnover time would drop to
4 years, which is close to the observed values for N. zetterstedtii
(3 years) in the soft-water Lake Varsjo (Sand-Jensen and Mller,
2011). Also, the turnover time of the biomass of N. pruniforme in
laboratory experiments at 15 25 8C and in the shallow water of
the hard-water Lake Esrum (2.5 mM DIC) during early summer
was 10 14 d (Mller et al., 2014; C. Mller and K. Sand-
Jensen, unpubl. data) and resembled the theoretical estimate of
11 d mentioned above. These calculations suggest that account-
ing for DIC concentrations, DBL thickness and CMA permits
sound predictions of growth and biomass turnover of Nostoc
colonies.
23
In addition to DIC, the metabolism and growth of Nostoc col-
onies could be constrained by the supply rate of dissolved inor-
ganic phosphorus (DIP). Nitrogen can be fixed as elementary
N2 and is less likely to be limiting, although growth stimulation
by high external concentrations may occur, particularly for ter-
restrial species with unrestricted access to atmospheric CO2
(see later). Lakes and ponds with Nostoc species mostly have
DIP concentrations in the water column ranging from undetect-
able by standard chemical methods to 0.3 mM (Sand-Jensen
et al., 2009b), though higher concentrations can occur at the sedi-
ment surface. For a DIP gradient of 0.3 mM across a 1 mm thick
20 25 DBL, the estimated influx was 0.22 nmol P cm 2 h 1 for small
colonies (radius 1 mm) and 0.12 nmol P cm 2 h 1 for large col-
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onies (radius 10 mm). These fluxes should permit a turnover time
of the P pool within 6 d for small and 72 d for large colonies of
N. zetterstedtii, supporting the hypothesis that growth is more
likely to be constrained by the supply rate of DIC across gradients
of 0.1 mM HCO3 . These calculations agreed with the observation
that growth was unaffected by P richness in the sediments of soft-
water Lake Varsjo (Sand-Jensen and Mller, 2011).
DIC supply is also important in dense epilithic communities
growing on inert substrata and covered by relatively thick bound-
ary layers (Sand-Jensen, 1983). Thus, short epilithic algal com-
munities in marine waters have DBL thicknesses similar to those
applied for Nostoc colonies and they appear to be constrained by
the DIC flux to photosynthesis despite high HCO3 concentration
(2 mM) in seawater. Larkum et al. (2003) showed that DBLs of
epilithic algal communities covering dead coral surfaces were
0.18 0.59 mm thick under moderate flow (8 cm s 1) and
.2 mm under quasi-stagnant conditions, and they generated
high resistance to DIC influx, limiting primary production.
Independent experiments with epilithic algal communities in
other coral reef environments appeared to support the conclusion
that DIC is a main limiting factor, as elevated nutrient levels had
no effect on primary production and growth (Larkum and Koop,
1997; Miller et al., 1999), while large macroalgae in such
nutrient-poor waters were stimulated by nutrient amendment
(Lapointe et al., 1987).
Resource supply solely by diffusion
The flux to Nostoc colonies may be controlled entirely by dif-
fusion in a stagnant water layer overlying the sediment. A stag-
nant surface layer may exist almost permanently in deep water
with restricted turbulence and periodically in shallow water
during calm weather. The colonies could be buried in a viscous
boundary layer a few centimetres thick overlying the sediment
(Boudreau and Jrgensen, 2001). When the surrounding water
is virtually stagnant and the solute concentration is reduced to
zero at the colony surface, the flux per unit surface area is inverse-
ly scaled to colony radius, emphasizing the immense advantage
of being small (eqn 3 in Table 3). The calculated flux is 720 nmol
C cm 2 h 1 for small colonies (radius 1 mm) in water containing
2 mM HCO3 or half the flux that occurs when DBL is 1 mm thick
(eqn 1). For large colonies (radius 10 mm), however, the flux in
stagnant water is only 72 nmol C cm 2 h 1, or 11-fold lower than
in stirred water with a DBL of 1 mm. The potential biomass turn-
over time for large colonies as described above would then be 120
d for N. pruniforme under stagnant conditions in hard-water lakes
and 32 350 d (89 years) for N. zetterstedtii in soft-water lakes.
24 Sand-Jensen Ecophysiology of Nostoc colonies
Both values are very high, stressing that the water overlying the
sediment is either not entirely stagnant or is enriched with DIC
(CO2 and HCO3 ) from sediment decomposition. Maberlys
(1985) measurements 10 cm above the sediments in a shallow
bay covered by the moss Fontinalis antipyretica showed elevated
CO2 concentrations relative to surface waters between 0.05 and
0.26 mM on 14 different dates. Concentrations are higher directly
on the sediment surface and a near-bed CO2 concentration of, for
example, 0.3 mM CO2 could permit a 6-fold higher influx of CO2
than a HCO3 gradient of 0.1 mM because the diffusion coefficient
of CO2 is approximately twice that of HCO3 (Denny, 1993). The
resulting turnover time could then be 6.3 years for large colonies
of N. zetterstedtii if we assume that the granulated surface
doubles the influx and 3.2 years if the inward flux continues in
both light and darkness. Thus, we cannot exclude that large col-
onies of N. zetterstedtii could survive in an entirely diffusive en-
vironment provided available concentrations of CO2 and HCO3
were markedly higher than 0.1 mM. Small colonies a few milli-
metres in diameter can perform well by diffusive fluxes alone
and are likely to be fully embedded in a diffusive boundary
above the sediment surface.
Resource supply by diffusion relative to colony volume
The diffusive flux through stagnant water to a smooth, spheric-
al colony follows the well-known formula often used for bacteria
(eqn 4 in Table 3; Fenchel et al., 1998) because the colonies are
usually so small relative to the thickness of DBL that the supply
rate is entirely determined by diffusion. When metabolic activity
and the demand for external resources are evenly distributed
throughout the colony volume, the flux relative to unit volume
(Fv) will be inverselyscaled to the second power of radius, empha-
sizing the likelihood of extreme limitation as the organisms grow
in size (eqn 5 in Table 3). The solutions to cope with this rapidly
increasing risk of resource limitation with larger size would be
either to reduce volume-specific requirements for metabolism
(in the case of N. zetterstedtii) or develop a hollow sphere
where all activity is restricted to an outer shell of thickness Ls
and volume 4/3p (r 3 (r Ls)3) 4p (r 0.5Ls)2 Ls. When r
is large relative to Ls, volume is close to 4p r 2 Ls and the flux rela-
tive to volume of the outer shell (Fvs) declines linearly with
colony radius (eqn 6 in Table 3).
Thus, a 10-fold increase in colony radius will reduce the flux
relative to the surface area and volume of a hollow spherical
colony by the same order of magnitude, while the flux relative
to the volume of a homogeneous, solid spherical colony declines
100-fold, emphasizing that if large colonies are indeed solid then
their central parts must be composed of recalcitrant matrix mater-
ial and either have no cells or cells of low metabolism. Because
N. zetterstedtii colonies with a diameter of 10 20 mm absorb
96 % of incident irradiance from the surface to the centre
(Sand-Jensen et al., 2009b), there is no scope for photosynthesis
of Nostoc trichomes in the centre, but there is organic matter
present here that could potentially be used by heterotrophic bac-
teria. The colony matrix is persistent and protected by bacteri-
cides (Sand-Jensen et al., 2009a), however, implying that there
are no or very few active heterotrophic bacteria living within
N. zetterstedtii colonies. The year-long persistence and low res-
piration rates of the colonies in complete darkness support this
implication (see below).
Efflux of solutes
While the large size of gelatinous Nostoc colonies constrains
the influx of external resources, it will also limit the efflux of
valuable dissolved products derived from metabolism within
the colonies. The possible implications of variations in this
efflux have been overlooked so far. When the diffusive loss of
respiratory CO2 and inorganic nutrients (e.g. phosphate) from
the colony is constrained, they can better be retained and recycled
within the colony (see section Size effects, diffusive supply and
loss of solutes). Organic compounds that are produced and to a
great extent released from the trichomes to form the extensive
colony matrix can also be lost to the surrounding water and this
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loss can presumably be reduced in large colonies. Perhaps
more importantly, antibiotics and toxins produced by Nostoc to
reduce the risks of being attacked by harmful bacteria, viruses,
mixotrophic algae and animals can be retained within the
colony and here reach highly effective concentrations for a
limited rate of production. Thus, with increasing colony size a
constant production rate of the protective substance per colony
volume will lead to increasing internal concentrations, or a con-
stant internal concentration can be attained despite a declining
production rate of the protective substance.
The formal analysis shows that a solute produced by a massive
spherical organism at a uniform specific rate per unit volume (Pv)
and lost by diffusion to stagnant surrounding water with a zero
background concentration will have an effective concentration
right at the surface of the colony that increases by the second
power of the radius (eqn 7 in Table 3). If the concentration is
kept constant, the production rate can decline by the second
power of the radius. The mathematical formulas have been
derived from Denny (1993), who treated an analogue biological
example.
Photosynthetic production is constant relative to the surface
area of N. pruniforme and N. zetterstedtii (Raun et al., 2009;
Sand-Jensen et al., 2009b), in accordance with the fact that
photosynthesis is confined to a surface shell of thickness Lsh
and an approximate volume of 4p r 2 Lsh. Because protective sub-
stances derive from photosynthetic products, they could have the
same scaling properties. Thus, inserting shell volume instead of
total colony volume in eqn 7 predicts that the concentration on
the colony surface would increase in proportion to colony
radius (eqn 8 in Table 3). Alternatively, the concentration
would remain constant if the production rate of protective sub-
stances declined in proportion to increasing colony radius.
Therefore, the incorporation rate of inorganic carbon into new
cells, colony matrix and extracellular products should all be
determined as a function of colony size. This would permit
direct determination of turnover rates of cells and colony
matrix and evaluation of the relative cost of producing and releas-
ing extracellular products. Despite extensive and elaborate work
on the biochemistry of Nostoc colonies, this analysis has not been
performed as yet.
FU NCT IO NA L CO NSEQ UE NC ES O F THE U SE
O F LI GH T AN D IN OR GA NI C CA RB ON
Nostoc concentrates and circulates inorganic carbon efficiently
within the large colonies, while the use of light suffers from
 Sand-Jensen Ecophysiology of Nostoc colonies
TA B L E 4. Photosynthesis light variables of three Nostoc species at 15 8C
Species Pmax
2 1 1 1
(nmol O2 cm h ) (mg O2 mg Chl
N. commune 353 376 2.4
N. pruniforme 552 654 2.1
N. zetterstedtii 206 409 0.7
Data are means of measurements in several series.
Pmax, maximum net photosynthesis at light saturation; Icomp, light compensation point.
Sources: N. commune (K. Sand-Jensen, unpubl. data); N. pruniforme (Raun et al., 2009); N. zetterstedtii (Sand-Jensen et al., 2009b).
extensive self-shading and absorption by non-photosynthetic
elements.
Use of light
The large size, long internal light paths and high densities of
trichomes within Nostoc colonies imply that light absorptance
and internal self-shading are high. Mean light absorptance
ranged from 55 % in N. commune to 96 % in N. zetterstedtii
(Table 4). Moreover, because the cells experience high O2 and
low CO2 concentrations within the colonies during photosyn-
thesis, maximum photosynthesis relative to cell chlorophyll
(i.e. the assimilation number), maximum quantum efficiency
of photosynthesis and the ratio of photosynthesis to respiration
should be much lower than among free-living unicells. To the
extent that photons are absorbed by non-photosynthetic pig-
ments and other coloured substances inside the colonies,
maximum quantum efficiency will decline further.
Quantum efficiency was indeed low for N. commune [average
19.1 mmol O2 (mol absorbed photon) 1] and N. zetterstedtii
(11.2 39.9 in three series; Table 4). A similarly low efficiency
[27 mmol O2 (mol photon) 1] was calculated for the thick-
walled, balloon-like green macroalga Codium bursa (Geertz-
Hansen et al., 1994) and microbial mats with extensive absorp-
tion (40 80 %) by structural components (Al-Najjar et al.,
2012). In contrast, quantum efficiencies are much higher for free-
living microalgae [70 120 mmol O2 (mol photon) 1], macroal-
gae and submerged plants [37 79 mmol O2 (mol photon) 1]
(Frost-Christensen and Sand-Jensen, 1992).
Nostoc zetterstedtii had particularly low light-saturated
photosynthesis relative to chlorophyll (average 0.5 0.7 mg O2
mg 1 chlorophyll a h 1) and values were also low for
N. commune (average 2.4) and N. pruniforme (average 2.1).
Likewise, photosynthesis relative to dark respiration (NP:R)
was low for N. zetterstedtii (2.0 5.8) and N. commune
(average 2.5), but not for N. pruniforme (average 10). Again free-
living unicells have higher rates of photosynthesis relative to
chlorophyll, typically between 2 and 20 (Harris, 1978) and
NP:R ratios from 5 to 20 (Harris, 1978; Geider and Osborne,
1992). High self-shading within Nostoc colonies is an important
constraint because photosynthesis at high irradiance increased
4-fold and NP:R increased 3-fold when self-shading was
reduced by cutting colonies of N. zetterstedtii into 1 2-mm
pieces (Sand-Jensen et al., 2009b).
Light attenuation by coloured substances, structural sub-
stances and dead or senescent cells within the colonies competes
25
Pmax/dark respiration Icomp (mmol m 2 s 1) Quantum efficiency Absorption (%)
(mmol O2/mol photons)
h )
2.5 9.5 19 55
10 90
2.0 5.8 19.3 20 96
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with photosynthetic pigments for photons, and thereby lowers
quantum efficiency and increases the light compensation point
(where photosynthesis outweighs respiration) compared with
free-living unicells (Krause-Jensen and Sand-Jensen, 1998;
Al-Najjar et al., 2012). Thus, light compensation points of
N. zetterstedtii and N. commune (9.5 19.3 mmol photon m 2
s 1) exceeded those of most unicells (typically 0.8 9 mmol
m 2 s 1; Langdon, 1988) and thin thalli and leaves of macroal-
gae and submerged plants (typically 2 12 mmol m 2 s 1;
Sand-Jensen and Madsen, 1991). This comparison supports the
hypothesis that inefficient light use in Nostoc colonies combined
with higher respiratory costs of producing and maintaining the
colony should lead to higher minimum light requirements for
survival than for unicells, characeans and plants with thin photo-
synthetic tissues. Observations of lower light availability at the
maximum depth limits of characeans, filamentous algae and
mosses (3.1 7.8 % of surface light) than of N. pruniforme and
N. zetterstedtii (9.4 12.5 % of surface light) in two Danish
lakes support this hypothesis (Table 7 in Sand-Jensen et al.,
2009b).
Use of inorganic carbon
The diffusive supply of DIC from outside is constrained by the
large size, low SA:V ratio and high density of filaments in the
1 3-mm-thick outer shells of gelatinous Nostoc colonies
without direct contact with the surrounding medium. The effect-
ive diffusion path involves both the diffusive boundary layer sur-
rounding the colony and the path through the gel to the sites of
fixation in the Nostoc filaments, but the internal resistance has
not been accounted for.
While increasing the resistance to influx of gas and solutes, the
gel will also slow effluxes and contribute to the retention of night-
time respiratory CO2 and facilitate accumulation of internal DIC
pools. To ameliorate carbon limitation of photosynthesis and
prevent excessive photorespiration, Nostoc colonies, like other
cyanobacteria (Badger and Price, 1992, 2003), must attain rea-
sonably high quotients of CO2 to O2 at the site of Rubisco activity
within the cells. To achieve this, Nostoc colonies must be able to
extract high proportions of the DIC pool in the water and have low
compensation points of CO2 and HCO3 (Sultemeyer et al.,
1998). The ability to accumulate DIC pools within the colony
above immediate needs for photosynthesis and to retain respira-
tory CO2 from night-time respiration for later use during daytime
photosynthesis could assist the carbon supply even further. Such
efficient extraction and accumulation of carbon are known for
26 Sand-Jensen Ecophysiology of Nostoc colonies

free-living cyanobacteria (Kaplan et al., 1980; Sultemeyer et al.,
1998) and phototrophs in lichen photosynthesis (Green et al.,
1994), and the potential may also exist for Nostoc trichomes
buried within the colony gel.
We found a high mean extraction capacity, of between 74 and
82 % of the initial DIC pool in the water (0.3 1.1 mM), during
20 h of photosynthesis in closed bottles with N. commune and
N. zetterstedtii where DIC was gradually depleted and pH and
O2 increased over time (Table 5). The mean extraction capacity
was lower (58 %), but probably not fully expressed, in experi-
ments with N. pruniforme, though it still reflected very active
DIC uptake. Efficient DIC use is also known for
quotient of 1.0, we determined that a DIC pool of 16.3 24.5
mmol g 1 FM was available for photosynthesis in colonies of
N. zetterstedtii and about half this pool size (5.8 10.2 mmol
g 1 FM) was available in N. commune and N. pruniforme
(Table 7). Direct measurements of the internal DIC pool in
N. zetterstedtii yielded 19.0 24.7 mmol g 1 FM for colonies
incubated in water containing 0.15 mM DIC and these measure-
ments fully corresponded with estimates derived from the
O2 DIC balance (Sand-Jensen et al., 2009b). Measured DIC
concentrations were 150-fold higher within the colonies
(average of cells and matrix) than in the external water and I
expect that DIC concentrations would be even higher within

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N. flagelliforme and edible Nostoc Ge-Xian-Me (Gao and Zou, the cells, resulting in a higher accumulation quotient than 150
2001; Qiu et al., 2004; Ye et al., 2012). Efficient active use of between cells and water. Accumulation quotients of 500 1000
external DIC by N. commune and N. zetterstedtii was also are known for free-living cyanobacteria (Kaplan et al., 1980;
reflected in the high final pH in the surrounding water (10.60 Badger and Price, 2003).
11.11) and the low final concentrations of HCO3 (6 75 mM) When I accounted for exchange of DIC during photosynthesis
and CO2 (0.3 7 nM) as measures of HCO3 and CO2 compensa- and respiration with both the colony matrix and the external
tion points (Table 6). Concentrations of HCO3 and CO2 at the water, I found mean molar exchange quotients of O2 relative to
sites of Rubisco within the cells must be markedly higher and DIC in the light (1.19) and in the dark (0.96) close to 1.0 in
molar quotients of O2 to HCO3 + CO2 lower than in the sur- experiments with N. zetterstedtii (Fig. 3). These direct measure-
rounding water (8 60) to ensure net carbon fixation and ments confirmed that a high proportion of DIC for photosyn-
reduce photorespiration (Kaplan et al., 1980; Price et al., 2008). thesis in the light was consumed from a DIC pool in the
The ability of all three Nostoc species to accumulate DIC to colony, while in the dark a high proportion of respiratory DIC
much higher concentrations within the colony than in the exter- accumulated within the colony (Fig. 3). The measured internal
nal water is supported by the relatively high rates of O2 release by DIC pools in N. zetterstedtii could support maximum photosyn-
photosynthesis for 20 h in water of low DIC (0.1 mM, 0.1 thesis for 11 and 23 h in 10- and 20-mm-diameter colonies, re-
mmol cm 3) without any appreciable uptake of external DIC. spectively, without uptake of external DIC. When N. commune
Assuming that O2 is produced and inorganic C consumed from was incubated for 20 h in relatively DIC-rich water containing
the colony itself during extended photosynthesis with a molar initially 1.1 mM, it accumulated 16.7 18.7 mmol DIC g 1 FM
of colony in excess of the immediate requirements for photosyn-
thesis, while it consumed 9 10.2 mmol DIC g 1 FM from the
colony to support photosynthesis when the initial DIC concentra-
TA B L E 5. Extraction capacity of the initial DIC pool (%) of three
tion in the water was only 0.2 mM (K. Sand-Jensen, unpubl. data).
Nostoc species during photosynthesis for 20 h in closed bottles
Photosynthetic ATP production provides ample energy for active
Mean + s.e. No. of
transport of HCO3 , and at least N. commune can consume DIC
Species (%) measurements Source for photosynthesis and at the same time accumulate DIC
within the colony in the light when sufficient external DIC is
N. commune 72 + 4 16 K. Sand-Jensen available. Thus, the species does not solely rely on accumulation
82 + 3 12 (unpubl. data) of respiratory DIC in the dark but can also take up external HCO3
N. pruniforme* 56 + 3 5 Raun (2006); Raun for use in the following light period.
et al. (2009)
N. zetterstedtii 74 + 9 16 Sand-Jensen et al. The ability of Nostoc colonies to build up substantial internal
(2009) DIC pools had been overlooked in the past. This ability means
that photosynthesis is less dependent on the immediate external
*Full extraction capacity probably not realized. DIC supply. Nonetheless, all three species were limited by the

TA B L E 6. Final pH, final DIC, HCO3 and CO2 and final O2/HCO3 ratio of the four Nostoc species after 20 h of photosynthesis in
closed bottles. Ranges are shown from several experimental series with many replicates

Initial DIC Final DIC Final HCO3 Final CO2

Species (mmol L 1) Final pH (mmol L 1) (mmol L 1) (nmol L 1) O2/HCO3 No. of series

N. commune 106310 10.6811.11 1290 6 34 1 7 960 5

N. pruniforme* 570 10.6 260 110 ,30 5 1
N. zetterstedtii 149162 10.9411.06 77174 13 35 3 10 18 29 4
N. flagelliforme 3300 10.8

*Maximum final pH and minimum final DIC, HCO3 and CO2 probably not attained.
Sources: N. commune (K. Sand-Jensen unpubl. data); N. pruniforme (Raun, 2006; Raun et al., 2009); N. zetterstedtii (Sand-Jensen et al., 2009); N. flagelliforme
(Gao and Zou, 2001).
 Sand-Jensen Ecophysiology of Nostoc colonies 27

TA B L E 7. DIC consumed from the colony volume of three Nostoc external DIC supply as photosynthetic rates at 0.1 mM DIC exter-
species during extended light periods (20 h) nally were 20 % of the maximum rate for N. commune, 11 %
for small, 13 % for medium-sized and 25 % for large colonies
DIC consumed of N. pruniforme and 60 % for N. zetterstedtii (Figs 4 and 5).
(mmol C g 1 fresh
weight)
As the internal DIC pool in N. zetterstedtii colonies is gradually
exhausted during 18 h of light incubation in closed bottles, rea-
Species Mean 95 % CL Source lized mean rates of O2 production are 1.8-fold lower in water ini-
tially containing 0.29 mM compared with water initially
N. commune 5.8 1.1 K. Sand-Jensen (unpubl. data) containing 0.99 mM DIC (Fig. 3). In N. pruniforme, which has
6.5 0.5 higher rates of photosynthesis and DIC requirements per
9.0 1.7
10.2 2.3 surface area than N. zetterstedtii and apparently 2-fold lower in-
N. pruniforme 8.67 1.33 Raun (2006), Raun et al. (2009) ternal DIC pools, the dependency of photosynthesis on external
DIC was stronger, showing half-saturation constants of 0.78 mM

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8.57 0.84
N. zetterstedtii 16.3 2.9 Sand-Jensen et al. (2009) for small colonies (0.1 g FM) and 1.24 mM for larger colonies
24.5 5.6
(2.5 g FM) in 2-h experiments under well-stirred conditions
(Fig. 4). Large colonies have higher rates of photosynthesis
(164 nmol O2 cm 2 h 1) at near-zero external DIC than small
2 0 colonies (55 nmol O2 cm 2 h 1), probably because of a larger in-
Small colonies ternal DIC pool (being scaled to colony volume) relative to
photosynthetic activity (being scaled to colony surface area).
1 5
The more favourable SA:V ratio in small colonies causes photo-
synthesis to increase more steeply with increasing external DIC
1 0 (a-CO2, 319 nmol O2 cm 2 h 1 for a 1-mmol L 1 increase in ex-
ternal DIC) than in large colonies [a-CO2, 197 nmol O2 cm 2 h 1
(mmol DIC L 1) 1] (Fig. 4).
0 5 The terrestrial and semi-terrestrial N. commune and
N. flagelliforme can use CO2 and HCO3 under water and CO2
Net photosynthesis (mg O2 mg chl1 h1)

alone in air. Under water, N. commune experiences limitation

2 0 of photosynthesis at low DIC as rates rise to at least 1 mM. The
Medium-sized colonies diffusion coefficient of CO2 in air is 2 104 times higher
than that of HCO3 in water, and for the same DBL thickness
1 5 and with complete DIC depletion at the colony surface the CO2
gradient is 3050 times lower in air (external CO2 0.02 mM)
than in water containing 0.6 1.0 mM HCO3 . Thus, the potential
1 0
flux would be substantially higher (500- to 700-fold) in air than in
water through a diffusive boundary layer of the same thickness
05 overlying the colony surface, but this is followed by the same re-
sistance through the colony to the Nostoc filaments in both terres-
trial and aquatic habitats. At high irradiance and twice daily
2 0
Large colonies
Net photosynthesis (nmol O2 cm2 h1)

1 5 800
N. commune
N. pruniforme
1 0 600
N. zetterstedtii

0 5 400

0 200
0 1 2 3
External DIC (mM)
0
F I G . 4. Mean net photosynthesis (+ s.d. of four replicates) of small (0.18 g 0 1000 2000 3000
FM), medium (0.84 g FM) and large (2.71 g FM) spherical colonies of
N. pruniforme as a function of external DIC concentration (.95 % HCO3 ). HCO3 concentration (mol L1)
The apparent half-saturation constant for small, medium and large colonies
was 0.78, 0.78 and 1.24 mM HCO3 , the initial linear slope of net photosynthesis F I G . 5. Mean DIC uptake during photosynthesis in high light as a function of
at low limiting DIC concentration was 319, 305 and 197 nmol O2 cm 2 h 1 (1 mean DIC concentration in the water for N. commune, N. pruniforme and
mmol DIC L 1) 1 and photosynthesis extrapolated to zero DIC was 55, 72 and N. zetterstedtii. Sources: N. commune (K. Sand-Jensen, unpubl. data);
164 nmol O2 cm 2 h 1. From Raun (2006). N. pruniforme (Raun et al., 2009); N. zetterstedtii (Sand-Jensen et al., 2009b).
28 Sand-Jensen Ecophysiology of Nostoc colonies
watering, growth of N. flagelliforme was doubled in
CO2-enriched air (1500 ppm) compared with atmospheric air
(350 ppm at the time; Gao and Yu, 2000). The realized photosyn-
thetic rate of N. commune was the same in stirred air-saturated
water of 1 mM DIC (0.98 mM HCO3 + 0.02 mM CO2) as in atmos-
pheric air with 0.02 mM CO2 (Sand-Jensen and Jespersen, 2012).
Thus, the optimal habitat of N. commune could be wet soils where
water and inorganic carbon and nutrients are supplied to the
lower colony surface, while CO2 and light are supplied to the
upper surface. This is the habitat in 1-cm-deep water-filled
depressions in the limestone pavements where we have observed
massive growth of N. commune (Sand-Jensen et al., 2010).
TOL E R A N C E O F E N V I RO N M E N TA L E X T R E M E S
Freshwater Nostoc species do not experience extreme tempera-
tures like those experienced by terrestrial species. Active growth
of N. commune, N. pruniforme and N. zetterstedtii is, nonethe-
less, confined to the same temperature range (0 35 8C).
Temperature and desiccation tolerance
The terrestrial and semiterrestrial species N. commune and
N. flagelliforme are exceptional in their tolerance of alternating
freezing and thawing and of desiccation and rehydration at
varying temperatures in their open habitats, located from the
Arctic to tropical regions (Li, 1991; Sand-Jensen and Jespersen,
2012). In contrast, the freshwater species N. pruniforme and
N. zetterstedtii are not exposed to freezing and drying or to
extreme temperatures in their temperate and sub-Arctic environ-
ments. Nonetheless, it appears that active growth of all four
species is confined to the same temperature interval between 0
and 35 8C and that the fastest growth takes place around 25 8C
(Mller et al., 2014).
Both N. commune and N. flagelliforme are widely distributed
species in arid and semiarid bare land throughout the world (Li,
1991; Dodds et al., 1995). Nostoc commune survives annual
temperature ranges of  60 to 25 8C under Arctic conditions
and 30 to 50 8C under temperate conditions (Davey, 1989;
Sand-Jensen and Jespersen, 2012). Surface temperatures in the
open habitats of N. flagelliforme can reach 78 8C in summer
and 40 8C in winter (Li, 1991). Both species can survive
months or years of frost and drought as inactive desiccated
crusts, and within minutes to hours can reactivate ion exchange,
respiration, photosynthesis and N fixation when water and suit-
able temperatures again become available (Scherer et al.,
1984; Satoh et al., 2002).
Experiments with the temperate species N. commune con-
firmed that photosynthesis and respiration were maintained
after 36 h of desiccation of wet or already dry specimens at tem-
peratures from 269 to 70 8C, while temperatures above 70 8C
led to death (Sand-Jensen and Jespersen, 2012). During repeated
daily cycles of drying for 18 h at 18, 20 and 40 8C and rewetting
for 6 h at 20 8C, N. commune retained its photosynthetic capacity
at 18 and 20 8C but died at 40 8C, presumably because of the
greater costs of repair of macromolecules at this higher tempera-
ture, which would generate stronger thermal damage than lower
temperatures (Sand-Jensen and Jespersen, 2012). In 24-day-long
experiments under permanently submerged conditions,
N. commune also died at 45 8C but survived at 35 8C; growth
peaked at 25 8C and declined at the lower temperatures of 15
and 5 8C (Mller et al., 2014). Annual carbon fixation of rehy-
drated populations of N. commune from an Antarctic dry valley
was also a strong positive function of increasing temperatures
above zero and up to 20 8C, the highest temperature tested
(Novis et al., 2007). Thus, there was no obvious sign of tempera-
ture adaptation or acclimation across geographical ranges.
Nostoc flagelliforme, like other terrestrial Nostoc species
(Davey, 1989; Dodds et al., 1995; Sand-Jensen and Jespersen,
2012), showed great heat resistance when dry, while it was
more susceptible when wet or immersed (Mei and Cheng,
1990). The regular protein pigment structure of photosystem I
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complexes was destroyed at 70 and 80 8C (Hu et al., 2005).
Pretreatment of wet N. flagelliforme at 65 8C led to death and tem-
peratures above 45 8C stopped photosynthesis (Mei and Cheng,
1990). The exact temperature effect on survival of N. commune
and N. flagelliforme apparently depends on the duration and fre-
quency of heat exposure and on the length of time available for
repair of cellular damage between heat exposures (Sand-Jensen
and Jespersen, 2012). Likewise, a longer time was required for
maximal photosynthesis and respiration to recover after rehydra-
tion of specimens subjected to extended periods of desiccation
(Scherer et al., 1984; Potts, 1994; 2000; Qiu and Gao, 1999).
The recovery of rehydrated N. flagelliforme was light-dependent
(Gao et al., 1998) and required potassium (Qiu and Gao, 1999),
stressing that a suite of energy-demanding metabolic processes
involving expensive protein and lipid synthesis and various cellu-
lar repair systems restores cellular structure and catalytic capacity
(Angeloni and Potts, 1986; Taranto et al., 1993).
Desiccation is not an issue for freshwater species of Nostoc
(except when they are washed ashore) and temperature variabil-
ity is also much lower in freshwater than in terrestrial habitats. In
Danish lakes, for example, the annual temperature range is typic-
ally 0 25 8C in shallow water and 2 15 8C in deep water (Mller
et al., 2014). Nonetheless, the temperature dependence of long-
term growth of N. pruniforme and N. zetterstedtii resembled that
of hydrated N. commune in that all three species grew fastest at
25 8C and many times slower at 5 8C, while 45 8C led to die-off
(Fig. 6 in Mller et al., 2014). So while terrestrial Nostoc
species possess extraordinary desiccation tolerance there is no
major difference in the relationship of active growth with tem-
perature in terrestrial and aquatic specimens from north-
temperate localities, though we cannot rule out that different
relationships of growth with temperature have evolved in speci-
mens living under Arctic or tropical climates. Because, under tem-
perate conditions, temperature ranges between winter and summer
are also profound it would not be a great surprise, however, if these
relationships remained constant across geographical ranges. High
temperature requirements (20 8C) of maximum photosynthesis
of N. commune from an Antarctic valley with very short frost-free
periods support the latter suggestion.
pH and salt tolerance
Being photosynthetic organisms capable of using both free
CO2 and HCO3 at high efficiency, all four Nostoc species can
push pH in the external water above 10.5 and in some cases
even above 11 (Table 6). Exposure of rehydrated N. commune
to a pH gradient for 36 h also showed that it tolerated pH
ranging from 3 to 10, while it died at pH 2 (Sand-Jensen and
 Sand-Jensen Ecophysiology of Nostoc colonies
Jespersen, 2012). Terrestrial Nostoc species should be able to
survive exposure to rainwater of low pH (typically 3.8 4.8)
and only very acidic habitats receiving sulphuric acid from the
oxidation of metal sulphides are likely to be below pH
3. Therefore, when it comes to direct pH tolerance most terrestrial
habitats should be open to colonization by Nostoc. Although
N. commune can grow between garden tiles and on open sand
soils fed by rainwater, it appears to prefer clay soils and calcar-
eous rocks of high pH, perhaps because available HCO3 can
support photosynthesis during prolonged illumination. The re-
quirement of HCO3 for photosynthesis of aquatic Nostoc is ac-
companied by a preference for a pH above 7 in the water
because of the buffering and alkalinization effect of HCO3 .
Thus, at air saturation, freshwaters with pHs of 6.5, 7.5 and 8.5
have HCO3 concentrations of 0.05, 0.5 and 5 mM, respectively
(Stumm and Morgan, 1981).
Because N. commune and N. flagelliforme often grow in arid
habitats with salt accumulation they must be tolerant. Indeed,
N. commune retained its photosynthetic capacity upon exposure
to alkaline freshwater enriched with NaCl to 20 g kg 1, but not
30 g kg 1 as in oceanic water (Sand-Jensen and Jespersen,
2012). The closely related N. flagelliforme was also salt-resistant,
but photosynthesis, respiration and photosystem II activity
declined upon exposure to .12 g NaCl kg 1 (Ye and Gao,
2004; Yong-Hong et al., 2005). Salt resistance is probably
based on the formation of sucrose and trehalose, both of which
accumulate under desiccation and exposure to low salt concen-
trations (Sakamoto et al., 2009) and therefore play a dual role.
Tolerance of freezing and desiccation is extraordinary, while
salt tolerance is modest. Other cyanobacteria are more salt-
tolerant, because of the synthesis of glucosylglycerol in
species of moderate tolerance and glycine betaine and glutamate
betaine in species showing high tolerance (Mackay et al., 1984).
E COLO GI CAL A DA P TAT I ON S AN D S T R AT E G IE S
Colonial Nostoc species live in resource-poor environments and
succumb in competition with tall macroalgae and plants under
richer conditions.
All four colonial Nostoc species discussed here live in
resource-poor environments with a limited supply of water and
inorganic nutrients on land and a limited supply of nutrients
and DIC under water. Phosphorus could be a limiting nutrient
for growth but experimental tests are lacking. All species
can fix elemental N2, but this is a costly process that requires a
rare co-factor (molybdenum). In suspension culture with
N. flagelliforme, the variable availability of nitrogen and phos-
phorus influences the production of new cells and extracellular
polysaccharides (Fei et al., 2012).
The terrestrial N. commune and N. flagelliforme grow on
nutrient-poor, sparsely vegetated or bare soils and rock surfaces
that alternate between being wet and dry. From April to
September on the open limestone alvar on Oland, Sweden, mea-
surements of humidity and temperature suggested that popula-
tions of N. commune were rehydrated and active at least 26 %
of the time (Sand-Jensen and Jespersen, 2012). From October
to March, populations were probably active during larger parts
of the frost-free time.
29
Maximum recorded growth rates of N. commune floating on
water and exposed to atmospheric air under laboratory conditions
were 0.0500.056 d 1 at 1525 8C and markedly lower, at
0.0200.025 d 1, at 5 and 35 8C (Mller et al., 2014). These
growth rates correspond to doubling times of dry mass between
12 and 35 d. Growth rates are intermediate when compared with
the extremely low rates (0.0008 d 1) of oligotrophic
N. zetterstedtii and high rates (0.20.4 d 1) of nutrient-
demanding thin macroalgae (e.g. Cladophora spp. and
Enteromorpha spp.; Sand-Jensen and Borum, 1991; Nielsen and
Sand-Jensen, 1991). According to Grimes (1979) classification
of plant growth strategies in relation to resource richness and dis-
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turbance, the stress strategy (S) of N. zetterstedtii is linked to
resource-poor, stabile habitats, the ruderal strategy (R) of thin
green macroalgae is linked to resource richness and high disturb-
ance, and the competitive strategy (C) of thick macroalgae is
linked to resource richness and low disturbance. Resource-poor,
highly disturbed habitats do not, in Grimes concept, support a
viable strategy because the growth rate is very low when resources
are limiting and disturbance leads to frequent loss of biomass, thus
preventing long-term survival. However, N. commune (and
N. flagelliforme) can survive disturbance by desiccation, freezing
and extreme temperatures with restricted biomass loss in a quies-
cent stage and possesses a viable strategy in the resource-poor, dis-
turbed terrestrial habitats that Grime considered unoccupied, at
least by higher plants. A suite of drought-resistant lichens,
mosses and a few flowering plants are capable of surviving in
the same resource-poor, disturbed habitats as N. commune and
N. flagelliforme (Sand-Jensen et al., 2010) and could be classified
as belonging to a mixed SD strategy, where S represents toler-
ance of limiting resources and D represents resistance to disturb-
ance. Their biomass survives harsh physical conditions that are
detrimental to most other phototrophs. Likewise, in the marine en-
vironment, crust-forming coralline red algae are adapted to an ex-
tremely low supply of light or nutrients, tolerate high disturbance
by wave action and avoid intensive grazing, and thus have a viable
SD strategy with extremely slow growth and biomass turnover
(Littler and Littler, 1980; Littler et al., 1986).
The freshwater N. zetterstedtii grows in soft-water, oligotroph-
ic lakes and faces the strongest constraints on nutrients and DIC
supply. Nostoc pruniforme grows in mesotrophic, alkaline lakes
of higher DIC concentrations, where nutrients remain low from
late spring to early autumn due to nutrient uptake by phytoplank-
ton and other benthic phototrophs (Sand-Jensen et al., 2009b).
Because both species are perennial, we cannot dismiss the untest-
ed possibility that they can take up nutrients in excess during the
richer winter season for later use during summer when organic
production by photosynthesis is high. This mechanism has been
shown for perennial marine macroalgae (Chapman and Cragie,
1977; Lundberg et al., 1989; Lobban and Harrison, 1997). The
persistence of Nostoc colonies may therefore offer an opportunity
to withdraw external nutrients and produce photosynthates when
conditions are favourable and store them within the colony until
they are needed for growth and survival. The large size and low
SA:V of the colonies can promote internal circulation of valuable
substances and reduce their diffuse loss to the environment, as
previously discussed (see section Functional consequences of
the use of light and inorganic carbon).
Nostoc zetterstedtii is exceptional among aquatic phototrophic
organisms due to its exceedingly low rates of growth, respiration
30 Sand-Jensen Ecophysiology of Nostoc colonies
and mortality as a consequence of the large spherical colony size
and high CMA (Sand-Jensen and Mller, 2011). Because direct
selection for low growth rate is not a viable evolutionary strategy
(Lambers et al., 2008), low growth rate should, as emphasized, be
regarded as an indirect consequence of high CMA and invest-
ment in a large arsenal of organic compounds as protection
against environmental hazards, grazers and pathogens. The
maximum growth rate of N. zetterstedtii in laboratory experi-
ments was only 0.78 10 3 d 1, corresponding to a mean doub-
ling time of colony biomass of 2.4 years (Mller et al., 2014).
This value resembled the annual mean doubling time of 2.6
3.3 years in field experiments (Sand-Jensen and Mller, 2011).
With such low growth rates, the largest colonies, measuring
7 cm in diameter, are 24 31 years old. We found no senescence
and mortality in the examined population of colonies in the field
during 13 608 incubation days. This finding implies that the mor-
tality rate was ,7.3 10 5 d 1 and longevity .24 years, in ac-
cordance with the high age of the largest colonies. Macerated
N. zetterstedtii colonies diluted 10-fold in water and added to a
sample of lake bacteria immediately stopped all cell division
(Sand-Jensen et al., 2009a). Likewise, no grazing by small
macroinvertebrates has been observed, and large potential
grazers, such as crayfish, fish and swans, are probably unable
to consume the large, firm colonies. Recent experiments
showed that colonies survived for 14 months in complete dark-
ness at 5 8C with integrity of photosynthesis and fresh mass of
the colonies and extremely low daily respiration rates of
0.21 mmol C (mol organic carbon) 1 corresponding to a half-
time of the colony carbon biomass of 9 years (K. Sand-Jensen,
unpubl. data). I anticipate that Nostoc cells can utilize organic
compounds from the colony matrix for basal respiration and
long-term survival.
The ecological strategy of N. zetterstedtii is therefore an
extreme example of a stress-selected species living in a very
resource-poor environment, growing extremely slowly, being
highly persistent thanks to efficient physical and chemical pro-
tection against pathogens and grazers, and having efficient recir-
culation of inorganic carbon (Fig. 3) (Sand-Jensen et al., 2009b).
Measured growth rate in dry mass of N. zetterstedtii was
10-fold lower than rates predicted from general allometric rela-
tionships of growth rate with CMA of aquatic macroalgae
(Markager and Sand-Jensen, 1996) and, likewise, of predicted
growth rate with the thickness of photosynthetic tissue for a
broad range of terrestrial and aquatic plants (Nielsen et al.,
1996). This finding suggests that there are extra constraints on
the growth of N. zetterstedtii linked to limited DIC supply and/
or production of the colony matrix and the protective com-
pounds. The proposed reduced loss of organic solutes from
large colonies to the surrounding water has not been tested as
yet, though it is plausible given the ability of N. zetterstedtii to
maintain colony integrity and fresh mass during 14 months in
darkness (K. Sand-Jensen, unpubl. data).
Nostoc pruniforme grows in oligo-mesotrophic lakes richer in
DIC than the main habitats of N. zetterstedtii, and its growth,
mortality and ecological strategy are also markedly different.
Maximum growth rate of N. pruniforme in laboratory experi-
ments was 75-fold higher than that of N. zetterstedtii, corre-
sponding to a biomass doubling time of 12 d (Mller et al.,
2014). Growth rates in a clear-water, DIC-rich lake during
early summer resembled these laboratory rates (Mller and
Sand-Jensen, unpubl. data 2013). A high DIC (2.0 mM) in the
lake water was required to sustain a biomass turnover of 12 d,
while incubation in low-DIC water (0.2 mM) reduced net photo-
synthesis 4-fold and prolonged biomass turnover (Mller et al.,
2014). Field studies confirmed that N. pruniforme periodically
undergoes mass mortality, presumably by attack of microbial
pathogens. In terms of the ecological strategy, N. pruniforme
has species traits belonging to a mixture of ruderal-, stress- and
stress-disturbance-selected strategies. The expression of these
traits is plastic depending on whether the species lives in cold,
oligotrophic freshwaters and forms very large and old colonies
or colonies that live for shorter periods in warmer, mesotrophic
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lakes and reach smaller sizes.
All four Nostoc species have one important trait in common.
They live directly on the ground surface and are unable to
survive in competition with tall, dense terrestrial or aquatic vege-
tation. This is a main reason why they are primarily confined to
resource-poor habitats where tall, dense vegetation cannot
develop. The Nostoc species therefore face the risk of disappear-
ing when their habitats are subjected to nutrient enrichment. The
freshwater Nostoc organisms in Scandinavian lakes have been
further threatened by large-scale leaching of dissolved humic
substances from low-buffered, acidic soils.
O UT LOO K
Most studies on the ecophysiology of phototrophs have focused
on relatively fast-growing species from resource-rich habitats
(Larcher, 2003; Lambers et al. 2008). With this review on
large gelatinous Nostoc colonies I call attention to these competi-
tively inferior species from nutrient-poor habitats. I introduce
terrestrial and semiterrestrial Nostoc species possessing an
unprecedented ability to enter quiescent, desiccated or frozen
stages and rapidly resume metabolism when rehydrated. The
molecular, biochemical and physiological sequences leading
to quiescence and reactivation need to be fully explored and
the metabolic costs of frequent desiccation, freezing and rehy-
dration and substantial production of extracellular polysacchar-
ides need quantification in the future. There is strong
molecular, biomedical and ecological interest in these processes.
The rare freshwater species N. zetterstedtii has unprecedented
low rates of metabolism, growth and mortality in its DIC- and
nutrient-poor lake habitats. Its ability to take up external DIC
and recirculate DIC within the colony is profound. It is challen-
ging to determine whether the large colony size also creates
advantages for effective circulation of nutrients within the
colony and strong antibiotic effects for relatively low production
costs. Future studies should also unravel fluid dynamics and nu-
trient and DIC conditions in the neglected near-bed habitats of
N. pruniforme and N. zetterstedtii on lake sediments. This
effort is needed in order to put the presented diffusion models
for uptake and loss of solutes from spherical colonies of different
size into proper physical and chemical perspectives in nature.
As free-living organisms and as symbionts in lichens, Nostoc
species are both pioneers and permanent members of the vegeta-
tion of deserts, semideserts, dry grasslands and rock surfaces
ranging in geographical distribution from polar to tropical
regions. Their N input to these biomes can be of utmost import-
ance (Dodds et al., 1995; Holst et al., 2009; Caputa et al., 2013).
In a carefully mapped Low-Arctic tundra landscape, N2 fixation
 Sand-Jensen Ecophysiology of Nostoc colonies
by cyanobacteria (0.68 kg ha 1) was twice the annual wet depos-
ition of nitrogen (Stewart et al., 2011). It remains unexplored
how the high water-absorbing capacity and N2 fixation of
Nostoc can facilitate the colonization of bare or newly exposed
mineral surfaces by mosses and higher plants, thereby forming
more stable vegetation and more organic soils. With the exposure
of new mineral surfaces behind retreating glaciers on a warming
Earth, this ecosystem service of Nostoc deserves future attention.
ACK N OW L E DG E M E N T S
This work was supported by grants from the Willum Kann
Rasmussen Foundation to the Centre of Excellence for Lake
Restoration Research (CLEAR) and from the Carlsberg
Foundation. I thank Mikkel Rene Andersen, Michael Kuhl and
Claus Lindskov Mller for help and the referees for constructive
suggestions.
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