Cyanobacteria-dominated biofilms: A high quality food resource for intertidal grazers

Abstract

Hong Kong rocky shores are dominated by cyanobacterial biofilms composed of a diversity of species. Thirteen common species, belonging to seven genera, were isolated in pure culture in MN+ and MN– media under defined growth conditions from a semi-exposed shore in Hong Kong. The nutritional values (i.e., protein, carbohydrate and calorific value) of these 13 species were determined. All species showed high nutritional quality in terms of protein, carbohydrate and calorific value, however, overall nutritional value varied between the species. Species of Spirulina and Phormidium were most nutritious (highest nutritional values) whereas species of Calothrix and Lyngbya were the least nutritious. Microphagous molluscan grazer density and diversity were relatively high at the study site, despite the seemingly low biomass (as assessed by chlorophyll a concentration) of the biofilm. It is suggested that the high nutritional quality of cyanobacteria, together with their fast turnover rates can support high levels of secondary production (biomass of grazers). The high nutritional quality of cyanobacteria on tropical, cyanobacteria-dominated, rocky shores is therefore of great importance in the benthic food web.

Full text

Hydrobiologia 512: 89–95, 2004.
P.O. Ang, Jr. (ed.), Asian Pacific Phycology in the 21st Century: Prospects and Challenges.
© 2004 Kluwer Academic Publishers. Printed in the Netherlands.
89
Cyanobacteria-dominated biofilms: a high quality food resource for
intertidal grazers
Sanjay Nagarkar1, Gray A. Williams1,∗,G.Subramanian
2&S.K.Saha
2
1Department of Ecology & Biodiversity and The Swire Institute of Marine Science, The University
of Hong Kong, Hong Kong, SAR, China
2National Facility for Marine Cyanobacteria, Bharathidasan University, Tiruchirapalli, India
∗Author for correspondence; E-mail: snagarka@hkusua.hku.hk
Key words: cyanobacteria, biofilm, nutritional value, protein, carbohydrate, calorific value, tropical rocky shore
Abstract
Hong Kong rocky shores are dominated by cyanobacterial biofilms composed of a diversity of species. Thirteen
common species, belonging to seven genera, were isolated in pure culture in MN+ and MN−media under defined
growth conditions from a semi-exposed shore in Hong Kong. The nutritional values (i.e., protein, carbohydrate
and calorific value) of these 13 species were determined. All species showed high nutritional quality in terms of
protein, carbohydrate and calorific value, however, overall nutritional value varied between the species. Species
of Spirulina and Phormidium were most nutritious (highest nutritional values) whereas species of Calothrix and
Lyngbya were the least nutritious. Microphagous molluscan grazer density and diversity were relatively high at
the study site, despite the seemingly low biomass (as assessed by chlorophyll aconcentration) of the biofilm. It
is suggested that the high nutritional quality of cyanobacteria, together with their fast turnover rates can support
high levels of secondary production (biomass of grazers). The high nutritional quality of cyanobacteria on tropical,
cyanobacteria-dominated, rocky shores is therefore of great importance in the benthic food web.
Introduction
Intertidal, epilithic biofilms are 3-dimensional struc-
tures, mainly composed of bacteria, cyanobacteria,
diatoms, microalgae, protozoa and spores and sporel-
ings of macroalgae embedded in a mucopolysaccha-
ride matrix (Anderson, 1995). Rocky shores around
the world are typically covered with these biofilms
throughout the year. Temperate rocky shore biofilms
are dominated by diatoms or cyanobacteria (MacLu-
lich, 1987; Hill & Hawkins, 1991; Thompson et
al., 1996). On tropical rocky shores, in contrast, the
biofilms are mainly composed of cyanobacteria with
only sporadic patches of bacteria, diatoms, proto-
zoa and juvenile stages of macroalgae (Potts, 1980;
Whitton & Potts, 1982; Nagarkar & Williams, 1999).
Cyanobacteria are important primary producers, many
species of which are able to fix atmospheric nitrogen
(Stewart, 1973; Whitton & Potts, 1982). Cyanobac-
terial biofilms, therefore, form the energy base of the
benthic food web and are very important in terms of
overall productivity and community organization on
tropical rocky shores (Nagarkar, 1996).
Although the importance of cyanobacteria as a
food source for microphagous grazers such as inter-
tidal molluscs (Foster, 1964; Raffaelli, 1985; Quinn,
1988) and zooplankton (Schmidt & Jónasdóttir, 1997;
Repka, 1998) has long been realized, little attention
has been paid to the nutritional quality of cyanobac-
teria (but see Ahlgren et al., 1992; Kaehler & Ken-
nish, 1996). Many studies have found planktonic and
benthic cyanobacteria to be a poor food source due
to their nutritional inadequacy and toxicity (Lampert,
1987; Ahlgren et al., 1990; Thacker et al., 1997), but
no such information is available for intertidal, epi-
lithic cyanobacteria (but see Nicotri, 1977). The main
reason for this nutritional inadequacy of cyanobacteria
is the absence of long-chain, polyunsaturated, fatty
acids (PUFA), which are known to be essential com-
ponents of zooplankton diets and are an indicator of

90
high nutritional quality (Jónasdóttir & Kiørboe, 1996).
In contrast, no information is available on the nutri-
tional requirements of intertidal molluscs’ diet. The
importance of cyanobacteria as a high quality food
source, however, is mainly based on their protein con-
tent and the presence of essential amino acids, which
has become a focus of biotechnological exploitation
of cyanobacteria (Venkataraman, 1993). Chemical
screening of many laboratory grown, commercially
viable, marine cyanobacteria has revealed that they
have a high nutritional value, especially in terms of
protein (Venkataraman, 1993). The nutritional value
of ecologically significant cyanobacteria has, however,
received little attention.
Hong Kong rocky shores are dominated by cy-
anobacterial biofilms throughout the year (Nagarkar
& Williams, 1999). During winter, macroalgal growth
is common on Hong Kong shores (Kaehler & Willi-
ams, 1996). Several rocky shores, however, remain
free of macroalgae and are only covered with a cy-
anobacterial biofilm (Nagarkar, pers. obs.). On visual
observation, these shores appear bare, but chloro-
phyll aand microscopic analyses reveal the presence
of these cyanobacteria-dominated biofilms (Williams,
1994; Nagarkar & Williams, 1997). Cyanobacteria
are the only available food source on these shores
which support a wide variety of microphagous grazers
(Williams, 1993; Nagarkar, 1996). No information
is available, however, on the nutritional quality of
these cyanobacteria (except for one species, Kyrtu-
thrix maculans, Umezaki, 1996; Kaehler & Kennish,
1996) to their molluscan grazers. This paper, there-
fore, presents preliminary work on nutritional quality
(i.e., protein, carbohydrate and calorific value) of cy-
anobacteria present naturally in the intertidal, epilithic
biofilm on rocky shores in Hong Kong.
Materials and methods
Study site
The study site was an ∼100 m long, gently slop-
ping granodiorite heterogeneous rock platform, with
few crevices, on a semi-exposed rocky shore at Mo
Tat Wan (22 ◦13N, 114◦10E), Lamma Island,
Hong Kong. This site was located in a remote area
far from anthropogenic activities and was relatively
clean compared to other polluted shores. Absence of
visually conspicuous macroalgae and thick biofilm
at this site resulted in a bare appearance of the
shore. This site, however, supports a wide variety
and high density of intertidal grazers such as limpets,
Cellana grata Gould, Cellana toreuma Reeve, the
chiton Acanthopleura japonica Lischke and topshell
Monodonta labio Linné (Nagarkar, pers. obs.).
Isolation and purification of cyanobacteria
Samples were collected during March 1999, from the
intertidal zone between 1.25 m and 2.00 m above
Chart Datum. Rock chips (∼2cm
2) with firmly at-
tached cyanobacterial growth were removed using a
hammer and chisel and thin biofilms were scraped
from the rock with a single-sided razor blade. All
the samples were transferred into plastic vials with a
few drops of seawater. In the laboratory, within 6 h,
each sample was divided into four parts which were
inoculated into liquid MN+, MN−, ASN III+ and
ASN III−media (Rippka et al., 1979). To support
the growth of nitrogen fixing cyanobacteria, NaNO3
was omitted from the MN−and ASN III−media.
In all the media, 50 µgml
−1cycloheximide (antibi-
otic) was added to inhibit eukaryotic growth especially
macroalgal spores and microalgae. Bacterial growth
was reduced by adding 25 µgml
−1ampicillin and
15 µgml
−1tetracycline. Samples were incubated at
25 ◦C in continuous low white fluorescent, diffuse
light (<1000 Lux) for about seven days. Later, light
intensity was gradually increased to 1200–1500 Lux.
After 3 weeks, cyanobacterial growth was observed
in the respective media. Individual filaments or cells
were isolated using a long neck glass capillary pipette
under a dissecting microscope (Leitz Diaplan, U.S.A.)
and inoculated into respective fresh liquid MN and
ASN III (+ or −) media; incubated again for another
3–4 weeks. Pure cultures were obtained by repeating
this procedure several times.
Nutritional quality
Thirteen ecologically common cyanobacterial species
which were isolated in pure culture were selected for
nutritional analysis (i.e., protein, carbohydrate and
calorific value). Qualitative microscopic observations,
based on the number of filaments or cells under each
microscopic field of view at a fixed magnification
(×40), revealed that these 13 cyanobacterial species
were abundant at the study site. To investigate the nu-
tritional quality of laboratory cultured cyanobacteria,
all the non-heterocystous species (absence of hetero-
cyst, a special cell which performs nitrogen fixation)

91
were maintained in MN+ media and heterocystous
species (presence of heterocyst) in MN−media. Cy-
anobacterial cells were harvested at the log growth
phase (rapid cell multiplication stage) by centrifuging
at 10 000 rpm for 10 min. The resultant cyanobacterial
pellet was rinsed with distilled water several times and
blotted on blotting paper, air dried over night, ground
into a powder and then extracted for analysis.
Protein estimation
Protein was estimated by the Coomassie Brilliant Blue
(Bio-Rad Laboratories) dye-binding method of Brad-
ford (1976). One millilitre of 0.5 M NaOH was added
to 20 mg of dried powder of each species, incubated
for 10 min in a water bath at 80 ◦C and then centri-
fuged at 3000 rpm for 10 min. The supernatant was
removed, to which 4 ml of distilled water was ad-
ded and then 1 ml of the reagent Coomassie Brilliant
Blue and finally mixed with a vortex mixer. The ab-
sorbence of the sample was measured immediately
using a Pye Unicam spectrophotometer at 595 nm. A
similar procedure was repeated for the solid residue
and the absorbance of the supernatant was again meas-
ured at 595 nm. Protein was estimated by comparison
with a standard bovine serum-albumin curve (Kochert,
1978).
Carbohydrate estimation
Carbohydrate was estimated using the phenol sul-
phuric acid method (Dubois et al., 1956). Ten mil-
ligrams of sample were mixed with 3 ml of distilled
water, added to 50 µl of 90% phenol, mixed and then
finally 5 ml of concentrated H2SO4was added onto
the surface of the liquid. Treated samples were placed
in a water bath at 60 ◦C for 30 min and then centri-
fuged at 3000 rpm for 10 min. The absorbance of the
supernatant was measured spectrophotometrically at
485 nm. Carbohydrate was measured by comparison
with a standard curve prepared using glycogen (Sigma
Chemicals).
Calorific value
To estimate calorific value of each cyanobacterial
species, dry powder (800 mg) of each species was
combusted in a Parr 1261 semi-micro isoperibol bomb
calorimeter, calibrated using benzoic acid.
Data analysis
To investigate trends in the nutritional value of the 13
cyanobacterial species, Principal Component Analysis
(PCA) was performed on the appropriate correlation
matrix (MVSP, Ver. 3.01, 1998, Kovach Computing
Services, Wales).
Results
Thriteen cyanobacterial species (Calothrix contarenii
(Zanard.) Born. ex Flah., Calothrix crustacea Thuret,
Gloeocapsa crepidinum Thuret, Lyngbya martensiana
Menegh. ex Gomont, Lyngbya semiplena (C. Ag.)
J. Ag. ex Gomont, Oscillatoria formosa Bory ex
Gomont, Oscillatoria salina Biswas, Oscillatoria sub-
brevis Schmidle, Phormidium corium (Ag.) Gomont,
Phormidium tenue (Menegh.) Gomont, Spirulina
labyrinthiformis (Menegh.) Gomont, Spirulina sub-
salsa Oerst. ex Gomont and Synechococcus sp.) were
isolated in pure culture. Of the 13 species, only two
were unicellular forms (spherical or ellipsoidal shape,
i.e., G. crepidinum and Synechococcus sp.) and of
the remaining 11 filamentous forms (unbranched fil-
ament shape), two were heterocystous (i.e., C. crus-
tacea and C. contarenii) and the other nine were
non-heterocystous species (Table 1).
Protein
Most of the cyanobacteria species contained a high
percentage of protein (total dry weight). Protein values
ranged from 18.9% for L. martensiana to a maximum
of 70.8% for S. subsalsa (Table 1). Cyanobacteria be-
longing to Lyngbya and Calothrix genera contained
the lowest protein concentrations (<28%) as com-
pared to other species which contained >40% protein
(Table 1).
Carbohydrate
Carbohydrate values ranged from a minimum of 5.4%
for L. martensiana to a maximum of 16.6% of the total
dry weight for S. subsalsa (Table 1). Cyanobacterial
species belonging to Phormidium and Spirulina gen-
era contained the highest carbohydrate concentrations
(>14%) as compared to species belonging to other
genera (<11.5%; Table 1).

92
Table 1. Mean (±S.D) protein, carbohydrate and calorific values of 13 cyanobacterial
species isolated from a Hong Kong rocky shore (n=3). (HF) Heterocystous filament-
ous cyanobacteria; (F) Non-heterocystous filamentous cyanobacteria; (C) Unicellular
cyanobacteria
Species Protein Carbohydrate Calorific value
(% DW) (% DW) (kJ 10 g−1DW)
Calothrix crustacea (HF) 21.50 ±0.40 7.60 ±0.50 25.16 ±0.30
Calothrix contarenii (HF) 27.43 ±0.47 8.23 ±0.65 29.00 ±0.45
Gloeocapsa crepidinum (C) 56.46 ±0.25 7.63 ±0.55 20.53 ±0.55
Lyngbya martensiana (F) 18.86 ±0.65 5.43 ±0.41 21.73 ±0.56
Lyngbya semiplena (F) 27.50 ±0.45 8.93 ±0.15 23.30 ±0.36
Phormidium corium (F) 49.56 ±0.55 16.46 ±0.45 32.56 ±0.41
Phormidium tenue (F) 62.96 ±0.55 15.46 ±0.40 31.33 ±0.20
Spirulina subsalsa (F) 70.76 ±0.90 16.63 ±0.56 34.83 ±0.20
Spirulina labyrinthiformis (F) 68.03 ±0.85 14.73 ±0.66 34.16 ±0.37
Synechococcus sp. (C) 63.56 ±0.60 8.56 ±0.56 27.60 ±0.45
Oscillatoria formosa (F) 50.85 ±0.79 9.46 ±0.45 15.30 ±0.36
Oscillatoria salina (F) 41.80 ±0.81 11.20 ±0.36 19.30 ±0.20
Oscillatoria subbrevis (F) 45.16 ±0.41 11.53 ±0.68 21.43 ±0.30
Calorific values
Calorific values for cyanobacteria ranged from 15.30
kJ 10 g−1DW for O. formosa to a maximum of 34.8
kJ 10 g−1DW for S. subsalsa (Table 1). Species be-
longing to Phormidium and Spirulina genera had the
highest calorific values (>31.3 kJ 10 g−1DW) as com-
pared to species belonging to other genera (<29 kJ
10 g−1DW).
Overall nutritional value
Principal Component Analysis revealed a clear sepa-
ration of three groups of species on axis 1 (Principal
Component 1) which accounted for 90.11% of the
total variance and axis 2 accounted only for 8.67%
of the variance (Fig. 1). The first Principal Compon-
ent has a high positive loading for protein and low
positive loading for carbohydrate and calorific value
(Fig. 1). Species on the positive side of axis one (a
group containing S. labyrinthiformis, S. subsalsa and
P. tenue), have the highest nutritional values, whereas
species (C. contarenii, C. crustacea, L. martensiana
and L. semiplena) on the negative side have the low-
est nutritional values whilst the remaining species (G.
crepidinum, O. formosa, O. salina, O. subbrevis and
P. c o r i u m ) fall in between these extremes (Fig. 1).
Discussion
A great diversity of cyanobacterial species, belong-
ing to various morphological and functional groups,
have been reported from various rocky shores around
Hong Kong (Nagarkar, 1998a,b). In the present study,
13 species belonging to seven genera were isolated in
pure culture from one shore, Mo Tat Wan. These 13
species were the most common cyanobacteria found
in the biofilm on the study site. Most of these spe-
cies are abundant components of the intertidal, epi-
lithic biofilm in Hong Kong (Nagarkar & Williams,
1999). Similar species have also been reported on
rocky shores from other geographical locations (Ja-
pan, Umezaki, 1961; Red Sea, Sinai Peninsula, Potts,
1980; India, Thajuddin & Subramanian, 1992; Red
Sea, Saudi Arabia, Hussain & Khoja, 1993).
The nutritional value of a species is known to
be influenced by culture media (Ben-Amotz et al.,
1985), cell harvesting stage (Whyte, 1987), temper-
ature (James et al., 1989), light intensity (Thompson
et al., 1990) and pH (James et al., 1989). To com-
pare the nutritional value of these cyanobacteria, all
13 species were grown under similar laboratory con-
ditions and harvested at the same growth phase. Two
different culture media were, however, used to support
nitrogen fixing (i.e., Calothrix spp.) and non-nitrogen
fixing cyanobacteria. Differences in protein, carbo-
hydrate and calorific values between Calothrix and the

93
Figure 1. Principal Component Analysis (PCA) based on nutritional values (i.e., protein, carbohydrate and calorific value) of 13 isolated
cyanobacterial species from a Hong Kong rocky shore.
other species may, therefore, be partially influenced by
the culture media and hence this comparison should be
interpreted with caution.
All the cyanobacterial species isolated from Hong
Kong rocky shores showed high nutritional quality.
Protein content was usually high and contributed up to
70.8% of the total dry weight. Calothrix crustacea had
the lowest percentage of protein whereas S. subsalsa
had the highest value. The protein content of Hong
Kong species was similar to values for planktonic cy-
anobacteria (30–40%, Ahlgren et al., 1992) and those
isolated in pure culture for biotechnology purposes
from various sources such as freshwater, terrestrial or
marine environments (65–71%, Venkataraman, 1993;
Subramanian, 1998; Sujatha & Kaushik, 1998). Pre-
viously, K. maculans (one of the dominant encrusting
cyanobacteria on tropical rocky shores) was, however,
reported as a poor source of protein (7.0%, Kaehler
& Kennish, 1996). The protein content of most of
the cyanobacteria (>41%) in the present study was
in contrast, much higher than planktonic microal-
gae (12–35%) used in mariculture except for species
of Calothrix and Lyngbya which contained protein
(21.5–27.5%) within the range of protein values of
microalgae (Brown, 1991).
Carbohydrate values ranged from 5.4 to 16.6%
of the total dry weight. Most of the isolates showed
<11.5% carbohydrate, except species belonging to
Phormidium and Spirulina genera.The maximum car-
bohydrate recorded in the present study was 16.6% for
S. subsalsa. Carbohydrate values previously reported
for K. maculans (>30%, Kaehler & Kennish, 1996)
were much higher than the values recorded for all the
13 cyanobacterial species in the present study. Cy-
anobacterial species cultured for biotechnological pur-
poses (Venkataraman, 1993) and microalgal species
used in mariculture (Brown, 1991), however, contain
a similar range of carbohydrate levels (6.0–16.0%) as
reported in the present study.
The calorific values of the 13 cyanobacteria species
ranged from 15.3 to 34.8 kJ 10 g−1DW which fall
within the range of values previously recorded (Ven-
kataraman, 1993; Kaehler & Kennish, 1996). The cal-
orific value of K. maculans, for example, was 17.5 kJ
10 g−1DW (Kaehler & Kennish, 1996). In the present
study, G. crepidinum,O. formosa and O. salina had
calorific values (15.3–20.5 kJ 10 g−1DW) close to that
of K. maculans. The calorific values of many commer-
cially viable species such as Spirulina, however, fall
towards the higher side of calorific values recorded
in the present study. Most of the Spirulina species,

94
for example, have calorific values between 30 and 36
kJ 10 g−1DW (Venkataraman, 1993). In the present
study, only P. c o r i u m ,P. tenue,S. subsalsa and S.
labyrinthiformis had calorific values between 31.3 and
34.8 kJ 10 g−1DW.
When considering protein, carbohydrateand calor-
ific values together, P. tenue,S. labyrinthiformis and S.
subsalsa contained the greatest concentration as com-
pared to the other species. These results suggest that
due to their high protein content and calorific values,
and in some cases high carbohydrate content, cy-
anobacterial species are nutritionally superior to other
micro- and macroalgal food resources available on
rocky shores (Dawes et al., 1974; McQuaid, 1985;
Kaehler & Kennish, 1996). The importance of inter-
tidal, epilithic cyanobacteria as a high quality food
source, however, should be interpreted with caution
because the present comparison is made on the basis of
nutritional values of laboratory cultured cyanobacteria
which may vary from the field situation. Although
cyanobacteria contained high nutritional values, nutri-
tional adequacy of cyanobacteria for intertidal grazers
needs further investigation based on their PUFA and
toxicity.
During winter, Hong Kong has favourable con-
ditions for macroalgal growth (Kaehler & Williams,
1996) and shores can support a dense cover of
filamentous cyanobacteria, macroalgae and diatoms
(Kaehler & Williams, 1996; Nagarkar & Williams,
1999) and chlorophyll avalues can reach 40 µgcm
−2
(Nagarkar & Williams, 1999). The study site at
Mo Tat Wan did not support macroalgae in March
1999 and had very low, patchy, chlorophyll avalues
(2.6 µgcm
−2±1.9 S.D., n=25) resulting in the
shore being visually bare (Nagarkar, pers. obs.). This
patchy, sparse biofilm might appear insufficient as a
food supply to support the wide variety and high dens-
ity of intertidal grazers which are present at this site.
The present results, however, show that all the cy-
anobacterial species were highly nutritious, especially
in terms of protein, although these species were not
tested for their nutritional adequacy and toxicity. Since
cyanobacteria were the only food source available on
the study site, this suggests the low biomass of the
biofilm together with the known high production rate
of cyanobacteria was probably able to supply enough
nutrition to support the large population of grazers
at this site (Nagarkar, 1996). Cyanobacteria are im-
portant primary producers and often form the energy
base of the benthic food web on tropical rocky shores,
therefore, the nutritional value of these species could
play an important role in the energetics of intertidal
coastal ecosystems.
Acknowledgements
We would like to thank Dr Richard Corlett and Mr
A. D. Weerasooriya (HKU) for their advice on stat-
istics. This work was supported by a RGC grant (RGC
project no. HKU 7231/98M) to Dr G. A. Williams.
Mr S. K. Saha was supported by a Department of
Biotechnology, Government of India, fellowship.
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