Oecologia 27, 275-283 (1977) Oecologia �9 by Springer-Verlag 1977

Nitrogen Fixation by Blue-Green Algal Communities in the Intertidal Zone of the Lagoon of Aldabra Atoll

M. Potts and B.A. Whitton*

Department of Botany, University of Durham, Durham City, England

Summary. All eight types of blue-green algal community sampled from the intertidal sediments of the lagoon of Aldabra showed detectable rates of acetylene reduction, and six of them showed at least some examples of rates considered by previous authors to be rapid. When the reduction rates are related to the chlorophyll a contents of the communities, the maximum rates recorded come in the following order: Hyella balani, Scytonema sp., Hyella balani- Schizothrix sp., Calothrix crustacea, Rivularia sp., Microcoleus chthonoplastes, Hyella balani-purple sulphur bacteria, Pleurocapsa-Chroo- coccus. With the exception of the mixed Hyella balani-purple sulphur bacte- rial community, the rate of acetylene reduction was in all experiments greater in the light than in the dark, the difference being significant (P< 0.05) in the majority of cases. The rates were similar in all experiments whether incubation was carried out with sea or brackish water. It seems probable that nitrogen fixation by blue-green algal communities makes an important addition to the lagoon ecosystem, and that not only heterocystous, but also some non-heterocystous, species are involved. Among the latter, the data for Hyella balani and Microcoleus chthonoplastes are especially convinc- ing.

Introduction

Blue-green algae form conspicuous growths in many areas of the intertidal zone of the lagoon at Aldabra Atoll in the Indian Ocean (Potts and Whitton, in press a). Typically these occur as visually obvious mats or crusts over the surface of sediments or rock, although sub-surface growths of Hyella balani growing endolithically in sand grains are also locally important. The commu- nities are usually clearly zoned, and often consist almost entirely of a single species, so that it is possible to carry out experiments in situ and be confident that any algal response observed is due to a particular species. Both heterocystous and non-heterocystous species are widespread.

* Corresponding author

276 M. Potts and B.A. Whitton

A l t h o u g h a n u m b e r o f s tudies have been r e p o r t e d concern ing n i t rogen fixa- t ion in mar ine hab i ta t s , the da ta are still very i nadequa t e for a n y genera l iza t ion a b o u t i ts quan t i t a t ive significance. In t empe ra t e l a t i tudes the fo l lowing mar ine species are k n o w n to fix a tmosphe r i c n i t rogen : Calothrix scopulorum, C. aeru- ginea, C. crustacea, Nostoc spp., Rivularia atra, R. biasolettiana, NoduIaria h'ar- veyana, N. spumigena, Microchaete sp. (Stewart , 1973). Us ing the acety lene re- duc t ion assay technique, Bunt et al. (1970) d e m o n s t r a t e d n i t rogen f ixa t ion in situ by Anabaena sp. off a F l o r i d a shore. M o r e de ta i led studies have recent ly been descr ibed for the seaward side o f Enewe tak (Eniwetok) Atol l . W e b b et al. (1975) a n d Wiebe et al. (1975) found tha t the algal reef flats f ixed n i t rogen at ra tes c o m p a r a b l e to those in m a n a g e d agr icul ture , the d o m i n a n t fixer p r o b a b l y being Calothrix crustacea. They suggested tha t such f ixat ion was ma in t a in ing the h igh p roduc t iv i t y o f cora l reefs and also a to l l lagoons . M a g u e and H o l m - Hansen (1975) r e p o r t e d h igh ra tes o f f ixa t ion by benth ic communi t i e s o f Caloth- rix, Hormothamnion and Nostoc f rom the same atoll . There was a slight r educ t ion o f acetylene by Oscillatoria, but this m a y have been due to epiphytes . Light was shown to have a very m a r k e d inf luence on acetylene reduc t ion in Nostoc, with the mean ra te for C2H 4 mg N 1 h - 1 p r o d u c e d in the l ight being 220 n mol, bu t only 4.7 n tool in the dark . In con t r a s t to the m a n y repor t s o f n i t rogen f ixa t ion by he te rocys tous b lue-green in mar ine env i ronments , the only non- he te rocys tous species for which there are convinc ing field da t a is the p l ank ton i c Trichodesmium erythraeum ( D u g d a l e et al., 1961 ; Tay lo r et al., 1973; Carpente r , 1973). The presen t s tudy examines the po ten t i a l n i t rogen fixing act iv i ty o f repre- sentat ives o f the m a j o r b lue-green algal communi t i e s o f the in te r t ida l zone o f the l agoon shore o f Wes t I s land , A l d a b r a .

Methods

The general method used was to select locally uniform areas of blue-green algae or mixed blue-green algae-purple sulphur photosynthetic bacteria community, and to measure acetylene reduction by samples of the community in the light and the dark. The rate of acetylene reduction was estimated in relation both to unit area of community and to chlorophyll a. A number of studies were also included on dried and re-wetted communities returned to the laboratory in Durham.

The literature about Aldabra (9 ~ 24' S, 46 ~ 20' E) has been listed by Stoddart (1967). An account of the lagoon intertidal blue-green algal and photosynthetic bacterial communities has been given by Potts and Whitton (in press a), and data concerning pH and Eh in the sediments beneath some of the communities used here for acetylene reduction assays by Potts and Whitton (in press b).

Field

The present studies were carried out between December 1974 and April 1975. This period all lies within the wet season, but our observations to date do not indicate very marked seasonal changes in the abundance of the lagoon intertidal blue-green algal communities. The areas for the assay studies were chosen to represent well-developed, locally uniform regions of widespread communities. All assays commenced between 1000 and 1100 h, and lasted 60 min. The algal commu- nities were not submerged by lagoon water at the time of assay, but in all cases assays were carried out within a few hours of having been submerged by lagoon water, and had received no more than minor wetting by rain water since the previous submergence.

Nitrogen Fixation in Lagoon of Aldabra 277

An at tempt was made to consider the factors known to influence the reduction of acetylene (Hardy et al., 1973) when conducting the experiments. Glass serum bottles (7 ml) with perforated serum caps were used for all incubations, and gases were introduced or removed through the rubber seal by a syringe. Cores of alga plus about 5 m m depth of associated sediment were introduced into each bottle; in the case of the communit ies with Hyella balani present a slightly greater depth of sediment was used, in order to make sure that all the algal cells in a vertical column were included. The alga was disturbed as little as possible in the transfer, in order to minimize any environmental change likely to induce a lag, but a slight compression and subsequent spreading out of the core were inevitable, due to the shape of the serum bottle. One ml water was then added, the lid closed and the bottle pushed into the sediment such that the algal sample received a light regime similar to that of the same communi ty outside, A pre-incubation period of 10 rain was allowed prior to the addition of acetylene. 1 ml acetylene gas (East African Oxygen Ltd) was injected (pC2H 2 =0.17 atm.), 1 ml gas removed to equalize the pressure, and the bottles then incubated for a further 60 rain.

At the end of the experimental period, gas samples were removed with multiple-sample vacu- tainer needles and stored in non-sterile, non-silicone coated, 5 ml draw vacutainers (Becton and Dickinson 3206U, formula 134). These were sealed in paraffin wax within a couple of hours of use. Experiments carried out in Durham at 35 ~ C, a temperature in excess of that ever encountered by the stored vacutainers on Aldabra, have shown no detectable change in gas mixtures after 15 weeks of storage. On return to Durham, samples were analysed for acetylene and ethylene, using a Varian aerograph series 1200 gas chromatograph.

The water added to each serum bottle was either sea or rain water, which had been passed through a sintered glass (Sinta No. 2) funnel. In most cases addition of the latter probably led to salinities in the range 10-15~ . Acetylene reduction assays for samples of filtered water showed negligible rates of reduction, the Nghest rate found for filtered sea water being about one-hundredth of the lowest rate of reduction in the dark shown for an algal communi ty in Table 1. From four to eight replicates were used for each combination of light v. dark, sea v. brackish water. Temperature measurements were made in the sediments adjacent to the algal commu- nity at the beginning and end of the experiment, and also in an extra serum bottle with algal core used as a control. Temperatures inside the bottles at the end of the experiment never differed by more than 2 ~ C from that of the surrounding sediments. Rather similar controls for pH and Eh (Potts and Whitton, in press b) were also used in some experiments, w i t h the exception that slightly larger bottles had to be used to receive the electrodes. The controls showed that in several experiments there were changes in pH which differed from those taking place in their surroundings. The experiment with Rivularia showed a sufficient rise in pH (e. 1 unit) to suggest that CO 2 deficiency might be important by the end of the experiment in the smaller serum bottles, whilst in contrast Seytonema showed a drop in pH even in the light. If allowance is made for changes in Eh associated with these pH changes, the controIs showed that Eh underwent no similar marked changes due to incubation in a confined volume.

After each experiment, the algal cores were dried carefully and sealed in polythene bags, for subsequent extraction of chlorophyll a a n d , where appropriate, also bacteriochlorophyll a.

Laboratory

Laboratory experiments were carried out on material which had been dried down in the field, following previous success (Whitton et al., in press) using a similar approach with terrestrial Nostoc commune. Most of the experiments were carried out about six months after collection from the lagoon.

Pigment Analysis

Chlorophyll a was extracted from the cores after return to Durham. The cores were first incubated with sea water for 6 h at 32 ~ C in the light, and then with 95% methanol in 30 ml McCartney bottles for 15 min at 70~ in the dark. Extracts were cleared using pressure filtration through glass-fibre discs, and their absorbance measured at 665 nm before and after acidification with

278 M. Potts and B.A. Whitton

Table 1. Rates of CzH 4 production by blue-green algal and blue-green algal photosynthetic bacterial recorded for a particular community. L=light , D = d a r k (note that if the chlorophyll a content

Community Whether Morphology Environment with hetero- cysts

Hyella balani -- endolithic medium coarse sand Hyella balani - endolithic medium coarse sand Seytonema sp. + juvenile mat medium coarse sand Seytonema sp. + mature mat among mangroves Scytonema sp. + mature mat medium coarse sand Seytonema sp. + mature mat medium coarse sand Seytonema sp. + mature mat medium coarse sand Seytonema sp. + mature mat medium coarse sand Scytonema sp. + mature mat among mangroves Scytonema sp. + mature mat on rock in sheltered area Seytonema sp. + mature mat medium coarse sand Hyella balani - Schizothrix sp. - endolithic crust medium coarse sand Hyella balani - endolithic crust medium coarse sand Calothrix erustaeea + thin mat binding coarse sand Rivularia sp. + hemispherical colonies on silt Mierocoleus ehthonoplastes - film on silt Mieroeoleus ehthonoplastes - film on silt Hyella balani - purple bacteria -- endolithic + epipsammic medium coarse sand Hyella balani - purple bacteria - endolithic + epipsammic medium coarse sand Hyella balani - purple bacteria - endolithic + epipsammic medium coarse sand Pleurocapsa - Chroococcus - dense suspension edge of sheltered tidal

pool

HC1. Chlorophyll a and phaeophytin a were calculated using the formulae given by Marker (1972), but with a different 'acid-factor' derived constant. Based on a study of four eukaryotes, Marker reported an'acid factor 'of 1.5 when using 95% methanol. We have found a mean value of 1.85 for marine blue-green algal samples from Aldabra. This value is used in the present account for all populations, although the mean values for individual species may differ slightly from this.

In the samples including both chlorophyll a and bacteriochlorophyll a, pigment extraction was carried out in the same manner as above, but the absorbance was read at 771 nm as well as 665 nm. Comparison of the spectrum for pure chlorophyll a with that of pure bacteriochlorophyll a given by Pierson and Castenholz (1974) permitted the calculation of the contribution to the spectrum obtained for the mixed pigments made by chlorophyll a at 665 nm, and by bacteriochloro- phyll a at 771 nm. The extinction coefficient of Cohen-Bazire and Sistrom (1966) was used to estimate the amounts of bacteriochlorophyll a from the absorbance values.

Results

T h e r e s u l t s o f t h e f i e ld e x p e r i m e n t s a r e s u m m a r i z e d in T a b l e 1. T h e f o l l o w i n g

p o i n t s s e e m w o r t h c o m m e n t .

Nitrogen Fixation in Lagoon of Aldabra 279

communities in lagoon intertidal zone. The communities are shown in order of maximum rate of an alga 1% dry weight, then 1 lag chl a cm- 2 = 1 g dry weight m- 2)

pg cht a lag nM Sea water Brackish water cm- 2 Bchl a C2H4 nM C2H4 lag chl a- 1 min- 1 nM C2H 4 lag chl a 1 min- 1

cm- 2 min- 1 cm- 2 L D Das whether L D Das L % L L > D % L

signif.

whether L > D signif.

10.7 0.416 0.034 0.032 94 8.87 0.044 0.0051 0.00015 29 +

13.2 0.455 0.030 0.028 93 287 2.09 0.0074 0.00095 13 + 185 0.747 0.0055 0.0020 36 + 207 0.721 0.0035 0.0019 54 + 233 0.572 0.0027 0.0016 59 293 0.821 0.0024 0.0016 67 179 0.476 0.0024 0.00065 27 + 231 0.405 0.0019 0.0015 79 297 0.554 0.0015 0.00076 51 +

22.1 0.461 0.019 0.012 63 + 11.6 0.215 0.016 0.014 88 34.7 0.538 0.016 0.010 63 + 93.9 1.24 0.012 0.0010 83 + 38.2 0.451 0.010 0.0059 59

104 0.023 0.0040 0.00003 0.8 § 13.6 2.2 0.084 137 11.8 1.6 0.074 128 9.37 2.5 0.014 85

121 0.021 0.0002 0.00004 20 +

0.049 0.028 57 +

0.035 0.022 63

0.0031 0.0011 35 + 0.0045 0.0019 42 + 0.0025 0.0007 28 + 0.0030 0.0018 60 0.0023 0.00076 33 0.0017 0.0011 65 + 0.0017 0.0009 53 § 0.0024 0.014 58 + 0.032 0.017 53 + 0.015 0.0088 61

0.017 0.0085 50 +

i) W i t h the e x c e p t i o n o f t w o o f the th ree e x p e r i m e n t s on the Hyella balani- p u r p l e su lphu r bac t e r i a l c o m m u n i t y , all e x p e r i m e n t s s h o w e d h i g h e r ace ty l ene

r e d u c t i o n ra tes in t he l ight t h a n in the dark . I n 21 ou t o f the 31 to t a l expe r imen t s ,

this d i f fe rence b e t w e e n l igh t a n d d a r k was s ta t i s t ica l ly s ign i f i can t ( P < 0 . 0 5 ) .

ii) A l t h o u g h the ra te o f ace ty l ene r e d u c t i o n was g rea t e r in the l igh t t h a n

in the d a r k wi th the seven c o m m u n i t i e s d o m i n a t e d on ly by b l u e - g r e e n algae,

the d a r k r a t e was at least 5 0 % o f t h a t in the l ight in 2 l o f the 3 l expe r imen t s .

iii) T h e r e was no o b v i o u s d i f fe rence b e t w e e n the ra tes o b t a i n e d wi th b r a c k i s h as o p p o s e d to sea wa te r (Fig. 2) .

A wide r a n g e o f l a b o r a t o r y e x p e r i m e n t s were ca r r i ed o u t w i t h r e - w e t t e d m a t e r i a l s o f Hyella balani, Scytonema sp. (Fig. 1 ) a n d Rivularia sp. V a r i o u s

t e m p e r a t u r e , l ight , m e d i a a n d gas r eg imes were t es ted fo r b o t h p r e - i n c u b a t i o n w i t h ace ty lene . H o w e v e r de t ec t ab l e ra tes o f ace ty l ene r e d u c t i o n were f o u n d

wi th o n l y o n e p o p u l a t i o n o f Scytonema sp. Tes t s w i th this p o p u l a t i o n s h o w e d m e a n ra tes o f r e d u c t i o n in the l ight o f a b o u t 10% m e a n ra tes fo r m a t u r e Scytonema m a t s o n the atol l .

280 M. Potts and B.A. Whit ton

Fig. 1. Thick mat of Scytonema sp. growing over rock near Rhizophora mucronata forest

.=,

~o

T = ,'7 ,_ o E

'7

? o

=E = o P

LD I 0

. � 9

1 0 - 5 10 - 4 1 0 - 3 1 0 - 2 1 0 - 1

n M C 2 H 4 /Jg c h l a "1 r a i n -1

S E A W A T E R

Fig.2 . Relationship between C2H r production by Scytonema sp. in the light with brackish water as compared with sea water

Nitrogen Fixation in Lagoon of Aldabra 281

Table 2. Rates of C2I-I 4 production by Calothrix crustacea found in acetylene reduction assays carried out by three different authors. Data of Mague and Holm-Hansen and Wiebe et al. have been converted to present form in order to make the comparison. (In order to convert the data of Mague and Holm-Hansen, the assumption is made that the alga contains 7% N and 1% chlorophyll a)

n mol C2H4 n tool C2H 4 lag min 1 c m - z chl a - ~ m i n -

Light Dark Light Dark

Aldabra, thin mat 0.54 0.34 Mague and Holm-Hansen Enctwetak, splash zone Wiebe et al. Enetwetak, upper intertidal 0.12 Wiebe et al. Enetwetak, intertidal 0.62 0.021

0.016 0.010 0.007

Discussion

Among the seven communities dominated only by blue-green algae, all showed rates of acetylene reduction which were above those that might have been expected from experimental error, and which were greater in the light than in the dark. All these communities showed at least one experiment where this difference between light and dark was statistically significant. With the exception of the Pleurocapsa-Chroococcus community, all the communities showed at least one experiment where the rates (based on unit area) were similar to those considered high by previous authors such as Wiebe et al. (1975). There are two obvious explanations for a higher rate in the light-nitrogen fixation asso- ciated with algal photosynthesis, or release of substances from the alga leading to stimulation of fixation by heterotrophs. Although laboratory experiments with axenic algal isolates would be required to settle this problem without ambiguity, we suggest that the former is likely to be much the more important. Many of the rates of acetylene reduction observed were high, and the great bulk of the biomass in these communities was algal.

The fact that only a minority of experiments showed a marked difference between the values obtained in the light and in the dark contrasts with the observations of Mague and Holm-Hansen (1975) and Wiebe et al. (1975) as shown for Calothrix crustacea in Table 2. It seems possible that this may in part be due to the fact that in our experiments samples were darkened only 10 min before the addition of acetylene, and presumably therefore still had high cellular levels of photosynthetic products. Some evidence to support this hypothesis comes from the observation that of the nine experiments (in sea water) with Scytonema sp., the two experiments with the biggest difference between light and dark came from shaded communities among mangroves.

On the assumption that most of the acetylene reduction observed in algal dominated communities was in fact by the alga, it seems probable that not only the heterocystous, but also some of the non-heterocystous communities, are fixing nitrogen. The rate for the Pleurocapsa-Chroococcus community is low, when related to chlorophyll, so the results for this community must be

282 M. Ports and B.A. Whitton

regarded as unconvincing. However it seems likely that bo th Hyella balani and Microcoleus chthonoplastes can fix ni t rogen at rates o f the same order as those for heterocystous species.

As it was not possible to find a suitable photosynthet ic bacterial communi ty on West Is land free f rom Hyella balani, it is uncertain whether any par t of the acetylene reduct ion in the mixed Hyella balani-purple sulphur photosyn- thetic bacterial commun i ty was due to the latter. I f it did contribute, inhibition o f bacterial acetylene reduct ion by oxygen evolved by Hyella might provide an explanat ion of the reduced rates of reduct ion observed with this communi ty in the light.

The fact that only one o f the many sets o f experiments carried out in the labora tory with re-wetted material showed a positive result was unexpected, in view of previous success with re-wetted Nostoc commune f rom Aldabra. The materials were not collected at the same time as the field experiments, but it seems probable tha t the reasons for lack o f success were in par t due to a more rapid loss o f viability o f the intertidal algae with storage, and in par t due to the difficulty of re-creating closely similar environmental condi- tions.

A l though the present account reports experiments f rom only one area of Aldabra , it seems probable that ni t rogen fixation by blue-green algae makes an impor tan t cont r ibut ion to the ni trogen budget o f the lagoon. Communi t ies domina ted by potential ni t rogen fixing algal communi t ies cover at least several square kilometres of the l agoon sediments. However as the l agoon (including mangrove forest) occupies an area o f 210 km 2, it will require detailed studies on other likely ni t rogen fixing communit ies , such as silts in mangrove forest and the bacterial epiphytes on submerged angiosperms, before it is possible to assess just how impor tan t is the blue-green algal contr ibution.

Acknowledgments. We are most grateful to the Natural Environment Research Council and the Royal Society for financial support, and to Mr. A, Donaldson for helpful advice about practical techniques.

References

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Carpenter, E.J. : Nitrogen fixation by Oscillatoria (Trichodesmium) thiebautii in the southwestern Sargasso Sea. Deep-Sea Res. 20, 285-288 (1973)

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Dugdale, V.A., Menzel, D.W., Ryther, J.H.: Nitrogen fixation in the Sargasso Sea. Deep-Sea Res. 7, 298-300 (1961)

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Nitrogen Fixation in Lagoon of Aldabra 283

Potts, M., Whitton, B.A. : Vegetation of the intertidal zone of the lagoon of Aldabra, with particular reference to the photosynthetic prokaryotic communities. In press a

Potts, M., Whitton, B.A.: The limits of pH and Eh associated with different environments and communities on Aldabra Atoll. In press b

Stewart, W.D.P.: Nitrogen fixation by photosynthetic microorganisms. Ann. Rev. Microbiol. 27, 283-316 (1973)

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Webb, K.L., DuPaul, W.D., Wiebe, W., Sottile, W., Johannes, R.E.: Enewetak (Eniwetok) Atoll: Aspects of the nitrogen cycle on a coral reef. Limnol. Oceanogr. 20, 198 210 (1975)

Whitton, B.A., Donaldson, A., Potts, M. : Nitrogen fixation by Nostoc colonies in terrestrial environ- ments of Aldabra Atoll. In press

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Received June 10, 1976