438 S.-Afr.Tydskr. Plantk., 1989, SS( 4): 438-446 Biological nitrogen fixation (acetylene reduction) associated with blue-green algal (cyanobacterial) communities in the Beachwood Nature Reserve. I. The effect of environmental factors on acetylene reduction activity

Fiona O. Mann* and T.O. Steinke Department of , University of -Westville, Private Bag X54001, Durban, 4000 Republic of South

Accepted 23 March 1989

Nitrogen fixation of blue-green algae (cyanobacteria) associated with Avicennia marina (Forssk.) Vierh. pneumatophores and wet and dry surface sediments was investigated in the Beachwood Mangrove Nature Reserve by means of the acetylene reduction technique. Studies revealed percentage moisture and tempera­ ture to be the prime factors influencing ARA (acetylene reduction activity) in these habitats. and rates were highest under submerged conditions and at 22°C. High concentrations of inorganic nitrogen (between 1 and 5 mg 1- 1) significantly depressed ARA in all habitats. Increases in ARA occurred with increase in light intensity up to 40 I-lE m-2 S -1, with negligible dark rates being recorded in association with the wet and dry surface sediments. Significant dark rates of ARA and stimulation of ARA by sucrose in association with the pneuma­ tophores indicated that bacteria may also be contributing to ARA in this habitat. No organic carbon stimulation was noted in the other sites. Salinity had little effect on ARA over the range generally experienced in each habitat.

Stikstofvaslegging deur blougroenalge (cyanobacteria) wat met Avicennia marina (Forssk.) Vierh .-pneumato­ fore en nat en droe sedimente geassosieer is, is in die Beachwood-Mangelietnatuurreservaat, deur middel van die asetileenreduksietegniek ondersoek. Ondersoeke het aangetoon dat die persentasie vog en tempera­ tuur die belangrikste faktore is wat ARA (asetileenreduksieaktiwiteit) in hierdie habitatte be'fnvloed en die waardes was die hoogste by onderwatertoestande en 22°C. Hoe konsentrasies van anorganiese stikstof (tussen 1 en 5 mg 1-1) het ARA in aile habitatte aansienlik onderdruk. Toename in ARA het voorgekom by lig­ intensiteite tot by 40 I-lE m-2 S-1, met weglaatbare waardes wat gedurende die donker in assosiasie met nat en droe oppervlakke van sedimente geregistreer is. Beduidende donker-ARA-waardes en die stimulering van ARA deur sukrose in assosiasie met die pneumatofore het aangedui dat bakteriee ook 'n bydraende rol tot ARA in hierdie habitat kan speel. Geen stimulering deur die teenwoordigheid van koolwaterstowwe is in die ander opname-gebiede aangetoon nie. Daar is bevind dat die soutgehalte 'n klein invloed op ARA, in elke habitat gehad het.

Keywords: Blue-green algae, environmental responses, , Mgeni Estuary, nitrogen fixation

'To whom correspondence should be addressed

Introduction specifically to salt marshes and estuaries (Stewart & The element nitrogen IS an important constituent of Pugh 1973 ; Jones ]974; Bohlool & Wiebe 1978) is proba­ proteins, amino acids and nucleic acids of all living bly much more important than the contribution by organisms. Biological nitrogen fixation is responsible for heterotrophic micro-organisms. about 60% of the nitrogen input into the worlds soils In natural ecosystems, especially intertidal and estua­ (Postgate 1982) and has been shown to be important in a rine environments, organisms are subjected to a variety wide range of ecosystems including aquatic systems of extreme and often rapidly fluctuating chemical , (Paul 1978). physical and biological conditions. Little is known of the Little is known about the potential role of nitrogen effects of varying environmental conditions on rates of fixers in mangrove swamps. Heterotrophic nitrogen fixa­ nitrogen fixation of mangrove micro-organisms. tion associated with mangrove sediments (Kimball & This study investigated the effects of certain environ­ Teas 1975; Zuberer & Silver 1975, 1978, 1979; van der mental conditions, namely percentage moisture, temper­ Valk & Attiwill 1984) and the warty lenticellate bark of ature, salinity, light intensity, organic carbon and Bruguiera gymnorrhiza (L.) Lam . has been studied. inorganic nitrogen on nitrogen fixation in selected blue­ Limited attention has been paid to blue-green algal green algal communities with the aim of (a) evaluating nitrogen fixation. Potts (1979) and Hicks & Silvester nitrogen fixation under different estuarine conditions (1985) linked high rates of nitrogen fixation on A vicennia and (b) gaining an understanding of seasonal and temp­ marina (Forssk.) Vierh. pneumatophores to blue-green oral variations in nitrogen fixation. algal communities. It has been suggested that the contribution of blue­ Materials and Methods green algae to the nitrogen status of natural ecosystems The study area (Dugdale & Dugdale 1962; Brooks et al. 1971) and more The study was conducted in the Beachwood Mangrove

S.Afr.J. Bot. , 1989, 55(4) 443

submerged (Mann 1987) and the potential therefore trends would possibly be evident under exposed exists for ARA, though somewhat reduced at times, conditions. This needs to be determined. most of the year. Shading by A . marina prevents rapid Estuarine environments are subjected to marked fluc­ evaporation of moisture. The dry mat habitat is similarly tuations in salinity as a result of tidal inundation, only submerged at spring high tides, but dries out rapidly evaporation and flooding. Micro-organisms in all three subsequently due to exposure to direct sunlight, causing study sites appeared to be euryhaline, as ARA was not increased surface temperatures. Summer rains also markedly affected by a range of salinities, supporting provide moisture and during these periods, a relatively work on intertidal blue-green algae (Potts & Whitton high rate of ARA would be expected. The lowest 1977) and salt marsh micro-organisms (Patriquin & moisture content recorded in July was 25% (Mann 1987) Keddy 1978; Ubben & Hanson 1980). The pneumato­ and this resulted in decreased rates of ARA. The most phore micro-organisms are subjected to a wide range of dominant blue-green algae in the dry mat habitat and on salinities between high and low tide (Mann 1987) and the pneumatophores possess thick mucilaginous sheaths appear well adapted to an intertidal habitat as they (Mann 1987) which possibly aid in water retention exhibited no significant differences in ARA with salin­ during exposed periods (Stewart 1977). In the Sinai ities ranging from 0 to 35%0. The effect of higher mangrove swamps, Dor (1975) noted that the inner part salinities was not recorded, although higher salinities of blue-green algal thalli were composed of thick, empty may be achieved on pneumatophores under conditions sheaths and suggested that this forms a capillary system of extreme evaporation during exposure at low tide. absorbing water at high tide and retaining it during However, increases in salinity above 35%0 would be periods of exposure. accompanied by dehydration and under such conditions, Tn the absence of desiccation, temperature is regarded ARA has been shown to be limited. Maximum rates of as a major factor controlling ARA in blue-green algal ARA in the wet and dry mat habitats occurred at in situ mats (Jones 1977a). Tn warm temperate climates, as salinities to which these organisms are most frequently exists along the Natal coast, maximum ARA usually exposed, supporting work of Ubben & Hanson (1980) occurs between 30 and 35°C (Jones 1977c, 1981 ; Smith and Dicker & Smith (1981) . Tn the wet mat habitat, & Hayaska 1982; Talbot 1982; Hicks & Silvester 1985). salinities generally varied from 10 to 35%0 (Mann 1987) However, optimal temperatures of 20 to 25°C were and maximum rates of ARA occurred within this range recorded for all sites. Such optima are commonly recor­ of salinities. (Salinities of 4 to 48%0 were occassionally ded in cool , temperate climates (Jones 1974; Blasco & recorded). Tn the dry mat habitat salinities ranged from 0 Jordan 1976), although optimum temperatures in this to 47%0 (Mann 1987). In this habitat rain has an impor­ range have been recorded in warm temperate climates of tant influence in decreasing the salinity. Relatively high the south-eastern U.S.A. (Hanson 1977a; Thomson & levels of ARA were still maintained at salinity levels of Webb 1984). The pneumatophore micro-organisms fix 0%0. High salinities of 85 %0 have been recorded in this nitrogen maximally under submerged conditions habitat (29/5/85), although such conditions would usually surrounding high tide. Creek water temperatures be accompanied by prohibitively low moisture levels for ranging from 15.5 to 27°C, with a mean of 21.25°C ARA. It is significant that micro-organisms in this have been recorded in the field (Mann 1987). This habitat are able to survive such high salinities, although corresponds to the optimum temperature at which ARA other factors are likely to exert a greater limiting effect occurs and the pneumatophore organisms appear to be on ARA at this stage. well adapted to in situ temperatures. The dry mat habitat Saturation of ARA occurred at low light intensities in is exposed to full sunlight and this results in high midday all sites. Tn the Swartkops Estuary, Talbot (1982) repor­ summer temperatures. Mud temperatures ranging from ted saturation at higher light intensities of between 300 12 to 36°C have been recorded in this area during the and 390 J-lE m-2 S-I in surface sediments. In the wet mat study (Mann 1987), although lower air temperatures are and pneumatophore habitats an adaptation to lower light experienced during winter nights (5°C has been intensities would be expected as a result of shading and recorded at the Durban Botanic Gardens as the absolute submergence. The wet mat micro-organisms occur in minimum for June). The maximum and minimum mud shaded areas under the A. marina canopy, where light temperatures are inhibitory to ARA. This could result in intensities of between 16 and 150 J-lE m-2 S-I have been a diurnal variation in ARA. Jones (1977a, b) demonstra­ recorded (Mann 1987). The pneumatophore micro­ ted that high midday temperatures of 40°C were organisms generally also occur in partly shaded areas, inhibitory to ARA in blue-green algal mats, causing a although they are subjected to full light intensities for decrease in rates at this time. Permanent shading by the limited periods each day. Light intensities during periods A. marina canopy in the wet mat habitat promotes lower of submergence are important as it is under these condi­ temperatures. Mud temperatures ranging from 13 to tions that maximum rates of ARA occur. Under such 26.5°C have been recorded (Mann 1987). These algae conditions low submerged light intensities of between 22 seem reasonably well adapted to in situ temperatures, and 45 J-lE m- 2 S- I have been recorded (Mann 1987). The although lower winter temperatures may be inhibitory. dry mat micro-organisms are subjected to full sun for The temperature optima of all 3 habitats are very sharp most of the day and would therefore be expected to show in comparison to those previously recorded in estuarine an adaptation to higher light intensities. Full sunlight areas (Hanson 1977a; Talbot 1982). Experiments were intensities of between 250 and 860 J-lE m- 2 S-I have been conducted under submerged conditions and different recorded at this site (Mann 1987). Saturation at lower 444 S.-Afr.Tydskr. Plantk., 1989,55(4)

light intensities is therefore surpnsmg and cannot be associated mangrove surface sediment were correlated fully explained. These micro-organisms possibly show a with the presence of plant-derived organic matter. tolerance to a wide range of light intensities. Various mangrove sediments have been shown to Unfortunately because of limitations imposed by the possess high levels of organic carbon (Walsh 1967; Hicks growth cabinet, it was not possible to determine & Silvester 1985). In the wet mat habitat, organic carbon experimentally the effect of higher light intensities on levels are high, ranging from 50.8 to 94.7 mg g dry mass-I ARA. The inhibitory effect of high light intensities on (Mann 1987) and these levels may not be limiting to ARA in blue-green algae has been suggested by Stewart ARA. However, in the dry mat habitat organic carbon is et al. (1978) and Smith (1984), although in other cases, likely to be limiting at times, as lower levels of 24.9 to such light intensities were shown to have no inhibitory 65.0 mg g dry mass-I have been recorded and other effects (Jones 1977b; Carpenter et al. 1978). Stewart factors must be responsible for the lack of stimulation of (1974) and Stewart et al. (1978) suggested that blue­ ARA by sucrose. The enhancement of ARA by organic green algae adapt to high light intensities by increasing carbon amendment is generally regarded as evidence of pigmentation or aggregating together to effect self­ heterotrophic nitrogen fixation in carbon-limited habi­ shading, resulting in an effective decrease in light tats (Jones 1974; Marsho et al. 1975; Pearson & Taylor intensity. Jones (1977b) reported that high light intensities experienced in southern Africa at midday 1978; Talbot 1982), although Fay (1976) reported were not responsible for inhibition of ARA in blue­ stimulation of ARA in blue-green algae by glucose green algal mats. Talbot (1982) reported no inhibitory amendment. The lack of stimulation of ARA in the wet effects of high light intensities in surface sediments of the and dry mat habitats is more likely to be related to the Swartkops Estuary. Contradictory reports appear on fact that blue-green algae are the predominant fixers in dark rates of ARA in blue-green algal communities and these areas and utilize carbon manufactured during surface sediments. A marked decrease in ARA in the photosynthesis. This has been suggested by a number of absence of light has been reported by workers on workers (Capone et al. 1977; Capone & Taylor 1977; seagrasses (Capone & Taylor 1977), macroalgae Jones 1977a; Talbot 1982). The stimulation of ARA by (Capone et al. 1977), intertidal lagoons (Bohlool & pneumatophore micro-organisms possibly indicates that Wiebe 1978), Trichodesmium sp. (Taylor et al. 1973) and bacteria are contributing to pneumatophore-associated salt marshes (Jones 1974; Hanson 1977b). An 80% fixation and that carbon is limiting to organisms in this decrease in light rates was noted by Hicks & Silvester habitat. It would appear that blue-green algae and (1985) on mangrove pneumatophores in the dark. A bacteria are important nitrogen fixers on the pneumato­ 50% decrease in light rates was recorded by Potts & phores. Whitton (1977) in an intertidal lagoon and by Kimball & It has been suggested that the high concentrations of Teas (1975) in mangrove surface sediments. Potts (1979) ammonium generally associated with mangrove soils observed only a slight decrease in mangrove blue-green may inhibit nitrogen fixation (Kimball & Teas 1975; algal ARA in the dark. Talbot (1982) reported a lag of Zuberer & Silver 1975). Van der Valk & Attiwill (1984) 24 h before light and dark rates differed significantly. It partly attributed low rates of ARA associated with has been suggested that the occurrence of high rates of mangrove roots to high ammonium levels in interstitial dark fixation is an indication of heterotrophic nitrogen waters. The results indicate that high ammonium and fixation (Kimball & Teas 1975; Thomson & Webb 1984; nitrate concentrations are limiting to ARA. The pneu­ Hicks & Silvester 1985). If this is the case, it would matophore micro-organisms fix nitrogen optimally under appear that ARA is primarily due to blue-green algae in the wet and dry mat areas, whereas a significant portion submerged conditions at high tide, and at this time, in of ARA associated with pneumatophores could be situ inorganic nitrogen levels in the creek water were accounted for by heterotrophic bacteria. However, high relatively low (Mann 1987). High values were recorded night-time rates of ARA have been recorded in blue­ in the creek at low tide, but during this period, the green algal communities (Carpenter et al. 1978). Dark pneumatophores are exposed and dehydration becomes fixation is strongly linked to the availability of organic the prime limiting factor. In situ levels of ammonium are compounds (Taylor et al. 1973; Fay 1976). extremely high (0.15 to 7.82 mg g w mass-I) (Mann 1987) Organic carbon is most frequently implicated as an when compared to inhibitory levels reported from salt important factor limiting ARA in salt marshes (Jones marshes of 20 to 160 f.Lg g-I (Dicker & Smith 1980b; 1974; Hanson 1977a; Talbot 1982) and mangroves Casselman et al. 1981) and it is likely that such levels (Zuberer & Silver 1975, 1978). In this study, the supply were inhibitory to ARA in this study. However, Kimball of organic carbon does not appear to be a major factor & Teas (1975) suggested that ammonium may be limiting rates of ARA, especially in the wet and dry mat immobilized by adsorption onto soil particles in habitats, where no response to sucrose supplementation mangrove soils and ARA may therefore not be so was shown. In habitats rich in organic carbon, such as severely limited. Severe inhibition of ARA is apparent mangrove roots and litter, limited response to carbon between 1 and 5 mg I-I and this is within the range amendment has been shown (Zuberer & Silver 1978). reported in some salt marshes (Carpenter et al. 1978; Zuberer & Silver (1978) and Hicks & Silvester (1985) Teal et al. 1979; Capone 1982), but much higher than in related ARA to in situ levels of organic carbon in others (Patriquin & Keddy 1978; Dicker & Smith 1980b; mangrove soil. Higher rates of nitrogen fixation in plant- Casselman et al. 1981). S.Afr.J.Bot. , 1989, 55(4) 445

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