ACETYLENE REDUCTION BY BLUE-GREEN ALGAE IN SUB TROPICAL GRASSLAND

Transcription

1 New Phytol. (1977)78, ACETYLENE REDUCTION BY BLUE-GREEN ALGAE IN SUB TROPICAL GRASSLAND BY KEITH JONES Department of Botany, University of Pretoria, Pretoria 2, South Afriea and Department of Biological Sciences, University of Lancaster, Lancaster LAI 4YQ, England* {Received 25 June 1976) SUMMARY In situ measurements using the acetylene reduction technique showed that mats of bluegreen algae found amongst kikuyu grass fixed nitrogen throughout the day and night. Daylight fixation increased throughout the morning, fell during the hottest part of the day, and reached a peak (19.2 mg C2H4 m~^ h~*) during the afternoon. Nitrogen fixation was higher on overcast days than on hot sunny days. Nocturnal fixation was also due to bluegreen algae and was sensitive to temperature changes. INTRODUCTION Information on nitrogen-fixing algae in the tropics and sub-tropics is fragmentary and most of it is for continents other than Africa (Eogg et al, 1973). Such information is primarily concerned with blue-green algae of arid soils or paddy fields. In this paper work is described relating to nitrogen fixation by mats of blue-green algae found in lawns around Pretoria where temperatures are high during the summer months but the soils are not arid. MATERIALS AND METHODS Field assays for assays for acetylene reduction Triplicate samples (the equivalent of 1 cm^) of the soil surface covered with bluegreen algae were placed in serum bottles (volume 6 ml). Care was taken to keep the algal mats intact. Acetylene (1 ml) was added via a rubber stopper without prior purging of the atmosphere (Stewart, 1971) and the acetylated samples replaced at the site of collection. After incubation for an hour samples of gas (1 ml) were removed from the serum bottles and analysed for ethylene production using a Beckman GC-5 gas chromatograph fitted with a 3-m stainless steel column (internal diameter 3 mm) packed with Poropak R (Waters Assoc. Inc.). The column temperature was 6 C and the flow rate of the helium carrier gas was 3 ml min"^ A flame ionization detector was used. Laboratory assays for acetylene Experiments were carried out in the laboratory with algal mats removed from the field and with suspensions of a culture of Nostoc sp. isolated from the algal mats. Acetylene reduction was assayed in serum bottles at a variety of light intensities and temperatures. * Permanent address. 421

2 422 KEITH JONES The algae which were unialgal but not bacteria-free were cultured routinely in liquid medium free of combined-nitrogen (Allen and Arnon, 1955) at room temperature and at 431 lux. RESULTS Mats of blue-green algae are found on soil amongst kikuyu grass (Pennisetum clandestinium Hochst, ex Chiou,) and are abundant in the grounds of the University of Pretoria, The algal mats are composed almost entirely of Nostoc sp. The effect of incubation time on acetylene reduction by Nostoc sp. (a) When in an algal mat The results illustrated in Fig. 1 show that acetylene reduction by algal mats was linear 'E o 3 2 ^ 2 3 TJ 2 E 1 1 ) <3~ V 25 I I 5 75 I 1 Time (h) 125 J I I 1 15 Time (h) 2 25 Fig. 1 Fig. 2 Fig. 1. The effect of time of incubation on acetylene reduction by blue-green algal mats. (Algal mats were acetylated at time and triplicate samples were analysed for ethylene production at various intervals.) Fig. 2. The effect of time of incubation on acetylene reduction by Nostoc sp. in culture. (1 ml suspensions of Nostoc sp. were acetylated at time and triplicate samples analysed for ethylene production at regular intervals.) with time for over 5 days. Similar results showing linearity of acetylene reduction over -5 h were obtained in short term experiments carried out at 3-min intervals using 3-min incubations with acetylene. Therefore, even in small serum bottles the duration of incubation with acetylene is not critical when measuring acetylene reduction by algal mats. Incubation times of one hour were normally used for in situ experiments. (b) When in culture Similar experiments were carried out using unialgal suspensions (1 ml) of Nostoc sp. The results (Fig. 2) show that the rate of acetylene reduction fell after a few hours. Addition of above ambient concentrations of carbon dioxide had little effect in the first 2 h but significantly increased acetylene reduction over a two day period (Table 1),

3 Acetylene reduction by blue-green algae 423 Table 1. The effect of increased carbon dioxide concentration on acetylene reduction by Nostoc sp. in culture Carbon dioxide concentration (% of total gases) (Mg C After 2 h Acetylene reduction mg"' dry algae) After 48 h Ambient (.3) Triplicate samples of algal suspension were acetylated at time and incubated in a phytotron at 27 C and 431 lux. In situ acetylene reduction by algal mats over a 24-h period (a) During daylight The results for acetylene reduction are shown in Fig. 3 and should be collated with those for light intensity and temperature Time of day (hours) Fig. 3. (a) Temperature (o) and light intensity ( ) at the soil surface during the experimental period, (b) In situ acetylene reduction by algal mats over a 24-h period. (2 February 1976). Triplicate samples of algal mats were collected at hourly intervals, acetylated and replaced in the field. After an hour gas samples were analysed for ethylene. Acetylene reduction increased steadily throughout the morning concommitant with increases in light intensity and temperature. It fell during the hottest part of the day (12. until 14. hours) and increased to a peak between 15. and 16. hours. Thereafter it declined until nightfall. Experiments carried out on successive days, the one hot and sunny and the overcast and cool, showed that acetylene reduction between 1. and 14. hours was higher on the overcast day (Table 2).

4 424 KEITH JONES Table 2. A comparison of acetylene reduction by mats of blue-green algae on a sunny and an overcast day Weather Sunny Cloudy Soil surface temperature CC) 4 23 Light intensity (lux) 13, 13 Acetylene reduction (Mg CjH^ m Triplicate samples of algal mats were taken on successive days, acetylated and replaced at the site of collection at 1. hours. Assays for ethylene production were carried out at 14. hours. Table 3. Acetylene reduction by algal mats amended with glucose Time after addition of glucose (h) Acetylene reduction (Mg CjH^ cm,-2 amended algal mat) 2,.1 13, Algal mats were placed in serum bottles with 1% glucose solution and incubated in the dark at 2 C. At various intervals triplicate bottles were acetylated and analysed for ethylene production after further incubation of 1 h. Table 4. Acetylene reduction by unialgal suspensions of Nostoc sp. Length of time in the dark (h) Acetylene reduction (Mg CjH^ mg-^ dry algae) Samples of algal suspension in serum bottles were placed in the dark at time and triplicate samples were acetylated at hourly intervals.

5 Acetylene reduction by blue-green algae 425 (b) During darkness Acetylene reduction continued throughout the night (1.5 h) and only decreased sharply when the temperature dropped prior to dawn. To determine whether any of the dark acetylene reduction was due to bacteria the algal mats (1 cm^) were amended with 1 ml glucose solution (1% w/v) and tested for acetylene reduction at regular intervals. The results, shown in Table 3, indicate that dark acetylene reduction by the algae is inhibited by glucose and that bacterial acetylene reduction can only be detected when sufficient numbers of bacteria have developed. Adjacent soils with no algal covering gave similar results with ethylene being detected 48 h after acetylation. Unialgal- cultures of Nostoc were tested for dark acetylene reduction by removing them from light and testing for acetylene reduction at hourly intervals. The results (Table 4) show that the algae continue to reduce acetylene for several hours after being placed in darkness. DISCUSSION Acetylene reduction by uni-algal suspensions of Nostoc sp. in small serum bottles increased linearly for several hours and then slowed down. Acetylene reduction may cease altogether within 24 h although this is dependent on such variables as age of the culture, temperature and light intensity and may continue into the second day (Table 1). There are three possibilities which could account for the decrease in rate: acetylene toxicity, build up of oxygen, and depletion of carbon dioxide. The first is unlikely as in short-term experiments acetylene has been shown to have little effect on general metabolism (Fogg et al., 1973). Oxygen, in concentrations above that normally found in air, has been shown to inhibit nitrogen fixation by blue-green algae (Stewart and Pearson, 197), but such conditions are unlikely to arise in the serum bottles as the amount of oxygen produced will be dependent on the original amount of carbon dioxide present in the bottles (.3% of the atmosphere). The probable reason for the decrease is that carbon dioxide has become limiting for photosynthesis and there is a shortage of energy for nitrogen fixation. An increase in the levels of carbon dioxide in the bottles to above ambient (.5 and.1%) had little short term effect on the rate of acetylene reduction but increased the amount of acetylene reduced over 48 h by 79 and 17% respectively. Similar results were not obtained with fresh algal mats from the field. Under identical conditions to those used with the algal isolate acetylene reduction increased linearly for over 5 days. It is possible to explain the difference by the presence of bacteria in the algal mats. The inference being that the bacteria utilize oxygen produced by the algae and release carbon dioxide which allows the algae to continue with photosynthesis and to fix nitrogen. Therefore the length of time that the algal mats are incubated with acetylene is not critical provided that conditions such as light intensity and temperature are not extreme. However, in the in situ experiments with algal mats incubation times of 1 h were used so that the effects of fluctuations in temperature and light intensity could be detected. Short exposure times to acetylene were also used with algal cultures as has been suggested by the pioneers of the method (Stewart, Fitzgerald and Burris, 1967; Hardy et al., 1968). The results for the field experiments showed that acetylene reduction occurred throughout the day and night giving a total for the 24-h period of 183 mg C2H4 m"^. This is the equivalent of 61 mg nitrogen fixed m~^ if the widely accepted conversion figure of 3:1 is used (Stewart et al., 1967; Hardy et al, 1968). Significantly lower rates were found during the hotter parts of the day and it would seem that soil surface temperatures of above 4 C

6 426 KEITH JONES inhibit acetylene reduction by the algal mats. Excessive light intensity may also play a part in reducing nitrogen fixation (Stewart, 1974) and it is possible that both high light intensities and temperatures limit activity by the algal mats used in this study. When both temperature and light intensity fell during the afternoon acetylene reduction reached its highest value for the day (19.2 mg C2H4 m~^ h~^). Acetylene reduction during the hottest part of a sunny day was only 1% of that during the same hours of an overcast day. Acetylene reduction by the algal mats continued through the night and such fixation was due to the algae and not bacteria. Uni-algal isolates from the mats also reduced acetylene in darkness and bacterial fixation in glucose-amended mats only occurred after long term incubation. Nitrogen fixation in the dark by terrestrial algae has also been noted by Henriksson (1971) and Paul, Myers and Rice (1971), but more is known from aquatic habitats (Horne and Fogg, 197; Lannergren, Lungren and Granhall, 1974; see reviews by Fay, 1973; Fogg et al, 1973; Stewart, 1973; Fogg, 1974). Unlike the algal mats from temperate soils that Henriksson (1971) worked with, acetylene reduction by algal mats from amongst kikuyu grass is effected by temperature. There is a correlation between the pre-dawn drop in temperature and decreased acetylene reduction by the algal mats. When the pre-dawn samples were placed in the dark at 2 C acetylene reduction was 13.9 mg C2H4 m~^ h"^ compared with.9 mg C2H4 m~^ h~^ for those mats in the field. ACKNOWLEDGMENTS The author is grateful to members of the department of Botany at the University of Pretoria and in particular to Professor N. Grobbelaar for his interest and for making the visit to Pretoria possible. The receipt of a post-doctoral bursary from the CSIR is gratefully acknowledged. REFERENCES ALLEN, M. B. & ARNON, D. I. (1955). Studies on nitrogen-fixing blue-green algae. L Growth and nitrogen fixation by Anabaena cylindrica Lemm. PI. Physiol., Lancaster, 3, 366. FAY, P. (1973). The heterocyst. The Biology of Blue-green Algae (Ed. by N. G. Carr & B. \. Whitton), pp Blackwell Scientific Publications, Oxford. FOGG, G. E. (1974). Nitrogen Fixation. Algal Physiology and Bioehemistry (Ed. W. D. P. Stewart), pp Blackwell Scientific Publications, Oxford. FOGG, G. E., STEWART, W. D. O., JACKSON, E. K. & BURNS, R. C. (1968). The acetylene-ethylene assay for Nj fixation: laboratory and field evaluation. PI. Physiol, Lancaster, 43,1185. HENRIKSSON, E. (1971). Algal nitrogen fixation in temperate regions. Biological Nitrogen Fixation in Natural and Agricultural Habitats (Ed. T. A. Lie & E. G. Mulder), pp Plant and Soil special volume. HORNE, A. J. & FOGG, G. E. (197). Nitrogen fixation in some English Lakes. Proc. R. Soc. B., 175, 351. LANNERGREN, C, LUNGREN, A. & GRANHALL, U. (1974). Acetylene reduction and primary production in Lake Erken. Oikos, 25, PAUL, E. A., MYERS, R. J. K. & RICE, W. A. (1971). Nitrogen fixation in grassland and associated ecosystems. Biological Nitrogen Fixation in Natural and Agricultural Habitats (Ed. by T. A. Lie & E. G. Mulder), pp Plant and Soil special volume. STEWART, W. D. P. (1971). Physiological Studies on Nitrogen-Fixing Blue-Green Algae. Biological Nitrogen Fixation in Natural arid Agricultural Habitats (Ed. by T. A. Lie & E. G. Mulder), pp Plant and Soil special volume. STEWART, W. D. P. (1973). Nitrogen Fixation. The Biology of Blue-Green Algae (Ed. by N. G. Carr & B. A. Whitton), pp Blackwell Scientific Publications. STEWART, W. D. P. (1974). Blue-green algae. The Biology of Nitrogen Fixation (Ed. A. Quispel), pp North Holland Publishing Co., Oxford. STEWART, W. D. P., FITZGERALD, G. P. & BURRIS, R. H. (1967). In situ studies on Nj fixation using the acetylene reduction technique. I^oc. nat. Acad. Sci. U.S.A. 73, STEWART, W. D. P. & PEARSON, H. W. (197). Effects of aerobic and anaerobic conditions on growth and metabolism of blue-green algae./^oc. R. Soc. B. 175,

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