NITROGENASE AND HYDROGENASE IN CYANOPHILIC LICHENS

Transcription

1 New Phytol. (1982) 92, NITROGENASE AND HYDROGENASE IN CYANOPHILIC LICHENS BY J. W. MILLBANK Department of Pure and Applied Biology, Imperial College, London SW7 2BB, U.K. {Accepted 1 May 1982) SUMMARY The in vivo evolution of hydrogen by three nitrogen fixing lichens, Peltigera membranacea, Peltigera polydactyla and Lobaria pulmonaria has been studied. The hydrogen evolved concomitant with nitrogen fixation was recycled by means of an uptake hydrogenase and in general tbe net evolution was zero or small at 5 and 15 C. At 25 C there was appreciable net evolution of hydrogen in the Petigera spp. but Lobaria showed no net evolution. Direct comparison of acetylene reduction and nitrogen fixation using ^^Na gave ratios of six and four for Peltigera and Lobaria; these figures imply a significant production of hydrogen and indicate an effective mechanism for countering energy loss under normal in vivo conditions. INTRODUCTION The multisubstrate characteristic of nitrogenase is well known and the production of hydrogen gas by diazotrophs in vivo has been demonstrated by Schubert and Evans (1976) and Smith, Hill and Yates (1976). Walker, Partridge and Yates (1981) observed that hydrogen production can represent a considerable waste of biological energy. The proportion of nitrogenase activity devoted to hydrogen production is always substantial and under certain conditions of ph, temperature and enzyme component ratio can become very large (Burris et al., 198). A undirectional ' uptake hydrogenase' mechanism has been demonstrated in a number of organisms, however, which enables some of the hydrogen gas to be recycled and thus ameliorate the energy loss (Dixon, 1972). Walker and Yates (1978) have shown that in Azotobacter hydrogenase activity was consistently high under carbon-limited conditions, and deduced that hydrogen was recycled to provide electrons and energy for nitrogen reduction, and also contributed to the respiratory activity concerned with protecting nitrogenase. In the cyanobacteria, hydrogen evolution has attracted the attention of investigators for a number of years (Benemann and Weare, 1974; Rao, Rosa and Hall, 1976; Bothe et al., 198). In general, rates of hydrogen evolution are very low (Jones and Bishop, 1976) but can be increased by the use of inhibitors such as carbon monoxide and acetylene. The optimum conditions for hydrogen evolution have not been established, and the only report of evolution in a symbiotic system is that of Newton (1976) using Azolla. The lichen symbiosis is notable for ability to withstand environmental stress (Smith, 198) and nutrient availability fluctuates widely; energy conservation mechanisms are therefore likely to be well developed and this communication reports the findings of a study of the effect of temperature and light intensity on hydrogen evolution by a number of nitrogen fixing lichens. Further, hydrogen production concomitant with nitrogen fixation, even though the hydrogen is recycled, determines the relationship between acetylene reduction and nitrogen X/82/ $3./ 1982 The New Phytologist

2 222 J. W. MiLLBANK fixation; the theoretical value of three is never observed in vivo, and this paper reports values obtained for the two processes under conditions as closely identical as was possible, to quantify the biological advantage of an effective energy conserving process. MATERIALS AND METHODS Lichens Peltigera membranacea, P. polydactyla and Lobaria pulmonaria were collected from Dunstaffnage, Argyll, (British National Grid reference NM 88344) and kept in a lighted (5 fie m"^ g-i) cooled incubator at 8 C with a daylength of 16 h. They were maintained in a moist state and used within 4 weeks of collection. Measurement of hydrogen evolution Samples of the atmosphere in the closed experimental vessels were analysed by vapour phase chromatography using a Pye instrument, model 14. The columns (1-5 m) were packed with molecular sieve 5A, 1-12 mesh, and used at a temperature of 6 C; the carrier gas was argon or oxygen-free nitrogen, at a flow rate of 1 ml s"\ The katharometer detector was held at 15 C with a filament current of 11 ma. The output was displayed on a Servoscribe Is multirange chart recorder, which enabled a high sensitivity to be obtained; a peak height of 32 mm resulted from the injection of 1 nmole of hydrogen gas into an argon carrier stream. Measurement of nitrogenase activity Nitrogenase activity was estimated by the acetylene reduction technique (see Postgate, 1971). Thallus material was incubated in glass vials fitted with rubber serum caps, under an atmosphere containing 1 % (v/v) acetylene. Samples of gas were analyzed for ethyelene by vapour phase chromatography using a 1 m 'Porapak' R column at 5 C and a flame ionization detector; the carrier gas was argon or oxygen-free nitrogen. Incubation procedure Thalli were thoroughly moistened by spraying with glass distilled water, the surface blotted, and they were maintained in this saturation state in the cooled illuminated incubator for 24 h before experimentation. Thallus samples of approximately 1 cm^ area were used, incubated in glass vials of c. 3 ml capacity, containing an atmosphere of air + 5 % (v/v) carbon dioxide, as preliminary trials had established that carbon dioxide depletion could take place within 6 min at 25 C and 3 fie m~'^ s~^ illumination in the vessels used. The vials were illuminated at a quantum density of up to 3 /^E m~^ s~^ at the thallus surface, using a combination of mercury-fluorescent and tungsten filament lamps. Flux density was measured by means of a Lambda Instruments Inc. quantum meter. Substrate and inhibitor gases were added as appropriate by hypodermic syringe. Estimation of ^^N^ enrichment Replicate thalli after incubation were dried overnight at 1 C, weighed, and ground to powder in a pestle and mortar. Samples were taken for nitrogen estimation by a micro-kjeldahl technique (Umbriet, Burris and Stauffer, 1957) followed by room temperature distillation in Conway Microdiffusions Units

3 Nitrogenase and hydrogenase in cyanophilic lichens 223 (Conway, 1957). After titration the boric acid solution containing the nitrogen as ammonium chloride was acidified to ph 3 with 1 N sulphuric acid, transfered to glass vials and evaporated to dryness at 8 C. Nitrogen gas was generated from these vials by the addition of lithium hypobromite in the apparatus described by Ross and Martin (197) and mass analysis carried out in an AEl MS2 mass spectrometer. RESULTS AND DISCUSSION Hydrogen evolution by Peltigera membranacea Table 1 sets out the rates of production of hydrogen gas by P. membranacea thalh at three temperatures. At 5 C, it is evident that hydrogen evolution (column 2) is zero or below measurable level. Nitrogenase activity however, is appreciable at this temperature (column 5). Evidently uptake hydrogenase activity is sufficient to enable complete recycling of any hydrogen gas produced concomitant with Table 1. The rate of production of hydrogen gas by Peltigera membranacea. Each line represents one large thallus specimen Temperature Air H2 Nitrogenase activity evolution (nmol h"* g * thallus) (Ethylene production; - mmol h 1 g 1 thallus) Ar + Og Ar + O, + CO-HC,H * , Not determined. * No CoHo in this case. ANP 92

4 224 J. W. MiLLBANK nitrogen fixation. No production of hydrogen could be demonstrated in the presence of acetylene. At 15 C net hydrogen evolution in air (column 2) was normally negligible (with exceptions, see Table 2). Furthermore, in an atmosphere composed of argon, oxygen and carbon dioxide (column 3) in which hydrogen production by nitrogenase is maximal (up to six times the rate in air with this material as will appear later) net hydrogen evolution was again small (compare columns 3 and 5). The rate of ethylene production (column 5) is a good indication of the maximal rate of hydrogen production under argon. Potential uptake hydrogenase activity was clearly much greater than the normal requirement of the phycobiont Nostoc cells. Addition of acetylene showed the nitrogenase activity to be characteristically variable but substantial, with no simultaneous hydrogen evolution. The addition of 5 % (v/v) carbon monoxide, an inhibitor of nitrogen and acetylene reduction by nitrogenase, was expected to give rise to maximal production of hydrogen, probably equivalent to ethylene production. However rates of hydrogen evolution in the presence of carbon monoxide at concentrations of 5 or 1% (v/v) did not exceed 5% of the rates of ethylene production by the same thalli (compare columns 4 and 5). Evidently the uptake hydrogenase was not completely inhibited at these concentrations of carbon monoxide; Bothe et al (1977) also report an inability to achieve hydrogen production equivalent to that ethylene, using Anabaena cylindrica; clearly the optimum conditions for hydrogen evolution have yet to be established. At 25 C, production of hydrogen by P. membranacea thalli in air was consistent, though not large, and in an atmosphere of argon and oxygen very much greater than in air (see columns 2 and 3). This indicated that the uptake hydrogenase activity at this temperature was insufficient to recycle the hydrogen produced at maximum rate by nitrogenase. Again, carbon monoxide did not give rise to a rate of hydrogen production equivalent to that of ethylene in the presence of acetylene (columns 4 and 5). A noticeable feature referred to above, was that some specimens of lichen thalli had markedly deficient uptake hydrogenase activity. Such examples appeared to be random, being portions of large, apparently homogeneous thalli. Large variation in uptake hydrogenase activity in the cyanobacteria have been noted by a number of workers, and is emphasized by Bothe, Tennigkeit & Eisbrenner (1977). In the study reported here, of the 21 thallus samples examined at 15 C, six Table 2. The evolution of hydrogen by six specimens o/peltigera membranacea having reduced uptake hydrogenase activity Temperature - ( C) Air Hydrogen evolution (nmol h^' g"' thallus) Ar + O, Nitrogenase activity (Ethylene production; Ar + O, + CO + C,] H, nmoi Air n + g C,H, tnaiius^ , Not determined.

5 Nitrogenase and hydrogenase in cyanophilic lichens 225 showed evidence of reduced or deficient uptake hydrogenase activity, the figures of hydrogen evolution in air and argon being summarized in Table 2. It is emphasized that at 5 C in air, no hydrogen production was ever observed; and at 25 C, it was always observed although sometimes at very low rates. Hydrogen evolution by Peltigera polydactyla This is given in Table 3, and was measured at 5, 15 and 25 C, as with P. membranacea. The results were similar, no hydrogen evolution occurring at 5 C occasional thalli evolving hydrogen at 15 C, and consistent but small evolution occurring at 25 C. Table 3. The rate of production of hydrogen gas by Peltigera polydactyla. Each line represents one large thallus specimen Temperature - ( C) Air Hydrogen evolution (nmol h~* g~» thallus) Ar + O^ Ar + O^ + CO + C^lH2 Nitrogenase activity >«1_ 1 i (iitnylene production; nmol h~* g~' thallus) * * ~ * Just detectable., Not determined. Hydrogen evolution by Lobaria pulmonaria When this lichen was examined, at 5, 15 and 25 C in air at 3/^E illumination, no net hydrogen evolution was observed at any temperature; reduction of acetylene when added subsequently was very rapid, mean values (three estimates) of 172, 327 and 593 nmol h"^ g"^ thallus being obtained at the three temperatures. Assuming that hydrogen molecules were being produced at at least the same rate at nitrogen molecules were being reduced (i.e. at 25 % of the rate of production of ethylene) this hydrogen would have been easily demonstrable by the method employed, and must therefore have been recycled. The effect of light intensity on hydrogen evolution Two trials were carried out in which four replicate P. membranacea thalli were examined for hydrogen evolution in air at 25 C and 3 /le m~^ s'^ illumination. 8-2

6 226 J. W. MiLLBANK Any evolution was normally constant for up to 3 h. After 1 h the light intensity was reduced to 1 fie m.~^ s~^ by appropriately increasing the distance to the light source. No change in the rate of evolution of hydrogen was noted in either trial; a diminution of light intensity might be expected to give rise to carbon limitation in the long term, and thus possibly to reduce the rate of hydrogen evolution. However, the rates of evolution were in any case small, and several hours may well have been necessary to demonstrate the effect. The effect of complete darkness on a thallus evolving appreciable hydrogen (at 25 C) is envisaged as a future trial. The relation between acetylene reduction and ^^N^ incorporation under identical conditions The foregoing shows that the mechanisms for recycling hydrogen produced copcomitantly with fixation of dinitrogen are well developed and effective in the lichen algae tested. It is not therefore possible to deduce the corresponding rate of nitrogen fixation from observed rates of total electron flux (assumed to be very nearly identical to rates of acetylene reduction). Only sequential estimates of nitrogen fixed and acetylene reduced, by the same thalli under identical conditions. Table 4. The ratio of acetylene reduced to nitrogen fixed by Peltigera membranacea, P. polydactyla and Lobaria pulmonaria Lichen Ethylene production (nmol h~' g~') Nitrogen fixed (nmol h~' g^') CgHj reduced nitrogen fixed Electron allocation coefficient Peltigera membranacea P. polydactyla Lobaria pulmonaria can provide data for the 'electron allocation'. Baseline data for P. membranacea, P. polydactyla and Lobaria pulmonaria are presented in Table 4. The conditions used were 15 C, 3/*E m"^ s^^ light intensity and an atmosphere composed of 8% nitrogen, 19-5% oxygen and -5% carbon dioxide. High concentrations of carbon dioxide were used to ensure that depletion did not occur during the period of incubation for ^^Ng incorporation (5 h). A long period was essential as the quantity of nitrogen gas required to provide true atmospheric simulation demanded a rather low level of ^^Ng enrichment (3 %) in view of the expense of ^^Ng labelled materials. At the end of the incubation period acetylene was injected to a partial pressure of 1 (1 % v/v). This stopped nitrogen fixation, and ethylene production was measured by sampling every 5 min over the following 3 min. The thalli were then dried at 15 C and, after weighing, were ground to a fine powder in a pestle and mortar. Ten replicate samples were taken for total nitrogen and ^^Ng enrichment estimations. The ratios of four and six obtained indicate that hydrogen evolution accounted for a substantial portion of the electron flow via the nitrogenase systems, and thus emphasise the value of the uptake hydrogenase. A number of authors have discussed the function of uptake hydrogenases in diazotrophs, and normally cite three. These are: (1) protection of nitrogenase against possible hydrogen inhibition, (2) respiratory protection of nitrogenase by oxygen scavenging and (3) supplementary energy generation. A useful review is that of Robson and Postgate (198).

7 Nitrogenase and hydrogenase in cyanophilic lichens 227 The energetics of nitrogen fixation are such that mechanisms providing a significant economy of the overall energy requirement would impart worthwhile biological advantages to organisms inhabiting stressful environments with uncertain and irregular nutrient sources. Nutrient limitation would seem to be a normal feature in lichen phycobionts, where the non-photosynthetic (fungal) symbiont can be considered to exploit the synthetic abilities of its photosynthetic partner (Smith 1975). The tendency for the recycling mechanism to be less than completely effective at temperatures above 15 C is perhaps not unexpected in view of the temperate to sub-arctic habitats of the species investigated. Studies of nitrogen fixing lichens from warmer habitats would be illuminating. The proportion of electrons devoted to nitrogen reduction and proton production as revealed by the sequential estimation of i^ng fixed and acetylene reduced^ indicates that a potentially very important drain of energy resources can exist in nitrogen fixing lichen thalli, even under near-ideal metabolic conditions. That an effective mechanism for countering this loss exists can be demonstrated is further evidence of some adaptation to environment. ACKNOWLEDGEMENTS It gives me great pleasure to thank Mr J. D. Olsen for his valuable technical assistance. REFERENCES BENEMANN, J. R. & WEARE, N. M. (1974). Hydrogen evolution by nitrogen fixing Anabaena cylindrica Science, 184, BOTHE, H., TENNIGKEIT, J. & EISBRENNER, G. (1977). The hydrogenase-nitrogenase relationship in the blue green alga Anabaena cylindrica. Planta, 133, BOTHE, H., NEURER, G., KELBE, I. & EISBRENNER, G. (198). Electron donors and hydrogenase in nitrogen fixing microorganism. In: Nitrogen Fixation (Ed. by W. D. P. Stewart & J. R. Gallon), pp Acadennic Press, London. BURRIS, R. H., ARP, D. J., BENSON, D. R., EMERICH, D. W., HAGEMAN, R. V., JONES, T., LUDDEN, P. W. & SWEET, L. J. (198). The biochemistry of nitrogenase. In: Nitrogen Fixation (Ed. by W. D. P. Stewart & J. R. Gallon), pp Academic Press, London. CONWAY, E. J. (1957). Microdiffusion Analysis and Volumetric Error. Crosby, Lockwood & Son, London. DIXON, R. O. D. (1972). Hydrogenase in legume root nodule bacteroids - occurrence and properties Archives of Microbiology, 85, JONES, L. W. & BISHOP, N. I. (1976). Simultaneous measurements of oxygen and hydrogen exchange from the blue green alga Anabaena. Plant Physiology, 57, NEWTON, J. W. (1976). Photoproduction of molecular hydrogen by a plant-alga symbiotic system. Science, PosTGATE, J. R. (1971). The Chemistry and Biology of Nitrogen Fixation, pp Plenum Press London. ' RAO, K. K., ROSA, L. & HALL, D. O. (1976). Prolonged production of hydrogen gas by a chloroplast biocatalytic system. Biochemical and Biophysical Research Communications, 68, ROBSON, R. L. & POSTGATE, J. R. (198). Oxygen and hydrogen in biological nitrogen fixation. Annual Review of Microbiology, 34, Ross, P. J. & MARTIN, A. E. (197). A rapid procedure for preparing gas samples for nitrogen** determination. The Analyst, 94, SCHUBERT, K. R. & EVANS, H. J. (1976). Hydrogen evolution: A major factor affecting the efficiency of nitrogen fixation in modulated symbionts. Proceedings of the National Academy of Sciences, U.S.A., 73, SMITH, D. C. (1975). Symbiosis and the biology of lichenised fungi. In: Symbiosis; Symposia of the Society for Experimental Biology, 29, SMITH, D. C. (198). Mechanisms of nutrient movement between the lichen symbionts. In: Cellular Interactions in Symbiosis and Parasitism (Ed. by C. B. Cook, P. W. Pappas & E. D. Rudolph), pp Ohio State University Press, Ohio U.S.A.

8 228 J. W. MiLLBANK SMITH, L. A., HILL, S. & YATES, M. G. (1976). Inhibition by acetylene of conventional hydrogenase in nitrogen fixing bacteria. Nature, 262, UMBRIET, W. W., BURRIS, R.H. & STAUFFER, J. F. (1957). Manometric Techniques. Burgess Publishing Co., Minneapolis, U.S.A. WALKER, C. C, PARTRIDGE, C. D. P. & YATES, M. G. (1981). The effect of nutrient limitation on hydrogen production by nitrogenase in continuous cultures of Azotobacter chroococcum. Journal of General Microbiology, 124, WALKER, C. C. & YATES, M. G. (1978). The hydrogen cycle in nitrogen fixing Azotobacter chroococcum. Biochimie, 6,

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