Showing papers on "Nitrogen fixation published in 1975"
TL;DR: There was no evidence for the operation of the glutamine synthetase/glutamate synthase system in ammonia assimilation in root nodules, suggesting that ammonia produced by nitrogen fixation in the bacteroid is assimilated by enzymes of the plant system.
Abstract: SUMMARY: The enzymes involved in the assimilation of ammonia by free-living cultures of Rhizobium spp. are glutamine synthetase (EC. 6.3.1.2), glutamate synthase (L-glutamine: 2-oxoglutarate amino transferase) and glutamate dehydrogenase (EC 1.4.1.4). Under conditions of ammonia or nitrate limitation in a chemostat the assimilation of ammonia by cultures of R. leguminosarum, R. trifolii and R. japonicum proceeded via glutamine synthetase and glutamate synthase. Under glucose limitation and with an excess of inorganic nitrogen, ammonia was assimilated via glutamate dehydrogenase, neither glutamine synthetase nor glutamate synthase activities being detected in extracts. The coenzyme specificity of glutamate synthase varied according to species, being linked to NADP for the fast-growing R. leguminosarum, R. melitoti, R. phaseoli and R. trifolii but to NAD for the slow-growing R. japonicum and R. lupini.
Glutamine synthetase, glutamate synthase and glutamate dehydrogenase activities were assayed in sonicated bacteroid preparations and in the nodule supernatants of Glycine max, Vicia faba, Pisum sativum, Lupinus luteus, Medicago sativa, Phaseolus coccineus and P. vulgaris nodules. All bacteroid preparations, except those from M. sativa and P. coccineus, contained glutamate synthase but substantial activities were found only in Glycine max and Lupinus luteus. The glutamine synthetase activities of bacteroids were low, although high activities were found in all the nodule supernatants. Glutamate dehydrogenase activity was present in all bacteroid samples examined. There was no evidence for the operation of the glutamine synthetase/glutamate synthase system in ammonia assimilation in root nodules, suggesting that ammonia produced by nitrogen fixation in the bacteroid is assimilated by enzymes of the plant system.
335 citations
TL;DR: periodic sampling within a brachytic maize cultivar revealed that maximum nitrogenase activity occurred at about the 75% silking stage, and N(2)-fixing Spirillum sp.
Abstract: N2 fixation in field-grown maize (Zea mays L.) plants was estimated by a nondestructive acetylene reduction method which permitted the plants to continue growing and produce seeds. Samples from six areas revealed mean nitrogenase activities of 74-2167 nmol of C2H4/(g of dry roots × hr) for 10 plants. Among 276 S1 lines planted in two field experiments, 17 lines were selected for further nitrogenase activity assays after prescreening. Variability within lines was high but significant differences among lines were obtained in one experiment. The best lines showed mean nitrogenase activities of 2026, 2315, and 7124 nmol of C2H4/(g of dry roots × hr), whereas the original cultivar reduced only 313 nmol. The highest value approaches the nitrogenase activity of soybean. If the theoretical 3:1 (C2H4/N2 reduced) conversion factor is used, a potential daily N2 fixation of 2 kg of N2/hectare can be calculated. Periodic sampling within a brachytic maize cultivar revealed that maximum nitrogenase activity occurred at about the 75% silking stage. Soil effects also were pronounced. N2-fixing Spirillum sp. could be isolated from all active root pieces when they were surface sterilized. These organisms appear to be primarily responsible for root nitrogenase activity in maize.
278 citations
TL;DR: Several plant metabolites, including sugars known to favour rhizobial growth and citric acid cycle intermediates, were examined for their effect on this latter system and as possible direct inducers of nitrogenase activity in cultured rhizobia.
Abstract: IT has been widely believed that the legume root-nodule bacteria (Rhizobium spp.; rhizobia) fix N2 only within the tissues of the host plant1. There have however been recent reports that a strain of cowpea rhizobia, 32H1, developed nitrogenase activity when grown in association with legume or non-legume plant cells2,3. The induction of nitrogenase was apparently due to a diffusible factor(s) secreted by the plant cells, since nitrogenase activity was detected when strain 32H1 was grown adjacent to, but not in contact with, tobacco cells3. Several plant metabolites, including sugars known to favour rhizobial growth4,5 and citric acid cycle intermediates6,7, were examined for their effect on this latter system and as possible direct inducers of nitrogenase activity in cultured rhizobia. This led to the formulation of a defined medium on which strain 32H1 fixed N2 in the absence of plant cells.
217 citations
TL;DR: In this paper, a quantitative estimate of the amount of nitrogen symbiotically fixed by a legume crop growing under normal field conditions was made by simultaneous determinations of the AN values by the legume and a non-nodulating crop.
Abstract: A quantitative estimate is made of the amount of nitrogen symbiotically fixed by a legume crop growing under normal field conditions. The method involves simultaneous determinations of the “AN” values by the legume and a non-nodulating crop, using15N-labelled nitrogen fertilizer. The fertilizer is applied at a low rate to the legume crop to avoid interference with the symbiotic N fixation process, but at a normal rate to the non-nodulating crop. The amount of symbiotically fixed nitrogen expressed in kg N/ha is calculated by multiplying the difference in A value between the legume and the non-nodulating crop with the % utilization of fertilizer N by the legume crop.
171 citations
TL;DR: There is a close and competitive relationship between the processes of nitrate reduction and nitrogen fixation, with the latter process dominating as the major source of fixed nitrogen after the plants have flowered and initiated pods.
Abstract: The patterns of nitrate uptake, nitrate reductase activity in the leaves, and nitrogen fixation by the nodules were investigated in field-grown soybeans (Glycine max (L.) Merr.) over the growing season. The level of nitrate-reductase activity generally paralleled the concentration of nitrate in the leaf tissue over the entire growing season. A precipitous drop in both parameters was noted within 2–3 weeks after flowering. These parameters decreased by 80–95% at mid-pod fill, a stage where ovule (seed) development was in the logarithmic growth phase, placing a heavy demand on the plant for both energy and fixed nitrogen. The activity of nitrogen fixation of soybean root nodules bore a reciprocal relationship to that of nitrate reductase. The maximum levels of nitrogen fixation were reached at early pod fill when nitrate reductase activity had dropped to 25% of maximum activity. A rapid loss of nitrogen fixation activity occurred shortly after bean fill was initiated, again at a time when the ovules were developing at maximal rates. The total protein content of soybean leaves increased over the season to a maximum level at mid-pod fill. This was followed by a 50% drop over the next 3-week period when the plants approached senescence. This drop corresponded to that found for nitrogen fixation. A similar pattern was noted for watersoluble proteins in the leaf. These studies suggest that there is a close and competitive relationship between the processes of nitrate reduction and nitrogen fixation, with the latter process dominating as the major source of fixed nitrogen after the plants have flowered and initiated pods. At this transitional stage, both soil and environmental effects could cause pertrubation in these processes that could lead to a nitrogen stress causing flower and pod abscission. The rapid decay of nitrogen fixation at the time of midpod fill also suggests a competition between roots (nodules) and pods for available photosynthate. This competition appears to lead to the breakdown of foliar proteins and senescence.
141 citations
TL;DR: Nitrogen fixation in the rhizospheres of field grown tropical forage grasses was studied by the acetylene reduction method and values varied considerably between sites but indicate the possible economic importance of several of the species studied.
Abstract: Nitrogen fixation in the rhizospheres of field grown tropical forage grasses was studied by the acetylene reduction method Values varied considerably between sites but indicate the possible economic importance of several of the species studied Maximal nitrogenase activity measured (nmoles C 2 H 4 g −1 dry roots h −1 ) was 754 for Pennisetum purpureum , 750 for Brachiaria mutica , 341 for Digitaria decumbens , 299 for Panicum maximum , 283 for Paspalum notatum , 269 for Cynodon dactylon , 41 for Melinis minutiflora and 29 for Hyparrhenia rufa Nitrogenase activity varied considerably with season and was maximal during active vegetative growth of two of the grasses Significant differences between Paspalum notatum ecotypes and cultivars in Azotohacter paspali occurrence and nitrogen fixation, indicate the possibility of plant breeding to enhance nitrogen fixation in grass rhizospherc associations Other research lines of agronomic importance are fertilizer effects In intact soil plant cores with the Paspalum system 10 parts/10 6 NH 4 + J-N inhibited nitrogenase activity within 2 h and 10 parts/10 6 NO − 3 -N within 4 h but after 1 week these effects were negligible In the field, nitrogenase activity on roots of P purpureum and D decumbens , assayed 2 weeks after top dressings of 20 kg N ha −1 as NH 4 NO 3 was not affected even after eight such dressings
110 citations
TL;DR: Nitrogen fixation by free-living Rhizobium was obtained in a defined liquid medium, using microaerophilic conditions, and the rate of fixation was comparable to that of bacteroids prepared from soybean nodules.
106 citations
01 Jan 1975
TL;DR: The role of photosynthesis and transpiration in the desiccation-induced inhibition of acetylene reduction (nitrogen fixation) was investigated in soybean and it was concluded that the inhibition of shoot photosynthesis accounted forThe inhibition of nodule acetylene Reduction at low water potentials.
93 citations
91 citations
TL;DR: The production of nitrogenase (C2H2) activity in Rhizobium sp.
Abstract: THE genus Rhizobium is characterised by its ability to elicit nodule formation and fix dinitrogen in roots of legumes and its species or groups are classified according to their legume host range. There is only one non-legume association reported, that of Trema aspera with a ‘cowpea-type’ strain of Rhizobium1. Dinitrogen fixation by Rhizobium in the absence of a host plant has not yet been demonstrated. I report the production of nitrogenase (C2H2) activity in Rhizobium sp. ‘cowpea strain’ growing in association with callus cultures of non-legumes as well as with cultures of legume species which do not form nodules with the cowpea bacteria.
91 citations
TL;DR: This work has shown that nodules formed on the non-legume Trema canabina are caused by a strain of Rhizobium which nodulated Vigna unguiculata (cowpea), possess nitrogenase activity and fix atmospheric nitrogen.
Abstract: THE maxim that nitrogen fixation by the root nodule bacteria, Rhizobium, is restricted to a formal symbiotic association with specific legumes has recently been challenged. Trinick1 showed that nodules formed on the non-legume Trema canabina (previously identified as T. aspera; M. J. Trinick, personal communication) by a strain of Rhizobium which nodulated Vigna unguiculata (cowpea), possess nitrogenase activity and fix atmospheric nitrogen. Soybean tissue cultures inoculated with R. japonicum2–4, or with cowpea strains of rhizobia3, also possess apparently functional nitrogenase as determined by the acetylene reduction assay5. Several attempts have failed to demonstrate nitrogenase activity in cultured rhizobia6, including cowpea strains7.
TL;DR: Nitrogen fixation, as measured by the acetylene reduction method, has been detected in situ in stratified sediment cores from Kingoodie Bay, Tay Estuary, northeast Scotland and it is possible that these bacteria play an important role in supplying nitrogen to the nitrogen deficient Kingoodies Bay sediments.
TL;DR: An attempt is made to evaluate the phytochemical properties of the substance Na6(CO3)(SO4), which has the potential to be a raw material for nitrogenase synthesis.
Abstract: KLEBSIELLA . .. . .. . . . . . . . . . . . ... . . . ... . . . . . . . . . . . . . . Control of Nitrogen Fixation Repression by fixed nitrogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Repression by 0] . Requirement of Mo for nitrogenase synthesis . N] as an inducer of nitrogenase synthesis .
TL;DR: Nitrogen fixation activity appeared to be inversely related to the ability of shipworms to obtain combined-nitrogen compounds in their diet, and could be a significant source of nitrogen for shipwornms and perhaps other oceanic organisms that ingest terrestrial plant material.
Abstract: Nitrogen fixation is associated with four shipworl species A bacterium capable of fixing nitrogen under anaerobic conditions and of liquefying cellulose in culture has been isolated from the gut of one species High fixation rates (up to 15 micrograms of nitrogen per milligram dry weight per hour), which resulted in a doubling of cellular nitrogen in as little as 14 days, was associated with Teredora malleolus from the Sargasso Sea Three species from coastal waters were assayed, and of these juveniles showed the highest fixation rates Nitrogen fixation activity appeared to be inversely related to the ability of shipworms to obtain combined-nitrogen compounds in their diet It could be a significant source of nitrogen for shipwornms and perhaps other oceanic organisms that ingest terrestrial plant material
TL;DR: The nitrogen fixation proved to be more susceptible than photosynthesis, in both organisms, and in all cases treatments at pH 5.8 were more inhibitory than at higher pH-values.
Abstract: Responses of photosynthesis and nitrogen fixation to NaHSO3 (10−5−5 × 10−3M) were investigated in the lichen Stereocaulon paschale (L.) Fr. and the blue-green alga Anabaena cylindrica Lemmermann. The treatments were performed in buffered media with varying pH (5.8–8.1) and light conditions (0–32 W × m−2). The activities of the intact organisms were investigated, under the same environmental conditions, with 14C liquid scintillation and acetylene reduction techniques respectively.
The nitrogen fixation proved to be more susceptible than photosynthesis, in both organisms, and in all cases treatments at pH 5.8 were more inhibitory than at higher pH-values. Treatment with 5 × 10−4M NaHSO3 at pH 5.8 caused no reduction of photosynthesis in S. paschale, while the inhibition of nitrogen fixation was 97%. For A. cylindrica the corresponding values were 40% and 75% respectively. Short-time treatments of A. cylindrica showed that the nitrogen fixation was more rapidly affected than photosynthesis. The inhibition of nitrogenase activity and CO2-fixation was smaller in the dark and increased at higher light intensities. Both processes showed a good capacity for recovery after removal of the NaHSO3 solution. Also the clumping ability of A. cylindrica was disturbed by NaHSO3 treatments.
TL;DR: A mechanism of biological nitrogen fixation is formulated which is in accord with the available enzymological evidence and detected as intermediates in the reduction of molecular nitrogen under nonenzymatic conditions.
Abstract: The development of biologically relevant model systems of nitrogenase permitted the simulation of virtually all known reactions of the nitrogen reducing enzymes under nonenzymatic conditions. On the basis of these experiments, a mechanism of biological nitrogen fixation is formulated which is in accord with the available enzymological evidence. The key reactions of the substrates of nitrogenase occur at a molybdenum active site. The non-heme iron, which is bound to sulfur and protein-S⊝ groups, mediates the transport of electrons to the molybdenum active site but does not participate directly in the reduction of the substrates. ATP is required for the acceleration of the reduction and activation of the molybdenum site and is hydrolyzed to ADP and inorganic phosphate. Diimine and hydrazine were detected as intermediates in the reduction of molecular nitrogen under nonenzymatic conditions.
TL;DR: The distribution of radioactivity among the constituents of the basic fraction confirms the importance of glutamate, glutamine and aspartate as early products of the metabolism of the fixed nitrogen in broad bean nodules receiving ample supplies of new photosynthates.
Abstract: SUMMARY
Nitrogenase activity of the root nodules of field-grown Vicia faba increased during plant growth until 3–4 weeks after the onset of flowering. The subsequent decline in activity was never greater than to 20% of the maximum, until finally plants were killed by frost. Nodules of medium size (100–200 mg fresh weight) showed greatest nitrogenase activity.
After photosynthesis in 14CO2 for 30 min, maximum accumulation of 14C-assimilates in the nodules occurred within 90 min of synthesis. The relatively low levels of radioactivity associated with the organic acid fraction of the nodules, the absence of sucrose which is present in large quantities in the roots and the appearance of radioactivity in the nodule amino acids within 30 min of feeding the shoot 14CO2 show that photosynthates are rapidly metabolized on arrival in the nodules. The distribution of radioactivity among the constituents of the basic fraction confirms the importance of glutamate, glutamine and aspartate as early products of the metabolism of the fixed nitrogen in broad bean nodules receiving ample supplies of new photosynthates.
TL;DR: Several Bacillus strains from the rhizosphere of Ammophila arenaria appeared on ‘nitrogen-free’ agar plates and characterisation suggests that these strains are new nitrogen-fixing Bacillus species.
Abstract: Several Bacillus strains, from the rhizosphere of Ammophila arenaria, appeared on ‘nitrogen-free’ agar plates. They were able to grow in nitrogen-poor medium to which 0.1% yeast extract was added. Three of these bacilli were tested for their ability to fix nitrogen using the acetylene reduction assay. The C2H2-reducing activity was determined at 8-hour intervals during their growth cycle. C2H2 reduction (and accordingly N2 fixation) was greater under anaerobic than aerobic conditions. Additions of 0.1% CaCO3 significantly increased the C2H2-reducing activity under both conditions. Characterisation suggests that these strains are new nitrogen-fixing Bacillus species. re]19740121
01 Jan 1975
TL;DR: The main sources of nitrogen input are biological nitrogen fixation, precipitation and dry deposition in many tundra areas as discussed by the authors, and even a small nitrogen input from e.g. nitrogen fixation is important for the nitrogen balance of the ecosystem.
Abstract: Combined nitrogen is probably the limiting nutrient factor for primary production in many tundra areas. The removal of combined nitrogen from tundra areas by leaching or denitrification seems limited, and even a small nitrogen input from e.g. nitrogen fixation is important for the nitrogen balance of the ecosystem. The main sources of nitrogen input are biological nitrogen fixation, precipitation and dry deposition. Nitrogen-fixing organisms present in tundra areas are free-living, epiphytic and symbiotic blue-green algae, and free-living and symbiotic bacteria.
TL;DR: It can be concluded that Pleurotus ostreatus yields a suitable raw material in the food industry and possibly also a fodder based on the basis of solid, cellulose-containing wastes.
Abstract: The balance of nitrogen and nitrogen compounds in Pleurotus ostreatus, cultivated on waste materials, corn straw and maize residues, was investigated. The results show that this organism has a considerable ability to fix atmospheric nitrogen, fixing 312 g of total nitrogen per 100 kg dry weight. When recalculating with respect to a decrease of the substrate during growth of the organism a value of 9.7 mg per 1 g of the decrease in material is obtained. Fruiting bodies, as well as the produced substrate contain 17-19 amino acids. In the produced substrate the content of the protein nitrogen represents only 30% of the total. It can concluded that Pleurotus ostreatus yields a suitable raw material in the food industry and possibly also a fodder based on the basis of solid, cellulose-containing wastes.
01 Jan 1975
TL;DR: Nodulated plants of Coriaria arborea and Hippophaë rhamnoides were grown for a further 2½ months in the presence in the rooting medium of 0, 10 or 25 mg nitrate-nitrogen per litre of culture solution and the nitrate was labelled with 15N.
Abstract: Nodulated plants of Coriaria arborea and Hippophae rhamnoides, one year old and grown previously in a rooting medium essentially free of combined nitrogen, were grown for a further 2$\frac{1}{2}$ months in the presence in the rooting medium of 0, 10 or 25 mg nitrate-nitrogen per litre of culture solution. The nitrate was labelled with $^{15}$N. Plant growth was promoted by the supplied nitrogen, especially in Hippophae, but nodule growth and nitrogen fixation per plant were depressed, the latter, at the highest level of nitrate, being only 31% (in Coriaria) and 61% (in Hippophae) of the fixation in plants at zero nitrate level. Although the $^{15}$N penetrated (probably indirectly) into the nodules, in both species and at both levels of nitrate the enrichment shown by the nodule tissues as a whole was only about one-fifth of that shown by the rest of the plant. This finding would be explained if four-fifths of the nodule nitrogen was in the endophyte and was wholly unlabelled nitrogen fixed from the atmosphere, while the remaining one-fifth was in the uninfected cells and these were in equilibrium with the tissues of the rest of the plant and carried the same $^{15}$N label. The implications of this hypothesis are considered.
TL;DR: Root nodules of Alnus crispa (Ait.) Pursh were shown to possess a symbiotic nitrogen-fixing organism, and soil nitrogen measurements indicated that nitrogen fixation activity by A. crispa does not lead to an increase in soil nitrogen above levels in adjacent areas.
Abstract: Root nodules of Alnus crispa (Ait.) Pursh were shown to possess a symbiotic nitrogen-fixing organism. The reduction of acetylene to ethylene, as measured by gas chromatography, was used to determine the presence of the nitrogen-fixing system. Ethylene production was measured at 5.1 μmoles/g excised nodule · hr for both field and greenhouse plants. The nodules were found to consist of short nubs usually clustered in masses up to 4 cm in diam. Microscopic examination of nodules revealed some cortical cells fully packed with spherical endophyte cells. The outer cortex and radiating arms of cells in the inner cortex remained uninfected. Nodules examined during the winter were found to be shrunken, with a random distribution of endophyte cells. Soil nitrogen measurements indicated that nitrogen fixation activity by A. crispa does not lead to an increase in soil nitrogen above levels in adjacent areas.
TL;DR: The molecular mechanism by which leghemoglobin augments the oxygen consumption and nitrogen-fixing activity of bacteroids is inquired into, and facilitated diffusion makes oxygen more available to the terminal oxidases of subcellular organelles.
Abstract: A world increasingly short of protein may expend its limited supply of chemical or electrical energy t o fix atmospheric nitrogen into ammonia, or may rely in greater part on plants to harvest solar energy, some part of which is directed t o the support of bacterial nitrogen fixation. The rapid development of the nitrogen-fixing soybean as a major crop suggests that reliance on plants may already have become the more economical way t o regenerate the supply of fixed nitrogen. A hemeprotein, leghemoglobin, is an indispensable component of the system by which leguminous plants support the activity of nitrogen-fixing bacteria. We here inquire into the molecular mechanism by which leghemoglobin augments the oxygen consumption and nitrogen-fixing activity of bacteroids. That mechanism is leghemoglobin-facilitated oxygen diffusion brought about by translational diffusion of the oxygenated protein. The flux of oxygen is enhanced by facilitated diffusion. In addition, we now discover, facilitated diffusion makes oxygen more available to the terminal oxidases of subcellular organelles. We are concerned with the physicochemical definition of what we mean by “available.” Nitrogen-fixing nodules are formed on the roots of legumes in response t o invasion by bacteria of the genus Rhizobium. Rhizobia, modified for symbiotic life, are called bacteroids. The enzyme complex, nitrogenase, which is responsible for nitrogen fixation, is located wholly within the bacteroids. Nitrogenase does not utilize oxygen (in fact it is inhibited by even traces of oxygen) but depends for its activity of a supply of ATP formed (presumably) by bacteroidal oxidative phosphorylation. The bacteroids, which occur within the nodule cell, are in some ways analogous to muscle mitochondria. They occupy about the same fraction, approximately one-third,1,20f the cell volume and are responsible for the largest part of the oxygen consumption. Bacteroidal oxygen demand is vigorous; the oxygen consumption of the intact nodule is about one-tenth as great as that of the most active mammalian muscles. In fact, both
TL;DR: In this paper, the effects of light intensity and temperature on the rate of nitrogen fixation of two gelatinous lichens, Leptogium cyanescens (Ach.) Degel, and two foliichi lichens (L.) Hoffm.
Abstract: Nitrogen fixation by the lichens Lobaria pulmonaria, Sticta weigelii, Leptogium cyanescens and Collema subfurvum was measured by the acetylene reduction method. Fixation rates were maximum at 300C for all species except C. subfurvum which fixed maximally at 250C. Maximum fixation was observed at 200 lEinsteins m-2 sec-1 for the gelatinous lichens L. cyanescens and C. subfurvum. Saturating light intensities were not attained for L. pulmonaria and S. weigelii. Nitrogen fixation by living organisms is the most important source of reduced nitrogen in ecosystems. The process involves the reduction of atmospheric nitrogen to ammonia which is catalyzed by nitrogenase. Since nitrogen fixation is restricted to blue-green algae and certain bacteria, and combined nitrogen is essential for all living organisms, an assessment of the significance of the nitrogen fixing process is important to our understanding of the nitrogen balance in nature. Those lichens which have blue-green algae as part of the thallus have the capacity to fix atmospheric nitrogen (Milbank & Kershaw, 1969; Henriksson & Simu, 1971; Hitch & Stewart, 1973). Nitrogen fixing lichens have been shown to be important nitrogen contributors to their surroundings. In arctic and subarctic regions they are one of the primary sources of reduced nitrogen (Alexander & Schell, 1973). Estimates of nitrogen contribution for Lobaria oregana, found on tall Douglas fir trees, ranged from 2.2 to 11.2 kg ha-1 yr-1 (Denison, 1973). Nitrogen fixation rates of lichens have been examined; however, the effects of physical parameters such as light and temperature on the rate of nitrogen fixation have received little attention. Kallio, Suhonen and Kallio (1972) observed that nitrogen fixation rates in lichens are influenced by ambient temperature changes in arctic regions, with fixation starting at temperatures around 00C. Optimum temperature in the arctic for nitrogenase activity in Solorina crocea and Nephroma arcticum occurred at 150C, regardless of light intensity. In field studies Hitch and Stewart (1973) found hourly fluctuations in rates of nitrogen fixation depending upon temperature, moisture and light intensity. Light was required by Lichina confinis; however, after removal from the light it could fix nitrogen for up to 18 hours in the dark. This investigation dealt with the effects of light intensity and temperature upon the rate of nitrogen fixation of two gelatinous lichens, Leptogium cyanescens (Ach.) 1 This work was supported by grants from North Carolina Board of Science and Technology and from Wake Forest University Research and Publication Fund. 2 Department of Biology, Wake Forest University, Winston-Salem, NC 27109. This content downloaded from 157.55.39.120 on Mon, 05 Sep 2016 05:47:11 UTC All use subject to http://about.jstor.org/terms 1975] KELLY & BECKER: LIGHT & TEMPERATURE EFFECTS ON LICHENS 351 Korb and Collema subfurvum (Mull. Arg.) Degel., and two foliose lichens, Lobaria pulmonaria (L.) Hoffm. and Sticta weigelii (Insert ex Ach.) Vain. In L. pulmonaria the green alga, Myrmecia, is the primary phycobiont and is uniformly distributed across the thallus, whereas the blue-green alga, Nostoc, is located in cephalodia (Milbank & Kershaw, 1973). The other three lichens contain Nostoc as the phycobiont (Ahmadjian, 1967). MATERIALS AND METHODS Lichens for this study were collected from January through May 1974 in the Piedmont and mountains of North Carolina at elevations ranging from 275 to 1200 m. Specimens were dry when collected and were stored in envelopes in the dark at 220C up to 20 days. Storage longer than 20 days resulted in decreased nitrogen fixing ability. Mosses and other adhering materials were removed from the samples prior to determination of nitrogen fixation rates. Light intensity measurements were made with a light meter (LI-COR, Model LI-185, Lambda Instruments, Lincoln, Nebraska) in the photosynthetically active range (400-700 nm). The acetylene reduction method for measuring nitrogen fixation was used in this study (Hardy, Burns & Holsten, 1973). In this method nitrogenase catalyzes the conversion of acetylene to ethylene and the amount of ethylene provides a direct measure of nitrogenase activity. The stoichiometry between ethylene and ammonia is three molecules of ethylene formed to every two of ammonia. The qualitative validity of this method and its parallel to the reduction of N2 to ammonia has been well established (Hardy et al., 1973). The experimental procedure for measuring nitrogen fixation involved the following. Lichen thalli were allowed to imbibe water by submersion in distilled water for 3-5 min. After a rapid blotting on dry filter paper they were transferred to 15 cc glass serum bottles which were closed immediately with serum stoppers. Subsequently, the air atmosphere within the bottles was exchanged with 80% argon-20% oxygen. One-tenth of the argon-oxygen gas was removed with a syringe and was replaced with acetylene. Some samples were incubated at temperatures ranging from 0-400C for 1 hr. in a growth chamber at a light intensity of 200 AEinsteins m-2 sec-. The effects of light intensity were measured outdoors under bright sunlight and under several layers of window screening at 260C for 1 hr. Gaseous samples from the temperature and light experiments were trapped in "Vacutainers" according to the procedure of Schell and Alexander (1970). Ethylene levels were determined as described by Murphy and Becker (in press). The dry weight of each lichen sample was determined as reported by Hitch and Stewart (1973). Numbers of blue-green alga cells were measured as described by Kershaw (1974). Lichen samples were frozen in Tissue-Tek O.C.T. Compound (Miles Laboratories Inc., Elkhart, Indiana) and sectioned with a Model 880 American Optical microtome.