Perfusion Method for Assaying Microbial Activities in Sediments: Applicability to Studies of N2 Fixation by C2H2 Reduction

A perfusion method for assaying nitrogenase activity (acetylene reduction) in marine sediments was developed. The method was used to assay sediment cores from Spartina alterniflora (salt marsh), Zostera marina (sea grass), and Thalassia testudinum (sea grass) communities, and the results were compared with those of conventional sealed-flask assays. Rates of ethylene production increased progressively with time in the perfusion assays, reaching plateau values of 2 to 3 nmol · g of dry sediment⁻¹ · h⁻¹ by 10 to 20 h. Depletion of interstitial NH₄⁺ was implicated in this stimulation of nitrogenase activity. Initial acetylene reduction rates determined by the perfusion assay of cores from the Spartina community ranged from 0.15 to 0.60 nmol of C₂H₄ · g of dry sediment⁻¹ · h⁻¹. These rates were similar to those for sediments assayed in sealed flasks without seawater when determined over linear periods of C₂H₄ production. Initial values obtained by using the perfusion method were 0.66 nmol of C₂H₄ · g of dry sediment⁻¹ · h⁻¹ for sediments from Zostera communities and 0.70 nmol of C₂H₄ · g of dry sediment⁻¹ · h⁻¹ for sediments from Thalassia communities. In all cases, rates determined by simultaneous slurry assays were lower than those determined by the perfusion method.     

Biological dinitrogen fixation (acetylene reduction) associated with Florida mangroves

Biological dinitrogen fixation in mangrove communities of the Tampa Bay region of South Florida was investigated using the acetylene reduction technique. Low rates of acetylene reduction (0.01 to 1.84 nmol of C(2)H(4)/g [wet weight] per h) were associated with plant-free sediments, while plant-associated sediments gave rise to slightly higher rates. Activity in sediments increased greatly upon the addition of various carbon sources, indicating an energy limitation for nitrogenase (C(2)H(2)) activity. In situ determinations of dinitrogen fixation in sediments also indicated low rates and exhibited a similar response to glucose amendment. Litter from the green macroalga, Ulva spp., mangrove leaves, and sea grass also gave rise to significant rates of acetylene reduction.Higher rates of nitrogenase activity (15 to 53 nmol of C(2)H(4)/g [wet weight] per h were associated with washed excised roots of three Florida mangrove species [Rhizophora mangle L., Avicennia germinans (L) Stern, and Laguncularia racemosa Gaertn.] as well as with isolated root systems of intact plants (11 to 58 mug of N/g [dry weight] per h). Following a short lag period, root-associated activity was linear and did not exhibit a marked response to glucose amendment. It appears that dinitrogen-fixing bacteria in the mangrove rhizoplane are able to use root exudates and/or sloughed cell debris as energy sources for dinitrogen fixation.     

Nitrogen fixation (acetylene reduction) associated with roots of winter wheat and sorghum in Nebraska

Root segments and root-soil cores (6.5-cm diameter) from fields and nurseries of winter wheat and sorghum were tested for N2 fixation by using the acetylene reduction assay. Wheat samples (approximately 1,200) from 109 sites generally had low or no activity (0 to 3.1 nmol of C2H4 produced per h per g [dry weight] of root segments), even after 24 h of incubation. However, a commercial field of Scout 66, located in western Nebraska, exhibited appreciable activity (290 nmol of C2H4 produced per h per g [dry weight] of root segments). Of 400 sorghum lines and crosses, grain sorghums (i.e., CK-60A, Wheatland A, B517, and NP-16) generally exhibited higher nitrogenase activity than forage sorghums or winter wheats. CK-60A, a male sterile grain sorghum, was sampled at four locations and had the most consistent activity of 24 to 1,100 nmol of C2H4 produced per h per core. The maximum rate extrapolated to 2.5 g of N per hectare per day. Numerous N2-fixing bacterial isolates were obtained from wheat and sorghum roots that exhibited high nitrogenase activity. Most isolates were members of the Enterobacteriacae, i.e., Klebsiella pneumoniae, Enterobacter cloacae, and Erwinia herbicola.     

Biological Dinitrogen Fixation (Acetylene Reduction) Associated with Florida Mangroves

Biological dinitrogen fixation in mangrove communities of the Tampa Bay region of South Florida was investigated using the acetylene reduction technique. Low rates of acetylene reduction (0.01 to 1.84 nmol of C₂H₄/g [wet weight] per h) were associated with plant-free sediments, while plant-associated sediments gave rise to slightly higher rates. Activity in sediments increased greatly upon the addition of various carbon sources, indicating an energy limitation for nitrogenase (C₂H₂) activity. In situ determinations of dinitrogen fixation in sediments also indicated low rates and exhibited a similar response to glucose amendment. Litter from the green macroalga, Ulva spp., mangrove leaves, and sea grass also gave rise to significant rates of acetylene reduction.     

Nitrogen fixation (acetylene reduction) associated with roots of winter wheat and sorghum in Nebraska.

Root segments and root-soil cores (6.5-cm diameter) from fields and nurseries of winter wheat and sorghum were tested for N2 fixation by using the acetylene reduction assay. Wheat samples (approximately 1,200) from 109 sites generally had low or no activity (0 to 3.1 nmol of C2H4 produced per h per g [dry weight] of root segments), even after 24 h of incubation. However, a commercial field of Scout 66, located in western Nebraska, exhibited appreciable activity (290 nmol of C2H4 produced per h per g [dry weight] of root segments). Of 400 sorghum lines and crosses, grain sorghums (i.e., CK-60A, Wheatland A, B517, and NP-16) generally exhibited higher nitrogenase activity than forage sorghums or winter wheats. CK-60A, a male sterile grain sorghum, was sampled at four locations and had the most consistent activity of 24 to 1,100 nmol of C2H4 produced per h per core. The maximum rate extrapolated to 2.5 g of N per hectare per day. Numerous N2-fixing bacterial isolates were obtained from wheat and sorghum roots that exhibited high nitrogenase activity. Most isolates were members of the Enterobacteriacae, i.e., Klebsiella pneumoniae, Enterobacter cloacae, and Erwinia herbicola.     

Root-associated n(2) fixation (acetylene reduction) by enterobacteriaceae and azospirillum strains in cold-climate spodosols

N(2) fixation by bacteria in associative symbiosis with washed roots of 13 Poaceae and 8 other noncultivated plant species in Finland was demonstrated by the acetylene reduction method. The roots most active in C(2)H(2) reduction were those of Agrostis stolonifera, Calamagrostis lanceolata, Elytrigia repens, and Phalaris arundinacea, which produced 538 to 1,510 nmol of C(2)H(4).g (dry weight). h when incubated at pO(2) 0.04 with sucrose (pH 6.5), and 70 to 269 nmol of C(2)H(4). g (dry weight).h without an added energy source and unbuffered. Azospirillum lipferum, Enterobacter agglomerans, Klebsiella pneumoniae, and a Pseudomonas sp. were the acetylene-reducing organisms isolated. The results demonstrate the presence of N(2)-fixing organisms in associative symbiosis with plant roots found in a northern climatic region in acidic soils ranging down to pH 4.0.     

Dinitrogen fixation — acetylene reduction in soybeans during the reproductive growth period

In a greenhouse pot oulture experiment, a dinitrogen (N2) fixing — acetylene reduction activity profile was examined in detail as affected by plant age. Total [μmol C2H4 root-1 h-1] and speoifio nitrogenase [nmol C2H4 (mg nodule d. wt.)-1 min-1] activities peaked 63 days after sowing, near the end of flowering. The nitrogenase activities, nodule dry matter accumulation, top dry matter accumulation, and total nitrogen yield in the top dry matter were found to be highly correlated.     

Microbial production and consumption of dimethyl sulfide (DMS) in a sea grass (Zostera noltii)-dominated marine intertidal sediment ecosystem (Bassin d'Arcachon, France)

The relation between net dimethyl sulfide (DMS) production and changes in near surface (0-5 mm) oxygen concentrations in a sea grass (Zostera noltii Hornem)-covered intertidal sediment ecosystem was examined during a diel cycle. Sediment covered with Zostera was found to be more oxygenated than uncovered sediment during the period of photosynthesis. This phenomenon was probably caused by radial oxygen loss of the Zostera root-rhizome system. The population sizes of the three functional groups of microbes mainly responsible for the concentration of DMS, the dimethylsulfoniopropionate (DMSP)-demethylating, DMSP-cleaving and DMS-oxidizing bacteria, were quantified by most probable number (MPN) methodologies. Sediments with Zostera supported substantially higher populations of both aerobic (149x10(6) cm(-3) DMSP-utilizing and 0.4x10(6) cm(-3) DMS-oxidizing) and anaerobic (43x10(6) cm(-3) DMSP-utilizing and 0.4x10(6) cm(-3) DMS-oxidizing) microorganisms than sediments without Zostera (DMSP-utilizing aerobes and anaerobes both 2x10(6) cm(-3) and DMS-oxidizing aerobes and anaerobes both 0.2x10(6) cm(-3)). Experiments conducted with sediment cores and sediment slurries suggested that the net production of DMS in these sediments was significantly lower during oxic periods than during anoxic periods. Intact sediment cores with and without Zostera produced DMS when incubated under anoxic/dark conditions (97.0 and 53.6 nmol DMS m(-2) h(-1), respectively), while oxic/light-incubated cores did not produce detectable amounts of DMS. In addition, kinetic parameter values (V(max) and K(m)) for DMSP degradation in cell suspensions of isolated DMSP-demethylating and DMSP-cleaving bacteria were measured and compared to documented values for other strains. Both V(max) and K(m) values for DMSP-demethylating organisms were found to be relatively low (14.4-20.1 nmol DMSP mg protein(-1) min(-1) and 4.1-15.5 μM, respectively) while these parameter values varied widely in the group of the DMSP-cleaving organisms (6.7-1000 nmol DMSP mg protein(-1) min(-1) and 2-2000 μM, respectively). It was hypothesized that a diel rhythm in DMS emission occurred, with a relatively low net production during the day and a high net production during the night. Environmental changes which result in increased anoxic conditions in coastal sediments, such as an increase in eutrophication, may therefore result in increased atmospheric DMS emission rates.     

Nitrogen fixation associated with Suillus tomentosus tuberculate ectomycorrhizae on Pinus contorta var. latifolia

BACKGROUND AND AIMS: Tuberculate ectomycorrhizae are a unique form of ectomycorrhiza where densely packed clusters of mycorrhizal root tips are enveloped by a thick hyphal sheath to form a tubercle. The functional significance of such a unique structure has not previously been established. The purpose of the present study was to investigate and measure the potential nitrogenase activity associated with Suillus tomentosus/Pinus contorta tuberculate ectomycorrhizae in two stand ages, young and old, and across a range of nitrogen-poor soil conditions. METHODS: Short roots were compared with other mycorrhizae and non-mycorrhizal secondary roots using tuberculate ectomycorrhizae. Assessment of nitrogenase activity was determined and quantitative measurements were taken on tuberculate ectomycorrhizae in situ in a variety of different circumstances, by using an adaptation of the acetylene reduction assay. KEY RESULTS: Significant nitrogenase activity was measured associated with S. tomentosus/P. contorta tuberculate ectomycorrhizae whereas no nitrogenase activity was measured with non-tuberculate mycorrhizae or secondary roots without mycorrhizae. Average nitrogenase activity ranged from undetectable to 5696.7 nmol C2H4 g(-1) tubercle 24 h(-1). Maximum nitrogenase activity was 25,098.8 nmol C2H4 g(-1) tubercle 24 h(-1). Nitrogenase activity was significantly higher in young stands than in old stands of P. contorta. Season or some covariate also seemed to affect nitrogenase activity and there was suggestion of a site effect. CONCLUSIONS: Suillus tomentosus/P. contorta tuberculate ectomycorrhizae are sites of significant nitrogenase activity. The nitrogenase activity measured could be an important contribution to the nitrogen budget of P. contorta stands. Season and stand age affect levels of nitrogenase activity.     

Nitrogen Fixation Associated with Rinsed Roots and Rhizomes of the Eelgrass Zostera marina

Nitrogen fixation was associated with the rinsed roots and rhizomes of the seagrass, Zostera marina L. Nitrogenase activity (acetylene reduction) was greater on rhizomes compared to roots, and on older roots and rhizomes relative to younger tissue. Compared to aerobic assays, anaerobic or microaerobic conditions enhanced the rate of acetylene reduction by rhizomes with attached roots, with the highest activity (100 nanomoles per gram dry weight per hour) occurring at pO₂ = 0.01 atmosphere. Addition of glucose, sucrose, or succinate also increased the rate of acetylene reduction under anaerobic conditions, with glucose providing the most stimulation. In one experiment, comparison of acetylene reduction assays with ¹⁵N₂ incorporation yielded a ratio of about 2.6:1. Seagrass communities are thought to be limited by the availability of nitrogen and, therefore, nitrogenase activity directly associated with their roots and rhizomes suggests the possibility of a N₂-fixing flora which may subsidize their nutritional demand for nitrogen.     

Ammonia oxidation, denitrification and dissimilatory nitrate reduction to ammonium in two US Great Basin hot springs with abundant ammonia-oxidizing archaea

Many thermophiles catalyse free energy-yielding redox reactions involving nitrogenous compounds; however, little is known about these processes in natural thermal environments. Rates of ammonia oxidation, denitrification and dissimilatory nitrate reduction to ammonium (DNRA) were measured in source water and sediments of two ≈ 80°C springs in the US Great Basin. Ammonia oxidation and denitrification occurred mainly in sediments. Ammonia oxidation rates measured using (15)N-NO(3)(-) pool dilution ranged from 5.5 ± 0.8 to 8.6 ± 0.9 nmol N g(-1) h(-1) and were unaffected or only mildly stimulated by amendment with NH(4) Cl. Denitrification rates measured using acetylene block ranged from 15.8 ± 0.7 to 51 ± 12 nmol N g(-1) h(-1) and were stimulated by amendment with NO(3)(-) and complex organic compounds. The DNRA rate in one spring sediment measured using an (15)N-NO(3)(-) tracer was 315 ± 48 nmol N g(-1) h(-1). Both springs harboured distinct planktonic and sediment microbial communities. Close relatives of the autotrophic, ammonia-oxidizing archaeon 'Candidatus Nitrosocaldus yellowstonii' represented the most abundant OTU in both spring sediments by 16S rRNA gene pyrotag analysis. Quantitative PCR (qPCR) indicated that 'Ca. N. yellowstonii'amoA and 16S rRNA genes were present at 3.5-3.9 × 10(8) and 6.4-9.0 × 10(8) copies g(-1) sediment. Potential denitrifiers included members of the Aquificales and Thermales. Thermus spp. comprised <1% of 16S rRNA gene pyrotags in both sediments and qPCR for T. thermophilus narG revealed sediment populations of 1.3-1.7 × 10(6) copies g(-1) sediment. These data indicate a highly active nitrogen cycle (N-cycle) in these springs and suggest that ammonia oxidation may be a major source of energy fuelling primary production.     

Biological nitrogen fixation in acidic high-temperature geothermal springs in Yellowstone National Park, Wyoming

The near ubiquitous distribution of nifH genes in sediments sampled from 14 high-temperature (48.0-89.0°C) and acidic (pH 1.90-5.02) geothermal springs in Yellowstone National Park suggested a role for the biological reduction of dinitrogen (N(2)) to ammonia (NH(3)) (e.g. nitrogen fixation or diazotrophy) in these environments. nifH genes from these environments formed three unique phylotypes that were distantly related to acidiphilic, mesophilic diazotrophs. Acetylene reduction assays and (15) N(2) tracer studies in microcosms containing sediments sampled from acidic and high-temperature environments where nifH genes were detected confirmed the potential for biological N(2) reduction in these environments. Rates of acetylene reduction by sediment-associated populations were positively correlated with the concentration of NH(4)(+), suggesting a potential relationship between NH(4)(+) consumption and N(2) fixation activity. Amendment of microcosms with NH(4)(+) resulted in increased lag times in acetylene reduction assays. Manipulation of incubation temperature and pH in acetylene reduction assays indicated that diazotrophic populations are specifically adapted to local conditions. Incubation of sediments in the presence of a N(2) headspace yielded a highly enriched culture containing a single nifH phylotype. This phylotype was detected in all 14 geothermal spring sediments examined and its abundance ranged from ≈ 780 to ≈ 6800 copies (g dry weight sediment)(-1), suggesting that this organism may contribute N to the ecosystems. Collectively, these results for the first time demonstrate thermoacidiphilic N(2) fixation in the natural environment and extend the upper temperature for biological N(2) fixation in terrestrial systems.     

Nitrogen Fixation Associated with the New Zealand Mangrove (Avicennia marina (Forsk.) Vierh. var. resinifera (Forst. f.) Bakh.)

Nitrogenase activity in mangrove forests at two locations in the North Island, New Zealand, was measured by acetylene reduction and ¹⁵N₂ uptake. Nitrogenase activity (C₂H₂ reduction) in surface sediments 0 to 10 mm deep was highly correlated (r = 0.91, n = 17) with the dry weight of decomposing particulate organic matter in the sediment and was independent of light. The activity was not correlated with the dry weight of roots in the top 10 mm of sediment (r = −0.01, n = 13). Seasonal and sample variation in acetylene reduction rates ranged from 0.4 to 50.0 μmol of C₂H₄ m⁻² h⁻¹ under air, and acetylene reduction was depressed in anaerobic atmospheres. Nitrogen fixation rates of decomposing leaves from the surface measured by ¹⁵N₂ uptake ranged from 5.1 to 7.8 nmol of N₂ g (dry weight)⁻¹ h⁻¹, and the mean molar ratio of acetylene reduced to nitrogen fixed was 4.5:1. Anaerobic conditions depressed the nitrogenase activity in decomposing leaves, which was independent of light. Nitrogenase activity was also found to be associated with pneumatophores. This activity was light dependent and was probably attributable to one or more species of Calothrix present as an epiphyte. Rates of activity were generally between 100 and 500 nmol of C₂H₄ pneumatophore⁻¹ h⁻¹ in summer, but values up to 1,500 nmol of C₂H₄ pneumatophore⁻¹ h⁻¹ were obtained.     

Ecology of heterotrophic dinitrogen fixation in the rhizosphere of mangrove plant community at the Ganges river estuary in India

Summary Heterotrophic dinitrogen fixation in root associations of successional stages of the tropical mangrove plant community at the Ganges river estuary in India was investigated by excised-root acetylene reduction assay, and enumeration and identification of diazotrophic bacteria from sediment, root and tidal water samples. High to very high rates of nitrogenase activity (64–130 nmol C₂H₄/g dry root/h) were associated with washed excised roots of seven common early-successional mangrove species at the inundated swamps. Declining, late-successional mangroves at the occasionally inundated ridges had considerably lower values and the “declined” mangroves and other non-littoral species at embankment protected highlands had very low to insignificant values of root nitrogenase activity. Total and inorganic nitrogen contents of the mangrove sediments were low and were positively related to the stages of physiographic succession. Plant-associated sediments of particularly the old formation swamps had very high C/N ratios. Nine isolates of nitrogen-fixing bacteria belonging to all known O₂ response groups were distinguished from a large population of diazotrophs associated with roots of mangroves and other associate plant species of the community. The isolates differed with respect to their N₂-fixation efficiency and halotolerance in pure culture. There was no specificity of any of the bacterial isolates to any of the plant species of the community but a higher number of efficient isolates were seen to be associated with mangroves at the swampy succession. Sediment-free tidal water also contained a large population of microaerophilic and anaerobic N₂-fixing bacteria.     

N(2) Fixation Associated with Decaying Leaves of the Red Mangrove (Rhizophora mangle)

N(2) (C(2)H(2)) fixation was associated with decaying leaves of Rhizophora mangle. The process was predominantly anaerobic, with about two-thirds of the nitrogenase activity being light dependent. Average N(2) fixation rates in the light were 11 mug of N per g (dry weight) per h for leaves that had decayed for 2 to 3 weeks. This nitrogen input is probably significant in the estuarine, detrital food chains linked to R. mangle.     

Spatial and Temporal Assessment of Diazotroph Assemblage Composition in Vegetated Salt Marsh Sediments Using Denaturing Gradient Gel Electrophoresis Analysis

Abstract Diazotroph assemblage compositions were assessed in rhizosphere sediments from the tall and short form Spartina alterniflora growth zones over an annual cycle. Sediment cores were collected for DNA extraction and nitrogenase (acetylene reduction) activity assays, and porewater samples were analyzed for several chemical parameters in March, June, September, and December 1997. These data were collected to determine if within- or between-zone differences in the diazotroph assemblage composition correlated with differences in key environmental variables or acetylene reduction activity. Acetylene reduction rates differed between zones and within a zone over an annual period. Soluble sulfide concentrations were higher in the short form S. alterniflora zone on all dates except those in June and differed within both zones on different sample dates. nifH sequences were recovered from rhizosphere sediment DNA by PCR amplification using nifH specific primers. These amplimers were analyzed using denaturing gradient gel electrophoresis (DGGE), and the resulting patterns were compared by neural network and linear discriminant analyses. Ten prominent amplimers, four of which were apparent heteroduplexes, were observed. DGGE banding profiles showed minor differences among sampling dates and between sample zones, but the overall banding pattern was remarkably consistent. This reflects overall similarity between the amplifiable diazotroph assemblages in the tall and short S. alterniflora growth zones and substantial seasonal stability in assemblage composition. Received: 2 March 1999; Accepted: 4 May 1999     

Differential metabolism of dimethylsulfoniopropionate and acrylate in saline and brackish intertidal sediments

In anoxic Spartina altemiflora—dominated sediments along a naturally occuring salinity gradient (the Cooper River estuary, South Carolina, U.S.A.), dimethylsulfoniopropionate (DMSP) was metabolized to dimethyl sulfide (DMS) and acrylate by sediment microbes. The rate of DMSP degradation and acrylate mineralization by sediment microbes was similar at all sites along this 25-km transect. However, sediments amended with acrylate (or DMSP) showed significantly higher rates of N₂ fixation (measured as acetylene reduction activity) (ARA) in the saline sediments downstream than brackish sediments. These results are consistent with the fact that acrylate stimulated the rates of both denitrification and CO₂ production in the saline sediments at the mouth of the river more than tenfold over rates in brackish sediments. Enrichment experiments indicate that microbes capable of using DMSP or acrylate were not present in upstream sediments despite the fact that microbial biomass, percent organic matter, and both glucose-stimulated ARA and denitrification were highest upstream. It appears that acrylate utilizing, N₂ fixing, and denitrifying populations are insignificant in the lower salinity sediments of the estuary. These results may reflect the availability of DMSP, which averaged 10.3 nmol g wet wt^–1 of saline sediments and levels less than our detection limit (1 m) in brackish sediments. Correspondence to: D.C. Yoch.     

Pectin decomposition and associated nitrogen fixation by mixed cultures of Azospirillum and Bacillus species.

Cocultures of different Azospirillum species with Bacillus polymyxa or Bacillus subtilis allow the efficient utilization of pectin as carbon and energy sources for nitrogen fixation. The nitrogenase activity obtained with cocultures was as high as 30-80 nmol C2H4 h-1 mL-1, a much higher value than that obtained with pure cultures of either Azospirillum (up to 13 nmol C2H4 h-1 mL-1) or B. polymyxa (up to 2 nmol C2H4 h-1 mL-1) alone. To establish to what extent each partner contributed to nitrogenase activity, acetylene reduction was assayed as a function of time and it was also measured on Azospirillum cultivated in the cultures filtrates of the Bacillus. The results suggested that the nitrogenase activity was mostly produced by Azospirillum. The nitrogenase activity occurred at the expense of the degradation and fermentation products of the pectin. The new pectinolytic species, Azospirillum irakense, utilized both degradation and fermentation products of pectin, whereas the nonpectinolytic strains (Azospirillum brasilense, Azospirillum lipoferum, Azospirillum amazonense) utilized only the fermentation products of pectin, including acetic and succinic acids. These cocultures can be considered as metabolic associations, where the Bacillus produces degradation and fermentation products of pectin, which can be used by Azospirillum species.     

H2 metabolism in the photosynthetic bacterium Rhodopseudomonas capsulata: production and utilization of H2 by resting cells.

Photoproduction of H2 and activation of H2 for CO2 reduction (photoreduction) by Rhodopseudomonas capsulata are catalyzed by different enzyme systems. Formation of H2 from organic compounds is mediated by nitrogenase and is nto inhibited by an atmosphere of 99% H2. Cells grown photoheterotrophically on C4 dicarboxylic acids (with glutamate as N source) evolve H2 from the C4 acids and also from lactate and pyruvate; cells grown on C3 carbon sources, however, are inactive with the C4 acids, presumably because they lack inducible transport systems. Ammonia is known to inhibit N2 fixation by photosynthetic bacteria, and it also effectively prevents photoproduction of H2; these effects are due to inhibition and, in part, inactivation of nitrogenase. Biosynthesis of the latter, as measured by both H2 production and acetylene reduction assays, is markedly increased when cells are grown at high light intensity; synthesis of the photoreduction system, on the other hand, is not appreciably influenced by light intensity during photoheterotrophic growth. The photoreduction activity of cells grown on lactate + glutamate (which contain active nitrogenase) is greatly activated by NH4+, but this effect is not observed in cells grown with NH4+ as N source (nitrogenase repressed) or in a Nif- mutant that is unable to produce H2. Lactate, malate, and succinate, which are readily used as growth substrates by R. capsulata and are excellent H donors for photoproduction of H2, abolish photoreduction activity. The physiological significances of this phenomenon and of the reciprocal regulatory effects of NH4+ on H2 production and photoreduction are discussed.     

Nitrogen fixation (acetylene reduction) associated with duckweed (lemnaceae) mats

Duckweed (Lemnaceae) mats in Texas and Florida were investigated, using the acetylene reduction assay, to determine whether nitrogen fixation occurred in these floating aquatic macrophyte communities. N(2)-fixing microorganisms were enumerated by plating or most-probable-number techniques, using appropriate N-free media. Results of the investigations indicated that substantial N(2)-fixation (C(2)H(2)) was associated with duckweed mats in Texas and Florida. Acetylene reduction values ranged from 1 to 18 mumol of C(2)H(4) g (dry weight) day for samples incubated aerobically in light. Dark N(2) fixation was always two- to fivefold lower. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (7 to 10 muM) reduced acetylene reduction to levels intermediate between light and dark incubation. Acetylene reduction was generally greatest for samples incubated anaerobically in the light. It was estimated that 15 to 20% of the N requirement of the duckweed could be supplied through biological nitrogen fixation. N(2)-fixing heterotrophic bacteria (10 cells g [wet weight] and cyanobacteria (10 propagules g [wet weight] were associated with the duckweed mats. Azotobacter sp. was not detected in these investigations. One diazotrophic isolate was classified as Klebsiella.