Ecology and physiology of bacterial activity in a temperate saltmarsh lagoon, with an emphasis on nitrogen fixation

Heterotrophic bacterial activity and nitrogen fixation are fundamental to nutrient regeneration and nitrogen cycling in saltmarsh ecosystems. Ecological and physiological aspects of bacterial production and nitrogenase activity in marine sediments and water were examined in Langebaan Lagoon, a temperate saltmarsh ecosystem. Emphasis was placed on factors modulating rates and patterns of nitrogen fixation. Nitrogen fixation appeared to be dominated by heterotrophic bacteria. Rates of nitrogen fixation (estimated by the acetylene reduction technique), and bacterial production (estimated by tritiated thymidine incorporation, Tri) were higher in fine, muddy sediments near the head of the lagoon (Geelbek) than in coarser, sandy sediments near the mouth of the lagoon (Oesterwal). These comparisons (between sites) reflected the higher bacterial abundance and organic content of sediments from Geelbek. Examinations of five sedimentary microhabitats at each site (including those associated with beds of the seagrass Zostera capensis, burrows of the sandprawn Callianassa kraussi at Oesterwal, and burrows of the mudprawn Upogebia africana at Geelbek) showed that bacterial activity was higher in surface sediments than in subsurface sediments. Highest rates of nitrogen fixation (annual mean, 0.28 + 0.07 nmol C2H4 g-1 dry sediment h-1) were measured in Zostera bed sediments at Geelbek. Thymidine incorporation activity and nitrogenase activity were higher in burrow linings than in adjacent subsurface sediments, suggesting that burrow linings provided an improved subsurface environment for bacterial activity. Burrow linings also had a higher organic content than subsurface sediments away from burrows. Nitrogenase activity was not detected in lagoon water

Direct and indirect costs of dinitrogen fixation in Crocosphaera watsonii WH8501 and possible implications for the nitrogen cycle

The recent detection of heterotrophic nitrogen (N2) fixation in deep waters of the southern Californian and Peruvian OMZ questions our current understanding of marine N2 fixation as a process confined to oligotrophic surface waters of the oceans. In experiments with Crocosphaera watsonii WH8501, a marine unicellular diazotrophic (N2 fixing) cyanobacterium, we demonstrated that the presence of high nitrate concentrations (up to 800 μM) had no inhibitory effect on growth and N2 fixation over a period of 2 weeks. In contrast, the environmental oxygen concentration significantly influenced rates of N2 fixation and respiration, as well as carbon and nitrogen cellular content of C. watsonii over a 24-h period. Cells grown under lowered oxygen atmosphere (5%) had a higher nitrogenase activity and respired less carbon during the dark cycle than under normal oxygen atmosphere (20%). Respiratory oxygen drawdown during the dark period could be fully explained (104%) by energetic needs due to basal metabolism and N2 fixation at low oxygen, while at normal oxygen these two processes could only account for 40% of the measured respiration rate. Our results revealed that under normal oxygen concentration most of the energetic costs during N2 fixation (∼60%) are not derived from the process of N2 fixation per se but rather from the indirect costs incurred for the removal of intracellular oxygen or by the reversal of oxidative damage (e.g., nitrogenase de novo synthesis). Theoretical calculations suggest a slight energetic advantage of N2 fixation relative to assimilatory nitrate uptake, when oxygen supply is in balance with the oxygen requirement for cellular respiration (i.e., energy generation for basal metabolism and N2 fixation). Taken together our results imply the existence of a niche for diazotrophic organisms inside oxygen minimum zones, which are predicted to further expand in the future ocean

Biological nitrogen fixation (acetylene reduction) associated with blue-green algal communities in the Mgeni estuary mangrove swamp.

Thesis (M.Sc.)-University of Durban-Westville, 1987.Nitrogen fixation of blue-green algae associated with Avicennia marina (Forssk.) Vierh. pneumatophores and wet and dry surface sediments were investigated in the Mgeni Estuary mangrove swamp by means of the acetylene reduction technique. Optimum partial pressures of acetylene ranged from 0,15 to 0,2 atm. for the different habitats. A lag phase of 3 h was observed in all habitats, followed by a period of linear ethylene production of 42 h for the pneumatophores and 72 h for the wet and dry mat areas. An assay period of 24 h was employed in all habitats. Laboratory studies revealed percentage moisture and temperature to be the prime factors influencing ARA (acetylene reduction activity) in all habitats and rates were highest under submerged conditions and at 22˚C. Short-term variations in ARA and salinity in the wet and dry mat areas, measured at 6 day intervals, were also related to percentage moisture. High concentrations of inorganic nitrogen (between 1 and 5 mg 1-1) significantly depressed ARA in all habitats. Increases in ARA occurred with increase in light intensity up to 40 μE m -2 s-1, with negligible dark rates being recorded in the wet and dry mat areas. Significant dark rates of ARA and stimulation of ARA by sucrose in association with the pneumatophores indicated that bacteria may also be contributing to ARA in this habitat. No organic carbon stimulation was noted in the other sites. Salinity had little effect on ARA over the range generally experienced in each habitat. Field studies revealed a marked seasonal variation in ARA, with summer maxima of 78, 678 and 341 nmol C2H4 cm-2 24 h-1 associated with the pneumatophore, wet and dry mat areas respectively. This coincided with maximum nitrogen-fixing blue-green algal numbers, temperature, light intensity and day length. No seasonal variations in organic carbon, inorganic nitrogen, salinity, percentage moisture or bacterial numbers were apparent. Rates of bacterial ARA associated with decomposing litter of A. marina were highest under exposed conditions and reached a maximum of 25 935 nmol C2H4 g dry wt -1 24 h -1 after 3 weeks. Maximum rates of ARA under submerged conditions of 5394 nmol C2H4 g dry wt -1 24 h -1were reached after 4 weeks of decomposition. An increase in percentage nitrogen occurred during decomposition and was greatest under submerged conditions. Rates of decomposition were highest under exposed conditions. It was estimated that nitrogen fixation by blue-green algal communities supplies 23,8% of the annual nitrogen requirements of the mangrove swamp

Sodium requirement and metabolism in nitrogen-fixing cyanobacteria

Sodium affects the metabolism of eukaryotes and prokaryotes in several ways. This review collates information on the effects of Na+ on the metabolism of cyanobacteria with emphasis on the N2,fixing filamentous species. Na+ is required for nitrogenase activity in Anabaena torulosa, Anabaena L-31 and Plectonema boryanum. The features of this requirement have been mainly studied in Anabaena torulosa. The need for Na+ is specific and cannot be replaced by K+, Li+, Ca 2+ or Mg2+. Processes crucial for expression of nitrogenase such as molybdenum uptake, protection of the enzyme from oxygen inactivation and conformational activation of the enzyme are not affected by Na+. Mo-Fe protein and Fe protein, the two components of nitrogenase are synthesized in the absence of Na+ but the enzyme complex is catalytically inactive. Photoevolution of O2 and CO2 fixation, which are severely inhibited in the absence of Na+, are quickly restored by glutamine or glutamate indicating that Na+ deprivation affects photosynthesis indirectly due to deficiency in the products of N2 fixation. Na+ deprivation decreases phosphate uptake, nucleoside phosphate pool and nitrogenase activity. These effects are reversed by the addition of Na+ suggesting that a limitation of available ATP caused by reduced phosphate uptake results in loss of nitrogenase activity during Na+ starvation. Na+ influx in Anabaena torulosa and Anabaena L-31 is unaffected by low K+ concentration, is carrier mediated, follows Michaelis-Menten kinetics and is modulated mainly by membrane potential. Treatments which cause membrane depolarisation and hyperpolarisation inhibit and enhance Na+ influx respectively. These cyanobacteria exhibit rapid active efflux of Na+, in a manner different from the Na+/H+ antiporter mechanism found in Anacystis nidulans. Na+ requirement in nitrogen metabolism including nitrate assimilation, synthesis of amino acids and proteins, in respiration and oxidative phosphorylation, in transport of sugars and amino acids, cellular distribution of absorbed sodium, physiological basis of salt tolerance and prospects of reclamation of saline soils by cyanobacteria are the other aspects discussed in this review

The physiological-ecology of the cyanobacterium Microcoleus

A study was carried out to determine how widespread N(_2) fixation is in the cyanobacterium Microcoleus, both in the laboratory and in the field. The research was extended to compare the influence of environmental variables on both Ng fixing and non-fixing strains of Microcoleus isolated from a range of habitats. Since the morphology of Microcoleus strains is likely to influence their physiology, attempts were made to grow them in vitro in a form morphologically akin to that in the field i.e. with a communal sheath. Limited success was achieved with Microcoleus D634 on incubating in standard medium supplemented with 516 mM Na and 125 mM Ca (salinity of 30% as shown by hydrometer) , where a thin, communal sheath was only found intermittently surrounding 2-3 trichomes. All five strains were mixohaline growing at salinities of 0.5 – 30 (40) % and surviving periods under euryhaline (30 – 40%) and polyhaline (>40%) conditions. The shorter the time of exposure the higher the salinity tolerated. Growth varied according to the ratio of Na(^+) to Ca(^2+), Na(^+) to K(^+) and Na(^+) to Mg(^2+). Despite many changes in the nutrient status of the medium (Na(^+), Ca(^2+), Mg(^2+), K(^+)) at varying PAR and temperature, under oxic and micro-oxic conditions, only one of the five strains (Microcoleus D778) fixed N(_2) as shown by acetylene reduction activity (ARA) and growth in the absence of combined N. No ARA was detected for Microcoleus mats at Gibraltar Point, over two diel cycles in August 1986; however, when ARA was measured at Church Island, Anglesey (from whence Pearson et al., (1979) isolated Microcoleus D778), over 6 diel periods between June and October, 1987, ARA was detectable at all times between 0.1 and 3.4 nmol C(_2)H(_4) cm(^-2) h(^-1) using 4 h incubation periods. A different response in ARA was found on each occasion; generally, high activity (with > 70% in the dark) was found on sunny days and low, fairly constant ARA during cloudy, overcast days. On incubating Microcoleus D778 in 86 mM Na (salinity of 5%) at 20ºC under 16:8 and 8:16 light (50 µmol photon m(^-2) s(^-1)):dark, the majority (72 and 92% respectively) of ARA occurred in the dark, whereas in 20:4 and 16:8 light: dark only 19 and 40% ARA occurred in the light. However, ARA over a 16:8 light:dark cycle varied with PAR, salinity and temperature. In addition, the optimum temperature for ARA varied according to pH and salinity. When DCMU was added to Microcoleus mats and to axenic cultures, ARA increased markedly; the precise value depending on PAR and preincubation conditions

The marine biogeochemistry of molybdenum

Thesis (Ph. D.)--Joint Program in Marine Geology and Geophysics (Massachusetts Institute of Technology, Dept. of Earth, Atmospheric and Planetary Sciences, and the Woods Hole Oceanographic Institution), 2003.Includes bibliographical references.Prevailing wisdom holds that the vertical distribution of molybdenum (Mo) in the open ocean is conservative, despite Mo's important biological role and association with Mn oxides and anoxic sediments. Mo is used in both nitrogenase, the enzyme responsible for N2 fixation, and nitrate reductase, which catalyzes assimilatory and dissimilatory nitrate reduction. Laboratory culture work on two N2 fixing marine cyanobacteria, Trichodesmium and Crocosphaera, and a marine facultative denitrifier, Marinobacter hydrocarbanoclasticus, showed that Mo cell quotas in these organisms were positively correlated with Mo-containing enzyme activity. Mo concentrations in Crocosphaera dropped almost to blank levels when not fixing N2 suggesting daily synthesis and destruction of the entire nitrogenase enzyme and release of Mo. Trichodesmium cultures, however, retained a pool of cellular Mo even when not fixing N2. Colonies of Trichodesmium collected in the field have Mo:C tenfold higher than seen in culture, these Mo:C ratios were reflected in SPM samples from the same region. Fe:C ratios for Trichodesmium were between 12-160 pmol:mol in field and culured samples. The Fe:C ratio of Crocosphaera was established to be 15.8 =/+ 11.3 under N2 fixing conditions. Mo cellular concentrations in cultured organisms were too small to significantly influence dissolved Mo distributions, but may slightly affect Suspended Particulate Matter (SPM) distributions. Mean SPM Mo:C ratios were slightly elevated in regions of N2 fixation and denitrification.. A high precision (=/+ 0.5%) isotope dilution ICP-MS method for measuring Mo was developed to re-evaluate the marine distribution of Mo in the dissolved and particulate phase.(cont.) Mn oxides were not found to significantly influence either the dissolved or SPM Mo distribution. Dissolved Mo profiles from the Sargasso and Arabian Sea were conservative. However, dissolved Mo profiles from the Eastern Tropical Pacific showed both depletion and enrichment of dissolved Mo possibly associated with interaction of Mo with coastal sediments. Dissolved Mo profiles in several California Borderland Basins showed 1-2 nM Mo depletions below sill depth. A more focused study of water column response to sediment fluxes using the high precision Mo analyses is necessary to determine whether these phenomena are related.by Caroline Beth Tuit.Ph.D

Free-living Diazotrophs and the Nitrogen Cycle in Natural Grassland Revealed by Culture Dependent and Independent Approaches

Biological nitrogen fixation contributes to half of the global supply of nitrogen to the biosphere. It is carried out by a diverse group of prokaryotes called diazotrophs via the nitrogenase enzyme. Nitrogen fixation research is focused on the narrow group of symbiotic diazotrophs, and the vast majority of free-living diazotrophs which contribute significantly to fixed nitrogen are yet to be explored. The goal of this research was to access phylogeny of diazotrophs considering the most up-to-date genomic information and apply that knowledge to understand the diversity of free-living diazotrophs in a natural grassland ecosystem, both by culture dependent and independent methods. Phylogeny was reconstructed using the concatenated sequences of six core proteins of nitrogenase (NifHDKENB) from 963 prokaryotic genomes. The diversity of free-living diazotrophs in grassland was explored by isolation of putative diazotrophs on a solid nitrogen free medium (NFM) and diazotrophy confirmed by nifH PCR, acetylene reduction assay and 15N2 assimilation assay. Streptomyces, the most abundant bacteria, was further characterized by sequencing the genome of one prominent strain, and differential gene expression in nitrogen rich Vs nitrogen deficient medium. For culture independent study of nitrogen cycle activity, meta-transcriptomic sequencing of complete mRNA from a grassland soil sample was performed. Phylogeny of nif genes from the complete genomes of cultured isolates revealed that diazotrophs are distributed across Actinobacteria, Aquificae, Bacteroidetes, Chlorobi, Chloroflexi, Cyanobacteria, Deferribacteres, Firmicutes, Fusobacteria, Nitrospira, Proteobacteria, PVC group, and Spirochaetes, as well as the Euryarchaeota, providing a curated database of nif genes. Culturing yielded 474 bacterial isolates which belonged to the phyla Actinobacteria, Proteobacteria, Firmicutes, and Bacteroidetes. However, only 81 (17%) of isolates yielded nifH, and the most dominant genus isolated on NFM, Streptomyces did not provide biochemical and genomic evidence of diazotrophy. The meta-transcriptomic study revealed nitrogen fixation and nitrification are the least and nitrate reduction is the most expressed pathway among various nitrogen cycling pathways. In conclusion, although the culture-based approach showed diverse free-living nitrogen fixing bacteria, diazotrophy should always be confirmed by biochemical and genetic evidence, and limitations to culture independent study due to primer bias in nifH PCR can be overcome by meta-transcriptomic study