Nitrogen fixation in denitrified marine waters

Nitrogen fixation is an essential process that biologically transforms atmospheric dinitrogen gas to ammonia, therefore compensating for nitrogen losses occurring via denitrification and anammox. Currently, inputs and losses of nitrogen to the ocean resulting from these processes are thought to be spatially separated: nitrogen fixation takes place primarily in open ocean environments (mainly through diazotrophic cyanobacteria), whereas nitrogen losses occur in oxygen-depleted intermediate waters and sediments (mostly via denitrifying and anammox bacteria). Here we report on rates of nitrogen fixation obtained during two oceanographic cruises in 2005 and 2007 in the eastern tropical South Pacific (ETSP), a region characterized by the presence of coastal upwelling and a major permanent oxygen minimum zone (OMZ). Our results show significant rates of nitrogen fixation in the water column; however, integrated rates from the surface down to 120 m varied by ～30 fold between cruises (7.5±4.6 versus 190±82.3 μmol m(-2) d(-1)). Moreover, rates were measured down to 400 m depth in 2007, indicating that the contribution to the integrated rates of the subsurface oxygen-deficient layer was ～5 times higher (574±294 μmol m(-2) d(-1)) than the oxic euphotic layer (48±68 μmol m(-2) d(-1)). Concurrent molecular measurements detected the dinitrogenase reductase gene nifH in surface and subsurface waters. Phylogenetic analysis of the nifH sequences showed the presence of a diverse diazotrophic community at the time of the highest measured nitrogen fixation rates. Our results thus demonstrate the occurrence of nitrogen fixation in nutrient-rich coastal upwelling systems and, importantly, within the underlying OMZ. They also suggest that nitrogen fixation is a widespread process that can sporadically provide a supplementary source of fixed nitrogen in these regions.     

Nitrogen fixation by Baltic cyanobacteria is adapted to the prevailing photon flux density

N₂ fixation, measured as acetylene reduction, was studied in laboratory cultures and in natural assemblages (both as a mixed population and as individually picked colonies) of the heterocystous cyanobacteria Aphanizomenon sp. and Nodularia spp. from the Baltic Sea. During a diurnal cycle of alternating light and darkness, these organisms reduced acetylene predominantly during the period of illumination, although considerable activity was also observed during the dark period. In both laboratory cultures and natural populations N₂ fixation was saturated below a photon flux density of 600 μm⁻² s⁻¹. In cyanobacterial blooms in the Baltic Sea, nitrogenase activity was mostly confined to the surface layers. Samples collected from greater depths did not possess the same capacity for acetylene reduction as samples from the surface itself, even when incubated at the photon flux density prevailing in surface waters. This suggests that, with respect to N₂ fixation, Baltic cyanobacteria are adapted to the intensity of illumination that they are currently experiencing.     

Growth and nitrogen fixation by immobilized cyanobacteria

Summary The nitrogen-fixing photosynthetic cyanobacteria have significant potential for utilization as a biological system for the production of reduced nitrogen compounds, either by industrial fermentation or in the environment as soil inocula. In either system, the ability to immobilize cyanobacteria on the external surface of fibrous substrata would significantly improve the ease of manipulation of the cells, control of growth, and product recovery without the complications inherent in immobilization by entrapment. We have shown that the filamentous heterocystous species Nostoc muscorum is naturally able to attach to a variety of different fibres, both natural and artificial. Attached cells are able to grow and fix nitrogen in both liquid and plate culture. Nitrogen-fixing cells attach to the fibres much more readily than do non-fixing cells, suggesting that the physiological and morphological changes accompanying heterocyst differentiation result in the production of specific attachment sites. Scanning electron microscopy of attached cells shows that heterocysts act as attachment sites and that the external cell wall material specifically synthesized around the heterocysts may be acting as the biological glue for this attachment.Journal Paper No. J-13259 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project No. 2649     

Degradation of crude oil by marine cyanobacteria

The marine cyanobacteria Oscillatoria salina Biswas, Plectonema terebrans Bornet et Flahault and Aphanocapsa sp. degraded Bombay High crude oil when grown in artificial seawater nutrients as well as in plain natural seawater. Oil removal was measured by gravimetric and gas chromatographic methods. Around 45-55% of the total fractions of crude oil (containing 50% aliphatics, 31% waxes and bitumin, 14% aromatics and 5% polar compounds) were removed in the presence of these cultures within 10 days. Between 50% and 65% of pure hexadecane (model aliphatic compound) and 20% and 90% of aromatic compounds (anthracene and phenantherene) disappeared within 10 days. Mixed cultures of the three cyanobacterial species removed over 40% of the crude. Additionally, these cultures formed excellent cyanobacterial mats when grown in mixed cultures, and thus have the potential for use in mitigating oil pollution on seashores, either individually or in combination.     

Nitrogen fixation by Methanobacterium formicicum

Abstract: Methanobacterium formicicum utilized molecular nitrogen as the sole nitrogen source for growth as monitored by methane production and culture turbidity measurements. The rate of methane production by the bacteria was correlated to nitrogen gas concentrations. In the absence of nitrogen gas or any other nitrogen source, the bacteria completely stopped growing. The presence of selenium and molybdenum in the culture medium was vital for the growth of the bacteria under nitrogen fixing conditions.     

Nitrogen fixation by hydrogen-utilizing bacteria

Seventeen strains of nitrogen-fixing bacteria, isolated from different habitats on hydrogen and carbon dioxide as well as on other substrates, morphologically resembled each other. All strains, including Mycobacterium flavum 301, grew autotrophically with hydrogen. The isolate strain 6 was sensitive to oxygen when dependent on N₂ as nitrogen source, a consequence of the sensitivity of its nitrogenase towards oxygen. At the same time, strain 6 was sensitive to hydrogen when growing autotrophically on N₂ as nitrogen source, but hydrogen did not affect acetylene reduction by these cells.Abbreviations MPN Most probable number - BS medium basal salts medium     

Nitrogen fixation by photosynthetic bacteria

In 1949, Howard Gest and Martin Kamen published two brief papers in Science that changed our perceptions about the metabolic capabilities of photosynthetic bacteria. Their discovery of photoproduction of hydrogen and the ability of Rhodospirillum rubrum to fix nitrogen led to a greater understanding of both processes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Nitrogen fixation by Anabaena cylindrica

Summary Blending Anabaena cylindrica cultures results in a loss of nitrogenase activity which is correlated with the breakage of the filaments at the junctions between heterocysts and vegetative cells. Oxygen inhibition of nitrogen fixation was significant only above atmospheric concentrations. Nitrogen-fixation activities in the dark were up to 50% of those observed in the light and were dependent on oxygen (10 to 20% was optimal). Nitrogenase activity was lost in about 3 h when cells were incubated aerobically in the dark. Re-exposure to light resulted in recovery of nitrogenase activity within 2 h. Blending, oxygen, or dark pre-incubation had similar effects upon cultures grown under air or nitrogen and did not inhibit light-dependent CO₂ fixation. We conclude that heterocysts are the sites of nitrogenase activity and propose a model for nitrogen fixation by Anabaena cylindrica.     

Nitrogen fixation by methane-utilizing bacteria

Several pure cultures of methane-utilizing bacteria, including types I and II membrane representatives, were found to be capable of fixing nitrogen. One nitrogen-fixing isolate grew in liquid medium, but not on a solid agar medium. Apparently, the ability to fix nitrogen is common in methane-oxidizing bacteria.     

Genetic engineering in marine cyanobacteria

Many species of microalgae producing useful materials have been isolated from marine environments. For their industrial application, widely applicable and stable gene expression is required. It is necessary to establish gene transfer methods as an essential first step in genetic manipulation. Although gene transfer techniques for cyanobacteria have been developed, only naturally transformable strains have been used. Here, we describe recent progress made in developing gene transfer methods for marine cyanobacteria. The following are covered: (1) transformation, (2) electroporation, (3) conjugation, (4) particle gun. A plasmid from the marine cyanobacterium, Synechococcus sp., whose copy number is dependent on salinity, was characterized. This plasmid is being used to develop a stable and controllable gene expression system.     

Hydrogen formation by cyanobacteria cultures selected for nitrogen fixation

Seventy-one cyanobacteria containing cultures were enriched from various soil and water locations either under aerobic and/or anaerobic conditions on agar medium selective for nitrogen fixation. Kept under argon containing 1% CO₂ for 24 and 48 h most of these cultures evolved hydrogen at very variable rates up to 116 l per mg chlorophyll and hour as a mean value over a time period of 24h. Several samples evolved hydrogen more efficiently compared with known hydrogen producing pure strains from culture collections. Thirty-one of the investigated cultures showed a hydrogen formation higher than 10 l per mg chlorophyll and hour measured over 24 or 48 h. Among these all the morphological forms of cyanobacteria i.e. unicellular and filamentous with or without heterocysts are found. Hence, selecting for nitrogen fixing cyanobacteria seems to be a practical method to find efficient hydrogen producers.     

Nitrogen fixing cyanobacteria potential uses

The large scale production of nitrogen fixing cyanobacteria is discussed and the use of ammonia excreting mutants ofAnabaena siamensis is described.Gloeotrichia natans is considered for use as a biofertilizer and for the production of phycobiliproteins.     

Nitrogen fixation by a marine non‐heterocystous cyanobacterium requires a heterotrophic bacterial consort

Cultures of the non‐heterocystous cyanobacterium, Leptolyngbya nodulosa, could be grown indefinitely in media devoid of combined nitrogen. Acetylene reduction assays showed that these cultures fixed nitrogen in the dark period of a diurnal cycle under micro‐oxygenic or anaerobic conditions. Addition of DCMU to cultures induced much higher rates of nitrogenase activity, most of which occurred in the light. Measurements of activity in the presence of chloramphenicol indicated that nitrogenase is synthesized in darkness and probably destroyed in the subsequent light period. Neither the dark‐mediated nitrogenase in the absence of DCMU nor light‐mediated activity in the presence of DCMU could be sustained for more than 3 days without a photoperiodic light/dark cycle. Axenic cultures could not be grown in the absence of combined nitrogen and did not demonstrate any acetylene reduction activity. An identical nifH gene sequence was found in axenic and non‐axenic cultures of L. nodulosa. RT‐PCR demonstrated that this gene was expressed only in non‐axenic cultures. Western blotting showed that the Fe‐protein of nitrogenase is absent in cultures that are incapable of acetylene reduction, indicating that the lack of nitrogenase activity is likely due to the absence of the enzyme. These observations strongly indicate that L. nodulosa contains a functional nitrogenase which is not expressed in the absence of heterotrophic bacteria.