Study of nitrogen fixation in microbial communities of oil-contaminated marine sediment microcosms

Aerobic microbial degradation of pollutant oil (petroleum) in aquatic environments is often severely limited by the availability of combined nitrogen. We therefore studied whether the microbial community enriched in marine sediment microcosms with an added oil layer and exposure to light harboured nitrogenase activity. The acetylene reduction (AR) assay indeed indicated active nitrogenase; however, similar activity was observed in oil-free control microcosms. In both microcosms, the AR rate was significantly reduced upon a dark shift, indicating that enriched cyanobacteria were the dominant diazotrophs. Analysis of structural dinitrogenase reductase genes (nifH) amplified from both microcosms indeed revealed NifH sequences related mostly to those of heterocystous cyanobacteria. NifH sequences typically affiliating with those of heterotrophic bacteria were more frequently retrieved from the oil-containing sediment. Expression analyses showed that mainly nifH genes similar to those of heterocystous cyanobacteria were expressed in the light. Upon a dark shift, nifH genes related to those of non-heterocystous cyanobacteria were expressed. Expression of nifH assignable to heterotrophs was apparently not significant. It is concluded that cyanobacteria are the main contributors of fixed nitrogen to oil-contaminated and pristine sediments if nitrogen is a limiting factor and if light is available. Hence, also the oil-degrading heterotrophic community may thus receive a significant part of combined nitrogen from cyanobacteria, even though oil vice versa apparently does not stimulate an additional nitrogen fixation in the enriched community.     

Distribution of nitrogen-fixing cyanobacteria (Nostocaceae) during rice cultivation in fertilized and unfertilized paddy fields

This study describes the development of nitrogen-fixing cyanobacteria (Nostocaceae) during different stages of rice seedlings plantation in fertilized and unfertilized paddy fields in north Bihar, India. A heterogeneous population of cyanobacteria was randomly harvested around day 20, 40 and 60 of rice seedlings plantation, and the diversity was analyzed. Thirtytwo species (7 genera) were identified from unfertilized fields, of which Anabaena was represented by 12 species, Anabaenopsis and Aulosira by 3 each, Cylindrospermum by 4, Nostoc by 8 and Aphanizomenon and Nodularia by 1 species each. However, fertilized fields contained only 25 species (7 genera), of which 8 each belonged to Nostoc and Anabaena , 3 to Cylindrospermum , 2 to each Anabaenopsis and Aulosira and 1 to each Aphanizomenon and Nodularia . Although Nostoc and Anabaena were dominant in both fertilized and unfertilized paddy fields, a marked decrease in nitrogen-fixing cyanobacteria was recorded from fertilized fields. In both treatments, the diversity of nitrogen-fixing cyanobacteria was at a maximum around day 60 after seedling plantation. The current study concludes that there is a negative effect of nitrogenous fertilizers on the development of heterocystous cyanobacteria in rice fields. It is proposed that early appearing efficient nitrogen-fixers should be used as nitrogen fertilizers in the management for better establishment and exploitation of heterocystous cyanobacteria for sustainable agricultural practices.     

The use of nickel to probe the role of hydrogen metabolism in cyanobacterial nitrogen fixation

The hydrogenase activities of the heterocystous cyanobacteria Anabaena cylindrica and Mastigocladus laminosus are nickel dependent, based on their inability to consume hydrogen with various electron acceptors or produce hydrogen with dithionite-reduced methyl viologen, after growth in nickel-depleted medium. Upon addition of nickel ions to nickel-deficient cultures of A. cylindrica, the hydrogenase activity recovered in a manner which was protein synthesis-dependent, the recovery being inhibited by chloramphenicol. We have used the nickel dependence of the hydrogenase as a probe of the possible roles of H2 consumption in enhancing nitrogen fixation, and particularly for protecting nitrogenase against oxygen inhibition. Although at the usual growth temperatures (25 degrees for A. cylindrica and 40 degrees for M. laminosus), the cells consume H2 vigorously in an oxyhydrogen reaction after growth in the presence of nickel ions, we have not found that the reaction confers any significant additional protection of nitrogenase, either at aerobic pO2 (for both organisms) or at elevated pO2 (for A. cylindrica). However, at elevated temperatures (e.g., 40 degrees for A. cylindrica and 48 degrees for M. laminosus) a definite protective effect was observed. At these temperatures both organisms rapidly lost acetylene reduction activity under aerobic conditions. When hydrogen gas (10%) was present, the cells retained approximately 50% of the nitrogenase activity observed under anaerobic conditions (argon gas phase). No such protection by hydrogen gas was observed with nickel-deficient cells. Studies with cell-free extracts of A. cylindrica showed that the predominant effect of temperature was not due to thermal inactivation of nitrogenase.     

Nitrogen Fixation by Photosynthetic Bacteria in Lowland Rice Culture

Propanil (3′,4′-dichloropropionanilide) was a potent inhibitor of the nitrogenase activity of blue-green algae (cyanobacteria) in flooded soil, but the herbicide at comparable concentrations was not toxic to rice, protozoa, and nitrogen-fixing bacteria. Ethanol-amended flooded soils treated with propanil exhibited higher rates of nitrogenase activity than those not treated with the herbicide. The enhanced nitrogenase activity in propanil-treated soils was associated with a rise in the population of purple sulfur bacteria, especially of cells resembling Chromatium and Thiospirillum. By employing propanil and a means of excluding light from the floodwater to prevent the development of phototrophs during rice growth under lowland conditions, the relative activities of blue-green algae, photosynthetic bacteria, and the rhizosphere microflora were determined. The results suggest that the potential contribution of photosynthetic bacteria may be quite high.     

Role of oxygen limitation and nitrate metabolism in the nitrate inhibition of nitrogen fixation by pea

The impact of nitrate (5–15 m M , 2 to 7 days) on nitrogenase activity and nodule-oxygen limitation was investigated in nodulated, 21-day-old plants of a near-isogenic nitrate reductase-deficient pea mutant (A3171) and its wild-type parent ( Pisum sativum L. cv. Juneau). Within 2 days, 10 or 15 m M nitrate, but not 5 m M nitrate, inhibited the apparent nitrogenase activity (measured as in situ hydrogen evolution from nodules of intact plants) of wild-type plants; none of these nitrate levels inhibited the apparent nitrogenase activity of A3171 plants. Nodule-oxygen limitation, measured as the ratio of total nitrogenase activity to potential nitrogenase activity, was increased in both wild-type and A3171 plants by all nitrate treatments. By 3 to 4 days the apparent nitrogenase activity of A3171 and wild-type plants supplied with 5 m M nitrate declined to 53 to 69% of control plants not receiving nitrate. By 6 to 7 days the apparent nitrogenase activity of A3171 plants was similar to the control value whereas that of the wild-type plants continued to decline. From 3 to 7 days, no significant differences in nodule-oxygen limitation were observed between the nitrate (5 m M ) and control treatments. The results are interpreted as evidence for separate mechanisms in the initial (O₂ limitation) and longer-term (nitrate metabolism) effects of nitrate on nitrogen fixation by effectively nodulated pea.     

Immobilized cyanobacteria as a biofertilizer for rice crops; Intl. Conference on Applied Algology, Knysna, South Africa, April 1996.

N₂-fixing cyanobacteria (Anabaena azollae, symbiont strains) were immobilized in polyurethane foam and ammonia production by the cyanobacteria was investigated in the laboratory and rice field. The cyanobacterial symbiont, A. azollae - MPK-SK-AM-24 showed the highest growth rate and biomass production amongst the 5 isolates examined while A. azollae-AS-DS showed the highest nitrogenase activity followed by A. variabilis - SA₀ (wild type, non-symbiotic). Treatment of the foam-immobilized cyanobacteria with the systemic fungicide Bavistin stimulated nitrogenase activity while inhibiting glutamine synthetase (GS) activity. Free-living A. azollae-MPK-SK-AF-38, A. azollae - MPK-SK-AM-24 and A. azollae-MPK-SK-AM-27 excreted the highest amounts of ammonia into the growth medium; under foam - immobilized conditions the ammonia production increased further. Treatment of the foam - immobilized cyanobacteria with the fungicides Bavistin and Vitavax resulted in ammonia production at significantly higher rates. Rice seedlings (var. ADT 36) grown in the laboratory in conjunction with foam - immobilized A. azollae showed increased growth. A field experiment with paddy rice and foam - immobilized A. azollae strains indicated that the cyanobacteria excreted significant amounts of ammonia into the flood water in the rice fields resulting in increased chlorophyll content of the plants and increased the rice grain and straw yields. A combination of fertilizer nitrogen and inoculation with foam - immobilized cyanobacteria also significantly increased the rice grain and straw yield. Additionally, both A. azollae and A. variabilis were immobilized in sugarcane waste (bagasse), added to rice paddy and resulted in increased rice grain yield. This revised version was published online in September 2006 with corrections to the Cover Date.     

Diversity and functional traits of culturable microbiome members,including cyanobacteria in the rice phyllosphere

The diversity and abundance of culturable microbiome members of the rice phyllosphere was investigated using cv. Pusa Punjab Basmati 1509. Both diversity and species richness of bacteria were significantly higher in plants in pots in a semi‐controlled environment than those in fields. Application of fertilisers reduced both diversity and species richness in field‐grown plants under a conventional flooded system of rice intensification (SRI) and in dry‐seeded rice (DSR) modes. Sequence analyses of 16S rDNA of culturable bacteria, those selected after amplified ribosomal DNA restriction analysis (ARDRA), showed the dominance of α‐proteobacteria (35%) and actinobacteria (38%); Pantoea, Exiguobacterium and Bacillus were common among the culturable phyllospheric bacteria. About 34% of 83 culturable bacterial isolates had higher potential (>2 μg·ml^?1) for indole acetic acid production in the absence of tryptophan. Interestingly, the phyllosphere bacterial isolates from the pot experiment had significantly higher potential for nitrogen fixation than isolates from the field experiment. Enrichment for cyanobacteria showed both unicellular forms and non‐heterocystous filaments under aerobic as well as anaerobic conditions. PCR‐DGGE analysis of these showed that aerobic and anaerobic conditions as well as the three modes of cultivation of rice in the field strongly influenced the number and abundance of phylotypes. The adaptability and functional traits of these culturable microbiome members suggest enormous diversity in the phyllosphere, including potential for plant growth promotion, which was also significantly influenced by the different methods of growing rice.     

Molecular cloning and characterization of hetR genes from filamentous cyanobacteria

HetR, a serine type protease, plays an important role in heterocyst differentiation in filamentous cyanobacteria. We isolated and sequenced the hetR genes from different heterocystous and filamentous nonheterocystous cyanobacteria. The hetR gene in the heterocyst forming Anabaena variabilis ATCC 29413 FD was interrupted by interposon mutagenesis (mutant strain WSIII8). This mutant does not form heterocysts and shows no diazotrophic growth under aerobic conditions. However, under anaerobic N(2)-fixing conditions, the WSIII8 cells are able to grow, and high nitrogenase (Nif2) activity is detectable. Nif2 expression was demonstrated in each vegetative cell of the filament by immunolocalization 4 h after nitrogen step-down.     

Molecular typing and distribution of filamentous heterocystous cyanobacteria isolated from two distinctly located regions in North-Eastern India

The diversity of cyanobacteria in the North-Eastern region of India has not been studied except for a few sporadic and inconclusive reports. Loktak Lake is a huge reservoir for various kinds of organisms, including cyanobacteria. The present study describes the isolation and molecular diversity of 72 filamentous, heterocystous cyanobacterial strains isolated from samples collected from Loktak Lake, its adjoining rice fields and rice fields in Indian Council of Agricultural Research (ICAR) complex, Shillong, Meghalaya, India. The isolated strains belonged to the genera Anabaena, Nostoc, Calothrix, Cylindrospermum and Mastigocladus. The molecular analysis of isolates revealed the occurrence of certain strains being present in the sample collected from the rice fields falling in the catchment area of Loktak Lake, Manipur and rice fields in ICAR complex, Shillong, Meghalaya both. A polyphasic approach based on morphological features and PCR based molecular polymorphism revealed enormous level of molecular diversity. Out of three primers targeted regions used for determining genetic polymorphism, STRR1A produced best fingerprint profile of cyanobacterial strains. The morphological diversity of isolates was assured by light microscope whereas PCR based multiple fingerprint profile was used for molecular characterization. Molecular typing using short tandemly repeated repetitive STRR1A sequences as primer provided strain specific fingerprint profiles of the isolates.     

Ontogenetic Variation of Nitrogenase, Nitrate Reductase, and Glutamine Synthetase Activities in Oryza sativa

The relationship between the rates of nitrogenase, nitrate reductase, and glutamine synthetase activities, and plant ontogeny in rice (Oryza sativa L.), cultivar `M9', grown in salt marsh sediment with and without nitrate treatment was studied. In both treatments, nitrogenase activity measured as the immediate linear rate of acetylene reduction by bacteria associated with the roots varied with plant age. In control plants, the nitrogenase activity developed during the vegetative stage, peaked during early reproductive growth and then declined. The application of 10 kilograms N per hectare as KNO₃ once every 2 weeks delayed the development of and decreased the nitrogenase activity. The nitrogenase activity in both treatments developed as leaf nitrate reductase activity declined. The per cent nitrogen of roots was negatively correlated with the rates of acetylene reduction during the life cycles of control and nitrate-treated plants. This suggests that the concentration of combined nitrogen in the plants controlled the development and rate of root-associated nitrogenase activity. During reproductive growth, no nitrate reductase activity was detected in the roots from either treatment. In control plants, the patterns of nitrogenase activity and glutamine synthetase activity in the roots were similar. Thus, rice roots have the potential to assimilate ammonia while fixing N₂. During the vegetative and early reproductive stages of growth, the development of maximal rates of nitrogenase activity coincided with an increase of total nitrogen of the plants in both treatments.     

Boron requirement in the Discaria trinervis (Rhamnaceae) and Frankia symbiotic relationship. Its essentiality for Frankia BCU110501 growth and nitrogen fixation

The essentiality of boron (B) for nitrogen fixation in heterocystous cyanobacteria and rhizobial symbioses has been widely established. However, nothing is known about the possible involvement of the micronutrient in actinorhizal symbioses. Therefore, the effect of boron (B) deficiency on the establishment of the Discaria trinervis-Frankia BCU110501 symbiosis was investigated. Nodulation was diminished in B-deficient D. trinervis or in plants inoculated with Frankia grown in the absence of B. These poorly nodulated plants showed a reduction of shoot and root weight and small size. Because depletion of the micronutrient during growth of the actinomycete altered the infection capacity of Frankia , we also studied growth, structure and nitrogen fixation of free-living Frankia BCU110501. Growth was delayed in B-deficient BAP media (+N cultures), and completely inhibited in B-deprived N-free BAP media (–N cultures), suggesting that B is required to enhance growth of Frankia and essential for the development of nitrogen fixing activity. Ultrastructural study of B-deficient cells showed an alteration of filament walls both in +N and especially in –N cultures, indicating a possible role of the microelement in the maintenance of these structures. Moreover, the stability of vesicle envelopes was impaired in the absence of B and, hence, nitrogenase occurrence and nitrogen fixation were totally absent. The results show that B is required for both partners to establish an effective symbiosis.     

Regulation of nitrogenase levels in Anabaena sp. ATCC 33047 and other filamentous cyanobacteria

Incubation in the dark of photoautotrophically grown N₂-fixing heterocystous cyanobacteria leads to a loss of nitrogenase activity. Original levels of nitrogenase activity are rapidly regained upon re-illumination of the filaments, in a process dependent on de novo protein synthesis. Ammonia, acting indirectly through some of its metabolic derivatives, inhibits the light-promoted development of nitrogenase activity in filaments of Anabaena sp. ATCC 33047 and several other cyanobacteria containing mature heterocysts. The ammonia-mediated control system is also operative in N₂-fixing filaments in the absence of any added source of combined nitrogen, with the ammonia resulting from N₂-fixation already partially inhibiting full expression of nitrogenase. High nitrogenase levels, about two-fold higher than those in normal N₂-fixing Anabaena sp. ATCC 33047, are found in cell suspensions which have been treated with the glutamine synthetase inhibitor l-methionine-d,l-sulfoximine or subjected to nitrogen starvation. Filaments treated in either way are insensitive to the ammonia-promoted inhibition of nitrogenase development, although this insensitivity is only transitory for the nitrogen-starved filaments, which become ammonia-sensitive once they regain their normal nitrogen status.Abbreviations Chl chlorophyll - EDTA ethylenediaminetetraacetic acid - MSX l-methionine-d,l-sulfoximine     

Epilithic cyanobacterial communities of a marine tropical beach rock (Heron Island, Great Barrier Reef): diversity and diazotrophy

The diversity and nitrogenase activity of epilithic marine microbes in a Holocene beach rock (Heron Island, Great Barrier Reef, Australia) with a proposed biological calcification "microbialite" origin were examined. Partial 16S rRNA gene sequences from the dominant mat (a coherent and layered pink-pigmented community spread over the beach rock) and biofilms (nonstratified, differently pigmented microbial communities of small shallow depressions) were retrieved using denaturing gradient gel electrophoresis (DGGE), and a clone library was retrieved from the dominant mat. The 16S rRNA gene sequences and morphological analyses revealed heterogeneity in the cyanobacterial distribution patterns. The nonheterocystous filamentous genus Blennothrix sp., phylogenetically related to Lyngbya, dominated the mat together with unidentified nonheterocystous filaments of members of the Pseudanabaenaceae and the unicellular genus Chroococcidiopsis. The dominance and three-dimensional intertwined distribution of these organisms were confirmed by nonintrusive scanning microscopy. In contrast, the less pronounced biofilms were dominated by the heterocystous cyanobacterial genus Calothrix, two unicellular Entophysalis morphotypes, Lyngbya spp., and members of the Pseudanabaenaceae family. Cytophaga-Flavobacterium-Bacteroides and Alphaproteobacteria phylotypes were also retrieved from the beach rock. The microbial diversity of the dominant mat was accompanied by high nocturnal nitrogenase activities (as determined by in situ acetylene reduction assays). A new DGGE nifH gene optimization approach for cyanobacterial nitrogen fixers showed that the sequences retrieved from the dominant mat were related to nonheterocystous uncultured cyanobacterial phylotypes, only distantly related to sequences of nitrogen-fixing cultured cyanobacteria. These data stress the occurrence and importance of nonheterocystous epilithic cyanobacteria, and it is hypothesized that such epilithic cyanobacteria are the principal nitrogen fixers of the Heron Island beach rock.     

Salinity effects on growth,photosynthetic parameters,and nitrogenase activity in estuarine planktonic cyanobacteria

Salinity has been suggested as being a controlling factor for blooms of N2-fixing cyanobacteria in estuaries. We tested the effect of salinity on the growth, N2 fixation, and photosynthetic activities of estuarine and freshwater isolates of heterocystous bloom-forming cyanobacteria. Anabaena aphanizomenoides and Anabaenopsis sp. were isolated from the Neuse River Estuary, North Carolina, and Cylindrospermopsis raciborskii from Lakes Dora and Griffin, central Florida. Salinity tolerance of these cyanobacteria was compared with that of two Nodularia strains from the Baltic Sea. We measured growth rates, N2 fixation (nitrogenase activity), and CO2 fixation at salinities between 0 and 20 g L(-1) NaCl. We also examined photosynthesis-irradiance relation-ships in response to salinity. Anabaenopsis maintained similar growth rates in the full range of salinities from 2 to 20 g L(-1) NaCl. Anabaena grew at up to 15 g L-', but the maximum salinity 20 g L(-1) NaCl was inhibitory. The upper limit for salinity tolerance of Cylindrospermopsis was 4 g L(-1) NaCl. Nodularia spp. maintained similar growth rates in the full range of salinities from 0 to 20 g L(-1) . Between 0 and 10 g L(-1), the growth rate of Nodularia spumigena was slower than that of the Neuse Estuary strains. In most strains, the sensitivity of nitrogenase activity and CO2 fixation to salinity appeared similar. Anabaenopsis, Anabaena, and the two Nodularia strains rapidly responded to NaCl by increasing their maximum photosynthetic rates (Pmn). Overall, both Neuse River Estuary and Baltic Sea strains showed an ability to acclimate to salt stress over short-(24 h) and long-term (several days to weeks) exposures. The study suggested that direct effect of salinity (as NaCl in these experiments) on cyanobacterial physiology does not alone explain the low frequency and magnitude of blooms of N2-fixing cyanobacteria in estuaries.     

Acclimation of Cyanobacterial Communities in Rice Fields and Response of Nitrogenase Activity to Light Regime

Abstract The short-term and long-term effects of light regime on nitrogenase activity (NA) and cyanobacterial communities in rice fields (Valencia, Spain) were examined. Daily variation in nitrogen fixation was measured during three periods of the crop cycle: tillering (formation of secondary stems in the rice plants), heading (formation of reproductive structures), and maturity. Two locations were examined over two consecutive years (1994 and 1995). Despite differences in the crop-cycle periods, location, and year, a consistent pattern of nitrogen fixation was observed, with a main activity peak in the morning and another in the late evening. Short-term experiments, performed on two cyanobacterial blooms (Nostoc sp. and Anabaena sp.) exposed to natural light under plant canopy (7% incidence irradiance), and to different light intensities under neutral density screens without plant cover (full sunlight, 43%, 26%, and 13% of incident irradiance), indicated that nitrogenase activity (NA) was dependent on both light intensity and quality. In long-term experiments, where natural communities of cyanobacteria were exposed to one month of different light intensities, changes in the species composition of the three main genera of heterocystous cyanobacteria (Nostoc, Anabaena, and Calothrix) were observed. The light intensity at which communities were exposed for one month became the optimum irradiance for NA for each cyanobacterial community. Assays performed at higher or lower irradiances showed lower NA. Nitrogen fixation followed a pattern of seasonal variation along the crop cycle. Values were low at the beginning of the crop (May), reached a maximum value at the end of the tillering stage (June), and declined thereafter until the end of the cultivation cycle (September). Received: 25 February 1997; Accepted: 8 July 1997     

Effects of ultraviolet radiation on productivity and nitrogen fixation in the Cyanobacterium, Anabaena sp. (Newton’s strain)

The biological effects of ultraviolet radiation (UVR; 290–400 nm), especially the UV-B (320–400 nm) component of the spectrum, include both direct and indirect effects on many cellular processes. In cyanobacteria both photosynthesis and nitrogen fixation can be affected directly by UVR, and indirectly by UVR through the production of reactive oxygen species (ROS). For the heterocystous cyanobacterium, Anabaena sp. (Newton’s strain), exposure to UVR causes a significant decline in the quantum yields of photosystem II (PSII) fluorescence and maximum productivity despite an increase in UVR absorbing compounds, mycosporine-like amino acids (MAAs), in those cells exposed to UVR. Concurrent with these observations are significant increases in the activities of superoxide dismutase indicative of an increase in the level of oxidative stress in cells exposed to UVR. Additionally, measurements of nitrogenase activity (acetylene reduction) show a significant decrease in cyanobacteria exposed to UVR, which manifests itself as a decrease in cellular nitrogen and an increase in C:N ratios. These results show that these nitrogen-fixing cyanobacteria are particularly sensitive to UVR, both its direct and indirect effects. The effects of UVR reported here add to the increasing evidence that UVR effects on this important group of prokaryotes could affect the input of new nitrogen, and the biogeochemical cycling of this essential macronutrient in terrestrial, marine, and freshwater habitats. Handling editor: L. Naselli-Flores An erratum to this article can be found at     

Epiphytic cyanobacteria on Chara vulgaris are the main contributors to N(2) fixation in rice fields

The distribution of nitrogenase activity in the rice-soil system and the possible contribution of epiphytic cyanobacteria on rice plants and other macrophytes to this activity were studied in two locations in the rice fields of Valencia, Spain, in two consecutive crop seasons. The largest proportion of photodependent N(2) fixation was associated with the macrophyte Chara vulgaris in both years and at both locations. The nitrogen fixation rate associated with Chara always represented more than 45% of the global nitrogenase activity measured in the rice field. The estimated average N(2) fixation rate associated with Chara was 27.53 kg of N ha(-1) crop(-1). The mean estimated N(2) fixation rates for the other parts of the system for all sampling periods were as follows: soil, 4.07 kg of N ha(-1) crop(-1); submerged parts of rice plants, 3.93 kg of N ha(-1) crop(-1); and roots, 0.28 kg of N ha(-1) crop(-1). Micrographic studies revealed the presence of epiphytic cyanobacteria on the surface of Chara. Three-dimensional reconstructions by confocal scanning laser microscopy revealed no cyanobacterial cells inside the Chara structures. Quantification of epiphytic cyanobacteria by image analysis revealed that cyanobacteria were more abundant in nodes than in internodes (on average, cyanobacteria covered 8.4% +/- 4.4% and 6.2% +/- 5.0% of the surface area in the nodes and internodes, respectively). Epiphytic cyanobacteria were also quantified by using a fluorometer. This made it possible to discriminate which algal groups were the source of chlorophyll a. Chlorophyll a measurements confirmed that cyanobacteria were more abundant in nodes than in internodes (on average, the chlorophyll a concentrations were 17.2 +/- 28.0 and 4.0 +/- 3.8 microg mg [dry weight] of Chara(-1) in the nodes and internodes, respectively). These results indicate that this macrophyte, which is usually considered a weed in the context of rice cultivation, may help maintain soil N fertility in the rice field ecosystem.     

Influence of some environmental factors on nitrogen fixation in the rhizosphere of rice

Summary Nitrogen fixers make up a large percentage of the total microflora in the rhizosphere of lowland rice. There are more aerobic nitrogen fixers than there are anaerobic ones. When soil crumbs from the root zone were placed on a nitrogen free agar medium and inoculated at 0, 5, 10, and 21 percent oxygen concentration, colonies of aerobic nitrogen fixers reached their greatest diameter at 5 and 10 percent oxygen. In acetylene reduction assays rice plants grown in paddy fields and in solution culture were tested for the nitrogenase activities of their roots at different oxygen tensions. Nitrogenase activity was highest at 3 percent oxygen, lower at 0 percent, and far lower at 21 percent. When rice was grown in solution culture the redox potential of the nutrient solution strongly influenced nitrogenase activity. With declining redox potential, nitrogenase activity increased to a maximum value but dropped sharply as redox potential further decreased. Ten ppm of combined nitrogen as urea depressed nitrogenase activity on excised roots. Combined nitrogen applied to one part of the root system affected, to some extent, nitrogen fixation on other roots kept in a solution without nitrogen. Nitrogenase activity in a fertility trial with lowland rice, examined at several dates, showed no inhibitory effect of fertilizer nitrogen, however, presumably because the nitrogen concentration in the soil solution rapidly decreased. Instead, an overall stimulating effect of nitrogen dressing was noticeable. Diurnal fluctuations of nitrogenase activity in the rhizosphere, with a peak in the afternoon and low fixation rates after low solar radiation, suggest a photosynthetic effect on nitrogen fixation. re]19751208     

Microbiological, molecular biological and stable isotopic evidence for nitrogen fixation in the open waters of Lake Michigan

We have used a combination of microbiological, molecular biological and stable isotope methods to relate specific microbial populations to elemental cycling at an offshore site in Lake Michigan. Several lines of evidence suggest that atmospheric N₂ may be a significant source of nitrogen to the lake. Particulate organic nitrogen (PON) at ≈ 10–15 m depth in July and October had a δ¹⁵N of 0.5–1.5‰. These values closely reflect the ¹⁵N composition of atmospheric N₂, suggesting biological nitrogen fixation. Historical data show a developing late-summer N:P minimum at ≈ 15 m; low abundance of inorganic nitrogen relative to phosphorus favours species able to acquire atmospheric nitrogen. Microscopic examination of October water samples revealed abundant heterocystous cyanobacteria, including Nodularia sp. Potentially nitrogen-fixing Anabaena spp. have been found in Lake Michigan before but, to our knowledge, this is the first report of Nodularia . Finally, we have amplified both cyanobacterial and non-cyanobacterial nifH sequences (encoding the nitrogenase iron protein) from lakewater samples, evidence for the presence of bacteria capable of nitrogen fixation. The surface waters of Lake Michigan are considered to be phosphate limited in the stratified season and, under these conditions, energetically expensive nitrogen fixation is expected to be uncompetitive with assimilation of combined nitrogen. Our results suggest that, from both microbiological and biogeochemical perspectives, this may be an oversimplification.