Comparison of Nitrogen Fixation Activity in Tall and Short Spartina alterniflora Salt Marsh Soils

A comparison of the N₂ fixers in the tall Spartina alterniflora and short S. alterniflora marsh soils was investigated. Zero-order kinetics and first-order kinetics of acetylene reduction were used to describe the activity of the N₂ fixers in marsh soil slurries. It was found that the V_max values were approximately 10 times greater for the N₂ fixers in the tall Spartina than in the short Spartina marsh when raffinose was used as the energy source. In addition, the (K_s + S_n) values were approximately 4 to 15 times lower for the N₂ fixers in the tall Spartina than in short Spartina marsh. First-order kinetics of nitrogen fixation for several substrates indicate that the N₂ fixers in the tall Spartina marsh were two to seven times more active than those in the short Spartina marsh. Ammonium chloride (25 μg/ml) did not inhibit nitrogen fixation in the tall Spartina marsh, but there was a 50% inhibition in nitrogen fixation in the short Spartina marsh. On the other hand, sodium nitrate inhibited nitrogen fixation almost 100% at 25 μg/ml in both soil environments. Amino nitrogen (25 to 100 μg/ml) had little or no effect on nitrogen fixation. The results indicate that the N₂ fixers in the tall Spartina marsh were physiologically more responsive to nutrient addition than those in the short Spartina marsh. This difference in the two populations may be related to the difference in daily tidal influence in the respective areas and thus provide another explanation for the enhanced S. alterniflora production in the creek bank soil system.     

Populations of Methane-Producing Bacteria and In Vitro Methanogenesis in Salt Marsh and Estuarine Sediments

Most probable numbers (MPNs) of methanogens in various salt marsh and estuarine sediments were determined with an anaerobic, habitat-simulating culture medium with 80% H₂ plus 20% CO₂ as substrate. Average MPNs for the short Spartina (SS) marsh sediments of Sapelo Island, Ga., were maximal at the 5- to 7-cm depth (1.2 × 10⁷/g of dry sediment). Populations decreased to approximately 880/g of dry sediment at the 34- to 36-cm depth. There was no significant difference between summer and winter populations. In tall Spartina (TS) marsh sediments, average populations were maximal (1.2 × 10⁶/g of dry sediment) in the upper 0- to 2-cm zone; populations from the 5- to 36-cm zones were similar (average of 9 × 10⁴/g of dry sediment). Methanogenic populations for TS sediments of James Island Creek marsh, Charleston, S.C., were similar (average of 3 × 10⁶/g of dry sediment) for all depths tested (0 to 22 cm), which was comparable to the trend observed for TS sediments at Sapelo Island, Ga. Sediment grab samples collected along a transect of James Island Creek and its adjacent Spartina marsh had MPNs that were approximately 20 times greater for the region of Spartina growth (average of 10⁶/g of dry sediment) compared with the channel (approximately 5 × 10⁴ methanogens per g of dry sediment). A similar trend was found at Pawley's Island marsh, S.C., but populations were approximately one order of magnitude lower. In vitro rates of methanogenesis with SS sediments incubated under 80% H₂-20% CO₂ showed that the 5- to 7-cm region exhibited maximal activity (51 nmol of CH₄ g⁻¹ h⁻¹), which was greater than rates for sediments above and below this depth. SS sediment samples (5 to 7 cm) incubated under 100% N₂ and supplemented with formate exhibited rates of methanogenesis similar to those generated by samples under 80% H₂-20% CO₂. Replacing the N₂ atmosphere with H₂ resulted in an eightfold decrease in the rate of methanogenesis. In vitro methanogenic activity by TS salt marsh sediments, incubated under 80% H₂-20% CO₂, was similar for all depths tested (0 to 22 cm). TS sediment samples (0 to 7 cm) supplemented with formate and incubated under 100% N₂ had greater rates of methanogenesis compared with unsupplemented samples.     

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.     

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.     

Acetylene Reduction (Nitrogen Fixation) by Pulp and Paper Mill Effluents and by Klebsiella Isolated from Effluents and Environmental Situations

High rates of acetylene (C₂H₂) reduction (nitrogenase activity) were observed in woodroom effluent from a neutral sulfite semi-chemical mill under aerobic (up to 644 nmol of C₂H₄ produced per ml per h) and under anaerobic (up to 135 nmol of C₂H₄ produced per ml per h) conditions. Pasteurized effluent developed C₂H₂ reduction activity when incubated under anaerobic but not under aerobic conditions. Activities were increased by addition of 0.5 to 3.0% glucose or xylose. Enrichment and enumeration studies showed that N₂-fixing Azotobacter and Klebsiella were abundant, and N₂-fixing Bacillus was present. Of 129 isolates of Klebsiella from pulp mills, lakes, rivers, and drainage and sewage systems, 32% possessed nitrogen-fixing ability.     

Populations of Anaerobic Phototrophic Bacteria in a Spartina alterniflora Salt Marsh

Habitat-simulating media were used with the Hungate anaerobic roll tube technique to enumerate culturable anaerobic photosynthetic bacteria in sediment, tidal waters, and Spartina alterniflora plant samples collected from the salt marsh at Sapelo Island, Ga. No phototrophs were detected in samples of creekside (low marsh) sediment or in tidal waters in creekside regions. In the high marsh region, 90% of anaerobic phototrophic bacteria occurred in the top 5 mm of sediment and none were detected below 6 mm. There was a seasonal variation, with maximal populations occurring in summer and fall (mean, 4.4 × 10⁵ phototrophs g of dry sediment⁻¹) and minimal numbers occurring in winter (mean, 3.9 × 10³ phototrophs g of dry sediment⁻¹). During winter and late spring, phototrophs had a patchy distribution over the high marsh sediment surface. In contrast, during late summer they had a random uniform distribution. Tidal water collected over high marsh sediment contained an average of 8.7 × 10² phototrophs ml⁻¹, with no significant seasonal variation. Anaerobic phototrophic bacteria were also cultured from the lower stem tissue of S. alterniflora growing in both the high (4.3 × 10⁴ phototrophs g of dry tissue⁻¹) and creekside (4.9 × 10⁴ phototrophs g of dry tissue⁻¹) marsh regions. Chromatium buderi, Chromatium vinosum, Thiospirillum sanguineum, Rhodospirillum molischianum, and Chlorobium phaeobacteroides were the predominant anaerobic phototrophic species cultured from high marsh sediment. The two Chromatium species were dominant.     

Adhesion of Biodegradative Anaerobic Bacteria to Solid Surfaces

In order to exploit the ability of anaerobic bacteria to degrade certain contaminants for bioremediation of polluted subsurface environments, we need to understand the mechanisms by which such bacteria partition between aqueous and solid phases, as well as the environmental conditions that influence partitioning. We studied four strictly anaerobic bacteria, Desulfomonile tiedjei, Syntrophomonas wolfei, Syntrophobacter wolinii, and Desulfovibrio sp. strain G11, which theoretically together can constitute a tetrachloroethylene- and trichloroethylene-dechlorinating consortium. Adhesion of these organisms was evaluated by microscopic determination of the numbers of cells that attached to glass coverslips exposed to cell suspensions under anaerobic conditions. We studied the effects of the growth phase of the organisms on adhesion, as well as the influence of electrostatic and hydrophobic properties of the substratum. Results indicate that S. wolfei adheres in considerably higher numbers to glass surfaces than the other three organisms. Starvation greatly decreases adhesion of S. wolfei and Desulfovibrio sp. strain G11 but seems to have less of an effect on the adhesion of the other bacteria. The presence of Fe³⁺ on the substratum, which would be electropositive, significantly increased the adhesion of S. wolfei, whereas the presence of silicon hydrophobic groups decreased the numbers of attached cells of all species. Measurements of transport of cells through hydrophobic-interaction and electrostatic-interaction columns indicated that all four species had negatively charged cell surfaces and that D. tiedjei and Desulfovibrio sp. strain G11 possessed some hydrophobic cell surface properties. These findings are an early step toward understanding the dynamic attachment of anaerobic bacteria in anoxic environments.     

Effect of Inoculation with N2-Fixing Spirilla and Azotobacter on Nitrogenase Activity on Roots of Maize Grown Under Subtropical Conditions

Inoculated and non-inoculated seedlings of maize were grown in fertile clayloam soils of Egypt and Belgium under subtropical conditions provided in a greenhouse. Acetylene-reducing activity and microbial counts were determined during a period ranging from 6 to 12 weeks after sowing. Irrespective of soil origin, N₂-fixing spirilla and Azotobacter were common under maize cultivation. Inoculation resulted in a transitional increase in their numbers at early stages of growth. Nitrogenase activity was not detected in the rhizosphere of young plants. The maximum activities measured (81 to 1,436 nmol of C₂H₄ g⁻¹ h⁻¹) occurred close to the 50 to 70% silking stage. Inoculation with N₂-fixing spirilla, particularly in Nile Delta soil, doubled the amount of N₂ fixed in a late period of growth (12 weeks), whereas inoculation with Azotobacter had no noticeable effect.     

Nitrogen Fixation Associated with Sand Grain Root Sheaths (Rhizosheaths) of Certain Xeric Grasses

Nitrogen fixation (acetylene reduction) was found to be associated with sand grain root sheaths (rhizoseaths) occurring on the following xeric grasses: Oryzopsis hymenoides (Roem. and Shult.) Ricker, Agropyron dasystachyum (Hook.) Scrib., Stipa comata Trin. and Rupr., and Aristida purpurea Nutt. Acetylene reduction rates associated with whole plant specimens of these species varied from 515 to 920 nmol C₂H₄/(g dry wt.) × (6 days). Nitrogenase activity was shown to be associated with the rhizosheaths. Bacillus polymyxa-like nitrogen fixers were isolated from the rhizosheaths of each grass. The isolates reduced acetylene and assimilated ¹⁵N₂.     

Evaluation of Azotobacter and Azospirillum as Biofertilizers in Aquaculture

Summary A preliminary comparative evaluation of the two commonly encountered free-living nitrogen fixers in the aquatic system, Azotobacter and Azospirillum was carried out in the laboratory for use as biofertilizers in aquaculture considering the importance of eco-friendly and econo-friendly productivity optimization of freshwater aquaculture. The ammonium–nitrogen levels in water media in Azotobacter treatment varied in the range 2.59–34.34 μg-N/l and was found to be significantly different from that of Azospirillum treatment (p < 0.05). The viable population of the respective nitrogen fixers as colony forming units (c.f.u.) in water media in charcoal-immobilized Azotobacter treatment ranged from 0.39 to 2.48 × 10³ c.f.u./ml and were significantly higher (p < 0.05) than the counterparts. The nitrogenase activity in the same treatment similarly remained higher, at 8.3–12.15 nmol of ethylene/ml water/h followed by the alginate-immobilized Azotobacter treatment which was at 7.2–11.40 nmol of ethylene/ml water/h compared to 5.8–7.8 and 4.65–4.83 in the respective Azospirillum-treated counterparts. Hence, better performance of Azotobacter sp. over Azospirillum sp., and of charcoal immobilization over alginate immobilization were observed.     

Nitrogenase activity in the papyrus swamps of Lake Naivasha,Kenya

Nitrogenase activity (acetylene reduction activity) was found to occur universally in the Cyperus papyrus swamp in Lake Naivasha. Low rates of acetylene reduction activity (0.9–104.9 nmol C₂H₄ g d.wt. roots^-1 h^-1) were associated with excised roots of C. papyrus but higher rates of activity (89.0–280.4 nmol C₂H₄ g d.wt. roots^-1 h^-1) were associated with intact root systems of the plant. It was estimated that nitrogen fixation associated with young roots alone could supply about 26% of the nitrogen requirements of growing papyrus plants. Acetylene reduction activity in the lake bottom sediments was generally low and associated with adjacent papyrus stands. Plate counts of putative aerobic and facultatively anaerobic N₂-fixing bacteria associated with papyrus roots showed the presence of high numbers of diazotrophs (5.4 × 10⁶ CFU g d.wt. roots^-1). Fewer numbers of N₂-fixing bacteria were detected in the sediments (1.9 × 10³-3.2 × 10⁴ CFU g d.wt. sediment^-1).     

Dinitrogen Fixation by Cultures of Frankia sp CpIl Demonstrated by N(2) Incorporation

The filamentous bacterium Frankia of the Actinomycetales, isolated from the nitrogen-fixing root nodules of certain woody plants, has shown nitrogenase activity in culture, using the acetylene reduction method. In the present work, nitrogenase activity in pure cultures of Frankia sp. CpIl is confirmed using mass spectrometric measurements of ¹⁵N₂ incorporation. After addition of carrier NH₄⁺ to digested cultures, those exposed to ¹⁵N₂ (25 atom%) had a ¹⁵N content of 3.16 atom% compared to 0.354 atom% ¹⁵N in the controls.     

BACTERIAL ASSOCIATIONS WITH MARINE OSCILLATORIA SP. (TRICHODESMIUM SP.) POPULATIONS: ECOPHYSIOLOGICAL IMPLICATIONS1

On three separate occasions we investigated morphological and physiological aspects of bacterial associations with planktonic aggregates of the ubiquitous marine N₂ fixing cyanobacterium Trichodesmium sp. Close associations generally characterized Trichodesmium blooms; associations were present during day- and night-time. Colonization by both rod-shaped and filamentous heterotrophic bacteria occurred on Trichodesmiun aggregates actively fixing N₂ (acetylene reduction). Scanning electron and optical microscopy showed bacteria located both around and within aggregates. Microautoradiography demonstrated that associated bacteria largely mediated utilization of trace additions of ³H-labeled carbohydrates (fructose, glucose, mannitol) and amino acids, whereas Trichodesmium utilized amino acids only. Oxygen measurements using microelectrodes revealed high localized oxygen consumption among aggregates, with rapid (within a minute) changes from supersaturated to subsaturated oxygen following the transition from photosynthetic illuminated to dark periods. Stab culturing techniques confirmed the presence of heterotrophic N₂ fixers among aggregate-associated bacteria. Parallel deployment of oxygen microelectrodes, the tetrazolium salt 2,3,5 triphenyl tetrazolium chloride (TTC) and acetylene reduction assays demonstrated microaerophilic requirements for expression of nitrogenase activity among cultured bacteria. Trichodesmium aggregates are characterized by dynamic nutrient and oxygen regimes, which promote and maintain simultaneous and contiguous oxygenic photosynthesis and N₂ fixation. In part, the above-mentioned consortial interactions with a variety of heterotrophic bacteria facilitate Trichodesmium biomass production and bloom formation in nitrogen depleted, oligotrophic tropical/subtropical waters.     

Cyanobacterial (unicellular and heterocystous) biofertilization to wetland rice influenced by nitrogenous agrochemical

Comparative growth and N₂-fixation of cyanobacteria, namely Aphanothece sp. (unicellular) and Gloeotrichia sp. (heterocystous, filamentous), were studied after their inoculation to rice crop in the absence and presence of urea nitrogen fertilizer. In the absence of N-fertilizer application (control), inoculation of both cyanobacterial species showed significant increase in growth and acetylene reduction activity (ARA), but gradual reduction in these parameters was observed at 30 and 60 kg N ha^?1 of urea application. In inoculation of Gloeotrichia sp. at control, 30 and 60 kg N ha^?1 increased grain yield significantly over uninoculated control in both wet and dry seasons, but grain yield with Aphanothece sp. inoculation was statistically similar to the control at N levels during both seasons. The inoculation study showed that heterocystous cyanobacteria contributed better than unicellular ones, and application of N-fertilizer adversely affected both growth and N₂-fixation of native as well of inoculated cyanobacteria.     

Nitrogen fixation in coniferous bark litter

Summary Non-symbiotic heterotrophic N₂ fixation in coniferous bark litter was investigated with the acetylene reduction assay under aerobic and anaerobic conditions. The litter studied was composed essentially of bark, of pH 5 and a C/N ratio of 101; the ratio of available C to available N, which governs N₂ fixation, was considerably higher. The rate of N₂ fixation was estimated as 2.5–4.4 g N. g^–1 dry wt. day^–1. Nitrogenase activity was still evident after seven months of incubation under aerobic conditions. The N₂-ase activity was O₂ dependent: under anaerobic conditions no N₂-ase activity was found unless a fermentable C source was added. The importance of N₂ fixation in N-poor litter for the maintenance of soil fertility is emphasized.     

Propionate-Degrading Bacterium, Syntrophobacter wolinii sp. nov. gen. nov., from Methanogenic Ecosystems

A new genus and species of a nonmotile gram-negative rod, Syntrophobacter wolinii, is the first bacterium described which degrades propionate only in coculture with an H₂-using organism and in the absence of light or exogenous electron acceptors such as O₂, sulfate, or nitrate. It was isolated from methanogenic enrichments from an anaerobic municipal sewage digestor, using anaerobic roll tubes containing a medium with propionate as the energy source in association with an H₂-using, sulfate-reducing Desulfovibrio sp. which cannot utilize fatty acids other than formate. S. wolinii produced acetate and, presumably, CO₂ and H₂ (or formate) from propionate. In media without sulfate and with Methanospirillum hungatei, a methanogen that uses only H₂-CO₂ or formate as an energy source, acetate, methane, and, presumably, CO₂ were produced from propionate and only small amounts of Desulfovibrio sp. were present. Isolation in coculture with the methanogen was not successful. S. wolinii does not use other saturated fatty acids as energy sources.     

Enumeration and Localization of N2-Fixing Bacteria Associated with Roots of Spartina alterniflora Loisel

Numbers and possible locations of N₂-fixing bacteria were investigated in roots of Spartina alterniflora Loisel, which support nitrogenase activity in the undisturbed native habitat. N₂-fixing bacteria were recovered in cultures both from S. alterniflora roots and from the surrounding sediment, and they formed a greater proportion of the bacteria recovered from root homogenates than from salt-marsh sediment. N₂-fixing bacteria were recovered in high numbers from the rhizoplane of S. alterniflora after roots were treated with 1 or 5% chloramine-T for 1 h or with 1% NaOCl for 1 or 2 h. Immersing S. alterniflora roots in 5% NaOCl for 1 h was more effective in distinguishing bacteria inside the roots since this treatment nearly eliminated N₂-fixing bacteria recoverable from the rhizoplane, although high numbers of N₂-fixing bacteria were recovered from homogenates of roots treated with 5% NaOCl for 1 h. However, this treatment was less effective with roots of Zea mays L. (Funks G4646) and Sorghum bicolor (L.) Moench (CK-60 A), indicating that techniques to surface sterilize roots should be evaluated for different plants. Bacteria were observed by light and electron microscopy inter- and intracellularly in the cortex and in the aerenchyma of S. alterniflora roots. This study clearly shows that bacteria, including N₂ fixers, colonize the interior of roots of S. alterniflora growing in a Chesapeake Bay, Maryland, salt marsh.     

小兴安岭典型苔草和灌木沼泽N2O排放及影响因子

为了研究小兴安岭林区典型苔草和灌木沼泽N2O排放通量的季节动态、年际动态及其与环境因子的关系,并估算排放总量,2007和2008年在植物生长季采用静态箱-气相色谱法,对小兴安岭林区典型修氏苔草（Carex schmidtii）沼泽和油桦-修氏苔草（Betula ovalifolia-Carex schmidtii）灌木沼泽N2O排放进行了监测。结果表明,苔草和灌木沼泽2007年生长季N2O排放总量分别为0.14和0.29 kg/hm2;2008年分别为0.68和-0.10 kg/hm2。苔草和灌木沼泽N2O排放通量除灌木沼泽2008年变化规律性不明显外,均具有比较显著的季节变化,最大排放出现在夏季或夏、秋季节,其中2007年N2O排放平均通量为0.0037和0.0082 mg?m-2?h-1;2008年为0.016和-0.0025 mg?m-2?h-1。分析表明,苔草沼泽N2O排放年际差异不显著,灌木沼泽N2O排放年际差异显著;不同类型沼泽间N2O排放差异不显著;仅苔草沼泽2007年N2O排放通量与水位具有显著的负相关性（r=-0.52,P < 0.05,n=15）。     

Inoculation of forage grasses with N2-fixing Enterobacteriaceae

Summary Previous investigations indicated some forage grass roots in Texas are heavily colonized with N₂-fixing bacteria. The most numerous N₂-fixing bacteria were in the genera Klebsiella and Enterobacter. In the present investigation inoculation experiments were conducted using 18 isolates of these bacteria to determine if a N₂-fixing association could be established between the bacteria and the grassesCynodon dactylon andPanicum coloratum. Plants were grown in soil for approximately 5 months in a greenhouse and were measured periodically for dry matter, nitrogen accumulation, and acetylene reduction activity. Results of the investigation indicated that 25% of the plant-soil systems were active in acetylene reduction and the activity was high enough to indicate agronomically significant quantities of N₂ were being fixed (>8kg N ha⁻¹). However, plant systems extrapolated to fix>8 kg N ha⁻¹ contained less nitrogen and accumulated less dry matter than plants less active in acetylene reduction. Inocula could not be re-isolated from healthy grass roots indicating that the N₂-fixing activity may have not have been closely assiciated with plant roots. Future research is needed to determine factors limiting colonization of grass roots.     

Nitrogenase Activity Associated with Halodule wrightii Roots

Nitrogen fixation (acetylene reduction) associated with roots of the seagrass Halodule wrightii was measured offshore near Beaufort and Moorhead City, N.C. Rates of acetylene reduction were higher in aerobic than in anaerobic assays and were linear for up to 5 days. The temperature range for acetylene reduction was 15 to 35°C with a maximum activity at 35°C. Nitrogenase activity was shown to vary seasonally with highest activities occurring during warmer summer months (23 μg of N₂ fixed per m² per day). At in situ temperature, nitrogenase activities associated with surface-sterilized and non-surface-sterilized roots were similar. One morphological bacterial type was isolated from surface-sterilized roots and identified as Klebsiella pneumoniae type 4B.