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.     

Insecticidal effects on soil microorganisms and their biochemical processes related to soil fertility

Among the four insecticides under study, hexachlorocyclohexane (BHC) followed by phorate significantly stimulated the populations of (total) bacteria, actinomycetes, fungi, aerobic non-symbiotic N2-fixing bacteria and P-solubilizing microorganisms in soil. Carbofuran significantly stimulated total as well as N2-fixing bacteria. Fenvalerate had no effect on P-solubilizers. All the insecticides stimulated the proportion of Penicillium in soil. Similarly, Pseudomonas with BHC, Sarcina with phorate, Corynebacterium, Azotobacter and Streptomyces with fenvalerate were also stimulated. On the other hand, Erysipelothrix with BHC, Staphylococcus with phorate, Staphylococcus, Nocardia and Fusarium with fenvalerate were inhibited. Almost all the insecticides reduced the proportions of Micrococcus and Rhizopus in soil. Insecticides also augmented the non-symbiotic N2-fixing and P-solubilizing capacities of the soil and the augmentation was more pronounced with BHC followed by phorate.     

Isolation of free-living dinitrogen-fixing bacteria and their activity in compost containing de-inking paper sludge

Knowledge of the microbiology of dinitrogen (N2)-fixing bacteria in compost rich in de-inking paper sludge (DPS) is limited. Dinitrogen (N2)-fixing bacteria from DPS composts were isolated and studied for their N2-fixing activity in vitro and in vivo. Two Gram-negative N2-fixing isolates were identified as Pseudomonas. At 20 degrees C, both isolates revealed that N2-fixing activity was higher than that of three arctic Pseudomonas strains. Their N2-fixing activity was found to occur between 18 and 25 degrees C, a pattern that was similar to the reference isolate Azotobacter ATCC 7486. Composts successfully showed N2-fixing activity after carbohydrate amendments both with and without inoculation of a N2-fixing isolate. These results suggest that DPS composts support N2-fixing bacteria and that N2-fixing activity is dependent on a usable carbohydrate source.     

Cell morphology and flagellation of nitrogen-fixing spirilla

Twenty isolates of N2-fixing spirilla were isolated from the rhizosphere of maize and sugar cane grown in Egyptian and Belgian soils. Electron microscopy distinguished two morphological groups. The first includes short and thick curved rods with an unipolar flagellum while cells of the second group are much longer with the typical appearance of spiral cells and most probably possess a bipolar tuft of flagella.     

Selection of Rhizobium leguminosarum strains for lentil (Lens culinaris) under growth room and field conditions

Most of the production of lentil (Lens culinaris) on the Great Plains occurs on soils that are free of indigenous Rhizobium leguminosarum. Inoculation is required to increase yields through N₂ fixation. A screening program to evaluate the effectiveness of R. leguminosarum strains for lentil was initially carried out under controlled environments followed by an evaluation under field conditions. In two separate growth room experiments, the effectiveness of 185 and 24 different strains of R. leguminosarum were tested for Laird and Eston lentil. Significant differences between strains in number of nodules, shoot weight and nitrogenase activity (acetylene reduction activity, ARA) were found for lentil grown for 5 weeks. When lentil were grown for 7 weeks, significant differences between strains in number of nodules, total plant weight, total N, and % N were observed.Fourteen strains plus Nitragin C inoculant were selected for further field testing on Eston and Laird lentil at two locations in 1986 and one site in 1987. Inoculation increased yield up to 135%. Percent Ndfa and total N₂ fixed ranged from 0 to 76 and 0 to 105 kg ha^-1, respectively. N₂-fixing activity was site specific and higher spring soil NO₃-levels resulted in lower N₂-fixing activity. Depending on site and growing conditions, strains 99A1 and I-ICAR-SYR-Le20 appeared to be superior to the other strains tested. A good agreement was found between the estimates for N₂ fixation based upon the ¹⁵N-isotope dilution and the classical N difference methods. Number of nodules, dry weight of nodules and ARA of Eston and Laird lentil grown under growth room conditions failed to show positive correlations with total dry matter production, total N or total N₂ fixed of field grown lentil. However, total plant weight and total N of lentil grown under growth room conditions were highly correlated with field parameters, and were the most reliable screening parameters for the selection of superior rhizobial strains.     

Adenine nucleotide levels in and nitrogen fixation by the cyanobacterium Anabaena sp. strain 7120.

Adenine nucleotide levels were determined in whole filaments of Anabaena sp. 7120 grown under different N2-fixing or non-N2-fixing conditions. These were compared with levels in isolated heterocysts, Rhodospirillum rubrum, and Azotobacter vinelandii. Adenine nucleotides in whole filaments of Anabaena sp. do not reflect the energetic expense of N2 fixation as they do in R. rubrum and A. vinelandii. However, adenine nucleotide levels in heterocysts were similar to the levels found in N2-fixing R. rubrum, i.e., an ATP:ADP ratio near 1 and an energy charge between 0.5 and 0.7. Nitrogenase activity was only 50% of optimal in permeabilized heterocysts at an exogenous ATP:ADP ratio of 3.33. Hydrogen, which increases acetylene reduction activity, also causes a transient increase (2 to 5 min) in the ATP:ADP ratio. Hydrogen has little effect on energy charge.     

Influence of soil water potential on respiration and nitrogen fixation ofAzotobacter vinelandii

Summary Respiration and N₂-fixation (acetylene reduction) ofAzotobacter vinelandii have been studied at a variety of soil water potentials. Both processes were strictly linked and strongly reduced at water potentials between –0.6 and –1.3 MPa. Complete inhibition occurred below –2.1MPa. Osmotic potentials in soil compared to matric potentials of the same value were less inhibitory to respiration and acetylene reduction by Azotobacter. The N₂-fixing efficiency (mg N/g glucose) was not influenced by water potentials ranging from –0.1 to –2.1 MPa.     

Effect of ammonia on the synthesis and function of the N 2 -fixing enzyme system in Clostridium pasteurianum

The N(2)-fixing system of Clostridium pasteurianum operates under regulatory controls; no activity is found in cultures growing on excess NH(3). The conditions which are necessary for the synthesis and function of this system were studied in whole cells by using acetylene reduction as a sensitive assay for the presence of the N(2)-fixing system. Nitrogenase of N(2)-fixing cultures normally can fix twice as much N(2) as is needed to maintain the growth rate. When cultures that have grown for four or more generations on NH(3) exhaust NH(3) from the medium, a diauxic lag of about 90 min ensues before growth is resumed on N(2). Neither N(2)-fixing nor acetylene reduction activity can be detected before growth is resumed on N(2). N(2) is not a necessary requirement for this synthesis since under argon that contains less than 10(-8)m N(2), the N(2)-fixing system is made. If NH(3) is added to N(2)-dependent cultures, synthesis of the enzyme system is abruptly stopped, but the enzyme already present remains stable and functional for at least 6 hr (over three generations). Cultures grown under argon in a chemostat controlled by limiting ammonia have derepressed nitrogenase synthesis. If the argon is removed and replaced by N(2), partial repression of nitrogenase occurs.     

Olive mill waste as a substrate for nitrogen fixation

Olive mill wastewaters (OMWW) because of their low content in nitrogenous organic components and reachness in carbon sources offer a highly favourable environment for the growth of free-living dinitrogen fixing microorganisms. This property is manifested both in natural environments and in axenic cultures. Repetitive addition of OMWW to soil under aerobic conditions leads progressively to its enrichment with dinitrogen fixers, the activity of which is beneficial to soil fertility. The microbial consortium that develops in soil is dominated mostly by members of Azotobacter. A very efficient N₂-fixing and slime producing strain of Azotobacter vinelandii (strain A) was isolated from such an enriched soil sample. The isolate is deposited in the culture collection of our laboratory and its biochemical and molecular characteristics are investigated. The strain proved to be effective in bio-remediation processes of OMWW both in a laboratory-scale fermenter unit and a field pilot plant of ca 5 m³ capacity. The inhibitory growth-limiting components of the principal OMWW constituents and their impact on the duration of the lag period of N₂-fixing activity recovery is examined. The design of a multi-stream two stage process is described which provides a stable N₂-fixing system suitable for the bio-transformation of OMWW into an agrobiological product and/or for the production of extracellular polysaccharide ‘slime’ in high yields.     

Effects of Mannose on the Growth of N(2)-Fixing Azotobacter vinelandii

Mannose is not a suitable substrate for N(2)-fixing Azotobacter vinelandii. However, when H(2) gas is provided, A. vinelandii can grow mixotrophically with H(2) as the energy source and mannose as the carbon source (T.-Y. Wong and R. J. Maier, J. Bacteriol. 163:528-533, 1985). In this report, seven sugars were used to determine whether A. vinelandii could derive energy from these sugars for mannose utilization. Supplementation of fructose- or galactose-limited medium with mannose did not influence the biomass produced by N(2)-fixing A. vinelandii. The presence of mannose in glucose- or maltose-limited cultures increased cell yield slightly. The addition of mannose decreased the total biomass in the melibiose-limited culture slightly. Mannose was a potent inhibitor of growth when sucrose or turanose was used as the primary sugar. The inhibitory effect of mannose on utilization of sucrose and turanose seems to be related to the energy requirement of the N(2)-fixing processes.     

Tomato Growth in Soil Amended with Sugar Mill By-Products Compost

Sugar mill by-products compost may be a good soil amendment to promote tomato (Lycopersicon esculentum L.) growth. In addition, the compost may further promote plant growth by inoculation with N₂-fixing bacteria. Compost from sugar-mill waste was prepared with and without the N₂-fixing bacteria, Azotobacter vinelandii, Beijerinckia derxii and Azospirillum sp. and incubated for 50 days. Each compost type was added to 10 kg of soil in pots at rates of 0, 15, and 45 g with and without fertilizer N at rates of 0, 0.75, and 1.54 g. A blanket application of P and K was applied to all pots. Shoot and root dry weights and N content of the whole plant was measured at 55 days. Dry weight of tomato shoots was increased by 40% by addition of fertilizer N and root weight was increased by 66%. Without fertilizer N the high rate of inoculated compost increased shoot growth 180% and uninoculated compost increased shoot growth 112%. For most treatments with and without fertilizer N, inoculated compost enhanced shoot growth and nitrogen content more than uninoculated compost. Root weights were nearly doubled by addition of either compost in comparison to the 0 N treatment. At the low rate of compost addition without fertilizer N, root weight was the same for uninoculated and inoculated compost but at the high rate of compost addition root weight was 32% higher for inoculated compost. The N₂-fixing bacteria colonized roots when inoculated compost was used. Sugar mill by-products compost proved to be an effective soil amendment for promoting the growth of tomato plants.     

Effect of nitrogen starvation on ammonium-inhibition of nitrogenase activity in Azotobacter chroococcum

When Azotobacter chroococcum cells grown in batch culture under N₂-fixing conditions were transferred to a medium lacking a nitrogen source, the cellular C/N ratio, the amount of alginic acid released into the external medium and the rate of endogenous respiration increased appreciably after 6 h to the exclusion of dinitrogen, whereas nitrogenase activity did not undergo any significant change. Nitrogen deficiency caused a decrease in the ammonium inhibition of nitrogenase activity from 95% inhibition at zero time to 14% after 6 h incubation under dinitrogen starvation, with no difference in the rate of ammonium utilization by N₂-fixing and N₂-starved cells being observed. This suggests that a balance of nitrogen and carbon assimilation is necessary for the ammonium inhibition of nitrogenase activity in A. chroococcum to take place.     

In vivo energetics and control of nitrogen fixation: changes in the adenylate energy charge and adenosine 5'-diphosphate/adenosine 5'-triphosphate ratio of cells during growth on dinitrogen versus growth on ammonia.

The effects of the intracellular energy balance and adenylate pool composition on N2 fixation were examined by determining changes in the energy charge (EC) and the ADP/ATP (D/T) ratio of cells in chemostat and batch cultures of Clostridium pasteurianum, Klebsiella pneumoniae, and Azotobacter vinelandii. When cells of C. pasteurianum, K. pneumoniae, and A. vinelandii in sucrose-limited chemostats were examined, in all cases the EC increased greater than or equal to 15% when the nitrogen source was switched from N2 to NH3 and decreased greater than or equal to 15% when the nitrogen source was switched from NH3 to N2. The D/T ratio of the same cultures decreased greater than or equal to 70% when they were switched from N2 to NH3. In such cultures the adenylate pools remained constant when the cells were grown on either NH3 or N2. In nitrogen (NH3)-limited cultures, the adenylate pool was two- to threefold higher than the adenylate pool in sucrose-limited cultures, and the nitrogenase content of such cells was two- to threefold greater than the nitrogenase content of sucrose-limited N2-fixing cells. The EC and D/T ratio of cells from batch cultures of C. pasteurianum growing on NH3 in the presence of N2 were 0.82 and 0.83, respectively, but when the NH3 was consumed and the cells were switched to a nitrogen-fixing metabolism, the EC and D/T ratio changed to 0.70 and 0.90, respectively. Conversely, when NH3 was added to N2-fixing cultures the EC and D/T ratio changed within 1.5 h the EC and D/T ratio of NH3-grown cells. The nitrogen content of N2-fixing cells to which NH3 was added decreased at a rate greater could be accounted for by cell growth in the absence of further synthesis. This decay of nitrogenase activity (with a half-life about 1.2 to 1.4 h) suggests that some type of inactivation of nitrogenase occurs during repression. The nitrogenase of whole cells was estimated to be operating at about 32% of its theoretical maximum activity during steady-state N2-fixing conditions. Similarities in the data from chemostat and batch cultures of both aerobic and anaerobic N2-fixing organisms suggest that low EC and high D/T ratio are normal manifestations of an N2-fixing physiology.     

粪产碱菌（Alcaligenes faecalis）对稻根氧化还原特性及水稻多元酚和内根际激素水平的影响

联合固氮粪产碱菌结合于水稻根表时能增强水稻根部还原力和稻根超氧化物歧化酶（ＳＯＤ）活性。实验室和田间试验证明粪产碱菌能提高水稻幼苗对高、低温不良环境的抗逆性。经浸种处理的水稻幼苗植株内多元酚含量增加了１２．５％。粪产以菌对接种水稻多元酚抽提物有强烈的趋化性,而该抽提物对粪产碱菌的固氮活性有明显的刺激作用。多元酚抽提物经双向纸层析和薄层层析表明接种诱导了至少一个特征组分含量提高。采用粪产减菌野生型Ａ１５０１（ｎｉｆ＋）和固氮缺陷型Ａ１５０６（Ｎｉｆ－）浸种能改变宿主水稻内源激素水平,提高内根际的ＩＡＡ和Ｚ的含量,促进植株及根系的生长发育,使其侧根和根毛数目明显增多。在根际联合体系形成过程中,多元酚或激素可能充当植物与细菌间相互作用的一类特异信号分子。     

Effect of inoculation and leaf litter amendment on establishment of nodule-forming Frankia populations in soil

High-N(2)-fixing activities of Frankia populations in root nodules on Alnus glutinosa improve growth performance of the host plant. Therefore, the establishment of active, nodule-forming populations of Frankia in soil is desirable. In this study, we inoculated Frankia strains of Alnus host infection groups I, IIIa, and IV into soil already harboring indigenous populations of infection groups (IIIa, IIIb, and IV). Then we amended parts of the inoculated soil with leaf litter of A. glutinosa and kept these parts of soil without host plants for several weeks until they were spiked with [(15)N]NO(3) and planted with seedlings of A. glutinosa. After 4 months of growth, we analyzed plants for growth performance, nodule formation, specific Frankia populations in root nodules, and N(2) fixation rates. The results revealed that introduced Frankia strains incubated in soil for several weeks in the absence of plants remained infective and competitive for nodulation with the indigenous Frankia populations of the soil. Inoculation into and incubation in soil without host plants generally supported subsequent plant growth performance and increased the percentage of nitrogen acquired by the host plants through N(2) fixation from 33% on noninoculated, nonamended soils to 78% on inoculated, amended soils. Introduced Frankia strains representing Alnus host infection groups IIIa and IV competed with indigenous Frankia populations, whereas frankiae of group I were not found in any nodules. When grown in noninoculated, nonamended soil, A. glutinosa plants harbored Frankia populations of only group IIIa in root nodules. This group was reduced to 32% +/- 23% (standard deviation) of the Frankia nodule populations when plants were grown in inoculated, nonamended soil. Under these conditions, the introduced Frankia strain of group IV was established in 51% +/- 20% of the nodules. Leaf litter amendment during the initial incubation in soil without plants promoted nodulation by frankiae of group IV in both inoculated and noninoculated treatments. Grown in inoculated, amended soils, plants had significantly lower numbers of nodules infected by group IIIa (8% +/- 6%) than by group IV (81% +/- 11%). On plants grown in noninoculated, amended soil, the original Frankia root nodule population represented by group IIIa of the noninoculated, nonamended soil was entirely exchanged by a Frankia population belonging to group IV. The quantification of N(2) fixation rates by (15)N dilution revealed that both the indigenous and the inoculated Frankia populations of group IV had a higher specific N(2)-fixing capacity than populations belonging to group IIIa under the conditions applied. These results show that through inoculation or leaf litter amendment, Frankia populations with high specific N(2)-fixing capacities can be established in soils. These populations remain infective on their host plants, successfully compete for nodule formation with other indigenous or inoculated Frankia populations, and thereby increase plant growth performance.     

Studies on the incorporation of a covalently bound disubstituted phosphate residue into Azotobacter vinelandii flavodoxin in vivo.

Previous studies have shown the flavodoxin from Azotobacter vinelandii (strain OP, Berkeley) to contain a covalently bound disubstituted phosphate residue [Edmondson & James (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3786-3789]. Phosphorylation of the protein in vivo was investigated by the addition of [32P]phosphate to cells grown under N2-fixing conditions, under conditions of nif-gene repression and under conditions of nif-gene de-repression. Rocket immunoelectrophoresis of cell extracts showed an approx. 5-fold decrease in the concentration of flavodoxin expressed in cells grown in the presence of NH4+ as compared with those grown under N2-fixing conditions. A similar increase in flavodoxin concentration was observed on nif-gene de-repression. Incorporation of [32P]phosphate occurs only into newly synthesized flavodoxin, as observed on SDS/PAGE of immunoprecipitates of cell extracts. Western blots demonstrated no observable precursor forms of flavodoxin. These data provide conclusive evidence for the phosphorylation of Azotobacter strain OP flavodoxin in vivo and suggest that the covalently bound phosphate residue does not exchange with cellular phosphate pools. Thus the role of this phosphodiester cross-link is proposed to be structural rather than regulatory.     

Effects of slow-release urea fertilizers on urease activity,microbial biomass, and nematode communities in an aquic brown soil

A field experiment was carried out at the Shenyang Experimental Station of Ecology (CAS) in order to study the effects of slow-release urea fertilizers high polymer-coated urea (SRU1), SRU1 mixed with dicyandiamide DCD (SRU2), and SRU1 mixed with calcium carbide CaC2 (SRU3) on urease activity, microbial biomass C and N, and nematode communities in an aquic brown soil during the maize growth period. The results demonstrated that the application of slow-release urea fertilizers inhibits soil urease activity and increases the soil NH4+-N content. Soil available N increment could promote its immobilization by microorganisms. Determination of soil microbial biomass N indicated that a combined application of coated urea and nitrification inhibitors increased the soil active N pool. The population of predators/omnivores indicated that treatment with SRU2 could provide enough soil NH4+-N to promote maize growth and increased the food resource for the soil fauna compared with the other treatments.     

Effects of slow-release urea fertilizers on urease activity,microbial biomass, and nematode communities in an aquic brown soil

A field experiment was carried out at the Shenyang Experimental Station of Ecology (CAS) in order to study the effects of slow-release urea fertilizers high polymer-coated urea (SRU1), SRU1 mixed with dicyandiamide DCD (SRU2), and SRU1 mixed with calcium carbide CaC2 (SRU3) on urease activity, microbial biomass C and N, and nematode communities in an aquic brown soil during the maize growth period. The results demonstrated that the application of slow-release urea fertilizers inhibits soil urease activity and increases the soil NH4+-N content.Soil available N increment could promote its immobilization by microorganisms. Determination of soil microbial biomass N indicated that a combined application of coated urea and nitrification inhibitors increased the soil active N pool. The population of predators/omnivores indicated that treatment with SRU2 could provide enough soil NH4+-N to promote maize growth and increased the food resource for the soil fauna compared with the other treatments.     

Possible mechanism of mannose inhibition of sucrose-supported growth in N2-fixing Azotobacter vinelandii

When mannose was added to a sucrose-supported culture of Azotobacter vinelandii under N2-fixing conditions, cell growth was inhibited. The degree of inhibition was proportional to the amount of mannose and to the aeration rate (T.-Y. Wong, Appl. Environ. Microbiol. 54:473-475, 1988). In this report, we demonstrate that once inside the cell, mannose was phosphorylated to mannose 6-phosphate. It was then isomerized to fructose 6-phosphate and to glucose 6-phosphate. Mannose inhibited sucrose uptake noncompetitively. The decrease in sucrose uptake after mannose addition coincided with a lower rate of respiration and a decrease in nitrogenase activity. The decrease in sucrose uptake and in the ATP pool may decrease the electron flow and reduce protection of the nitrogenase from O2. Cells became very sensitive to O2, and therefore, cell growth was inhibited under high aeration conditions.