Control of nitrogenase in chemostat cultures of Rhodobacter capsulatus grown on ammonium at different illuminations

Rhodobacter capsulatus strain 37b4 was grown phototrophically in chemostat cultures with 2 mM of ammonium chloride and 30 mM of malate at a constant dilution rate of 0.075 h^-1. When illumination was raised from 3000 to 30000 lx, steady state biomass levels as well as malate uptake increased linearly with increasing illumination. Yet, in no case external ammonium could be detected in the culture fluid. Specific nitrogenase activity increased by a factor of ten between 3000 and 15000 lx and approached constancy above 15 000 lx. When samples were anaerobically withdrawn from the chemostat and subsequently grown in batch cultures under saturating light conditions, biomass increased to a constant level, independently of the illumination used in the previous chemostat culture. In fact, the specific nitrogen contents of cells were 0.195 and 0.154 (g of N per g of protein) with chemostat cultures adapted to 3000 and 30000 lx, respectively. With the former cultures, specific nitrogen contents decreased to 0.142 g of nitrogen per g of cell protein upon incubation in a batch system. This suggests the existence of free nitrogen compounds in cells of chemostat cultures, the concentrations of which decrease while protein levels increase with increasing energy supply. Intracellular amino acid pools revealed slightly elevated levels of major amino acids in low-light cultures as compared to high-light cultures. On the basis of intracellular levels of ammonium, however, no significant differences could be detected. Since, in addition, malate consumption increased linearly with increasing illumination, it is proposed that light controls nitrogenase in Rhodobacter capsulatus via the C/N ratio, as represented by malate and ammonium consumption, rather than directly.     

The effect of nitrogen refeeding on the carbohydrate content into nitrogen-starved cells of Platymonas striata Butcher

Studies on the mean cellular carbohydrate contents of Platymonas striata Butcher under conditions of nitrogen-starvation, and after refeeding these starved cultures with either nitrate or ammonium ions (growing under continuous illumination or with an alternating light/dark regime) have shown that nitrogen-starved cells accumulated abnormal amounts of cellular carbohydrate and that nitrogen refeeding produced a marked drop in the cellular carbohydrate. Cells grown in a light/dark regime accumulated less carbohydrates than those grown in continuous light. The mean cellular carbohydrate levels 16 h after nitrogen refeeding were still much in excess of those of cells grown with normal nutrition. It was therefore suggested that the differences in nitrogen uptakes in this period — when comparing either the uptake of cells grown in continuous light with that of cells grown in a light/dark regime; or when comparing the uptakes of cells presented with either nitrate or ammonium ions and grown in a light/dark regime —cannot be directly due to shortages of carbohydrate for the provision of carbon skeletons for nitrogen assimilation.     

Control by light of whole-cell nitrogenase activity and of nitrogenase and bacteriochlorophyll a formation in Rhodobacter capsulatus strain B10S

Control of nitrogenase and bacteriochlorophyll a (BChl) by light was studied under steady-state conditions with continuous cultures of Rhodobacter capsulatus B10S supplied with malate and growth-limiting amounts of ammonium. Consumption of malate and, correspondingly, the C/N ratio at which malate and ammonium were consumed increased when illumination was increased from 3 to approximately 20 klx and became constant at higher illuminations of up to 40 klx. Essentially the same kinetics were observed with respect to nitrogenase activity of cells, contents of nitrogenase polypeptides, and nifH promoter activity. Substrate consumption was half-maximal at 8 klx and was independent of the presence of nitrogenase. Therefore, it is concluded that light controls the C/N ratio (a quantitative measure of the nitrogen status of cells), which in turn is involved in the control of nitrogenase at the level of nif promoter activity. Post-translational regulation of nitrogenase activity by ADP-ribosylation was not observed under steady-state conditions, but it took place when illumination was suddenly decreased to the range where malate consumption and, consequently, the C/N ratio decreased. Irrespective of the presence or absence of nitrogenase, specific BChl contents of the cultures were constant above 20 klx, and they increased at lower illuminations. These results do not confirm a recently proposed link between nitrogen fixation and photosynthesis as represented by BChl. Received: 29 October 1998 / Accepted: 30 December 1998     

高脱氮活性海洋着色菌YL28生物膜特性

【背景】细菌生物膜在废水处理领域显示出良好的前景,但目前应用于海水养殖水体处理的菌株主要源自淡水菌株,存在难以适应海水高盐环境的问题。源自红树林的海洋着色菌(Marichromatiumgracile)YL28应用于海水养殖水体处理,不仅具有高效除氮能力,而且趋光贴壁能力很强。【目的】阐明海洋着色菌(Marichromatium gracile) YL28的生物膜形成特性和规律,以期为海水养殖水体生物膜反应系统的开发和应用提供参考。【方法】以生物膜和游离菌体生物量、脱氢酶活性、生物膜多糖含量和蛋白含量、无机三态氮去除活性为测定指标,在光照厌氧环境中研究海洋着色菌YL28菌株的生物膜形成规律、生物活性和脱氮效果。【结果】随着时间延长,4 000 lx光照时游离菌体生物量逐渐升高,但在稳定期前快速降低,而成膜生物量经过延滞期后逐渐升高并趋于稳定,表明培养过程中游离菌体能趋光贴壁生长并形成生物膜。在0-5 000 lx光照范围内培养4 d,低光照强度(500 lx)时成膜率(71.21%)最高,1 000-4 000 lx光照强度下成膜率虽然不是最高(54.64%-68.66%),但适宜菌体成膜,膜生物量干重达到0.60-0.80 mg/cm2。除了5 000 lx光照对成膜菌体脱氢酶活性有不利影响外,成膜菌体和游离菌体脱氢酶活性随光照强度升高而升高,而且没有明显差异。生物膜的形成会导致光反应器内部光照受限,但反应器内部游离菌体的脱氢酶活性并没有降低,由此表明,培养液中的菌体主要在生物膜及其界面生长并游离扩散至培养液中。随光照强度(1 000-5 000 lx)和培养时间(4-10 d)的变化,胞外复合物(Extracellularpolymericsubstances,EPS)中蛋白含量变异较大,多糖含量变化较小;随时间延长,蛋白含量升高,其中3 000 lx时蛋白含量最高;4 000 lx时生物膜菌体与游离菌体脱氮活性相比,单位质量菌体的氨氮和亚硝氮去除活性未受到明显影响,而硝氮去除活性有所降低。【结论】海洋着色菌YL28具有良好的生物膜形成能力,其成膜过程主要是菌体趋光贴壁生长成膜,成膜菌体具有良好的脱氮活性,这为利用生物膜系统消除海水养殖水体氮污染奠定了基础。     

Responses of soil inorganic nitrogen to increased temperature and plant removal during the growing season in a Sibiraea angustata scrub ecosystem of eastern Qinghai-Xizang Plateau北大核心CSCD

Aims Little information has been available on the soil nitrogen transformation process of alpine scrubland under global warming and changing climate. This study aimed at clarifying seasonal dynamics of the soil nitrate and ammonium contents and their responses to increased temperature under different plant treatments. Methods We conducted a field experiment including two plant treatments (removal- or unremoval-plant) subjected to two temperature conditions (increased temperature or control) in Sibiraea angustata scrub ecosystem on the eastern Qinghai-Xizang Plateau. The contents of soil nitrate and ammonium were measured at the early, middle and late growing seasons. Important findings The results showed that soil nitrate and ammonium contents exhibited obvious seasonal dynamics. Throughout the entire growing season, the soil nitrate contents increased firstly and then decreased, while the soil ammonium contents increased continually. Particularly, in the early and middle growing season, the soil nitrate contents were significantly higher than those of ammonium, regardless of increased temperature and plant treatments; however, in the late growing season, the soil nitrate contents were significantly lower than those of ammonium. These results implied that soil nitrification was the major process of soil nitrogen transformation in the early and middle growing season; soil ammonification contributed mostly to soil nitrogen transformation in the late growing season. Furthermore, different responses of soil nitrate and ammonium contents to increased temperature and plant removal treatments were observed at the different stages in the growing season. The effects of increased temperature on soil nitrate contents mainly occurred in the middle and late growing season, but the effects varied with plant treatments. Increased temperature only significantly increased soil ammonium contents in the unremoval-plant plots during the middle growing season. The effects of plant treatments on soil nitrate contents only occurred in the control plots (controlled temperature). Plant removal only increased soil nitrate contents in the early and middle growing season, but significantly decreased soil nitrate contents in the late growing season. Plant removal significantly decreased soil ammonium contents in the increased temperature plots during the middle growing season. Probably, in the early and middle growing season, scrub vegetation mainly absorbed soil nitrate and the absorption process was not affected by increased temperature. These results would increase our understanding of the soil nitrogen cycling process in these alpine scrub ecosystems under global warming and changing climate. © 2018 Editorial Office of Chinese Journal of Plant Ecology. All rights reserved.     

How does glutamine synthetase activity determine plant tolerance to ammonium?

The wide range of plant responses to ammonium nutrition can be used to study the way ammonium interferes with plant metabolism and to assess some characteristics related with ammonium tolerance by plants. In this work we investigated the hypothesis of plant tolerance to ammonium being related with the plants’ capacity to maintain high levels of inorganic nitrogen assimilation in the roots. Plants of several species (Spinacia oleracea L., Lycopersicon esculentum L., Lactuca sativa L., Pisum sativum L. and Lupinus albus L.) were grown in the presence of distinct concentrations (0.5, 1.5, 3 and 6 mM) of nitrate and ammonium. The relative contributions of the activity of the key enzymes glutamine synthetase (GS; under light and dark conditions) and glutamate dehydrogenase (GDH) were determined. The main plant organs of nitrogen assimilation (root or shoot) to plant tolerance to ammonium were assessed. The results show that only plants that are able to maintain high levels of GS activity in the dark (either in leaves or in roots) and high root GDH activities accumulate equal amounts of biomass independently of the nitrogen source available to the root medium and thus are ammonium tolerant. Plant species with high GS activities in the dark coincide with those displaying a high capacity for nitrogen metabolism in the roots. Therefore, the main location of nitrogen metabolism (shoots or roots) and the levels of GS activity in the dark are an important strategy for plant ammonium tolerance. The relative contribution of each of these parameters to species tolerance to ammonium is assessed. The efficient sequestration of ammonium in roots, presumably in the vacuoles, is considered as an additional mechanism contributing to plant tolerance to ammonium nutrition.     

Essential metals for nitrogen fixation in a free-living N₂-fixing bacterium: chelation, homeostasis and high use efficiency

Biological nitrogen fixation, the main source of new nitrogen to the Earth's ecosystems, is catalysed by the enzyme nitrogenase. There are three nitrogenase isoenzymes: the Mo‐nitrogenase, the V‐nitrogenase and the Fe‐only nitrogenase. All three types require iron, and two of them also require Mo or V. Metal bioavailability has been shown to limit nitrogen fixation in natural and managed ecosystems. Here, we report the results of a study on the metal (Mo, V, Fe) requirements of Azotobacter vinelandii, a common model soil diazotroph. In the growth medium of A. vinelandii, metals are bound to strong complexing agents (metallophores) excreted by the bacterium. The uptake rates of the metallophore complexes are regulated to meet the bacterial metal requirement for diazotrophy. Under metal‐replete conditions Mo, but not V or Fe, is stored intracellularly. Under conditions of metal limitation, intracellular metals are used with remarkable efficiency, with essentially all the cellular Mo and V allocated to the nitrogenase enzymes. While the Mo‐nitrogenase, which is the most efficient, is used preferentially, all three nitrogenases contribute to N₂ fixation in the same culture under metal limitation. We conclude that A. vinelandii is well adapted to fix nitrogen in metal‐limited soil environments.     

Preferential Assimilation of Ammonium Ions from Ammonium Nitrate Solutions by Apple Seedlings

Apple seedlings, Pyrus malus L., were grown in complete nutrient solutions containing nitrate, ammonium, or ammonium plus nitrate as the nitrogen source. Uptake of nitrogen was calculated from depletion measurements of the nutrient solutions and by using ¹⁵N labelled nitrate and ammonium salts. If the plants received nitrogen as ammonium only or as nitrate only, the amounts of nitrogen taken up were similar. However, if the seedlings were supplied with ammonium nitrate, the amount of nitrate-nitrogen assimilated was only half that of ammonium. Nevertheless, if ammonium and nitrate were supplied to a plant with a split-root system, with each root half receiving a different ion, the uptakes were similar. The possibility of independent inhibition by ammonium of both nitrate uptake and reduction in the roots is discussed.     

Expression of the bacterial gdhA gene encoding a NADPH glutamate dehydrogenase in tobacco affects plant growth and development

We investigated the effects of genetic modification of nitrogen metabolism via the bacterial glutamate dehydrogenase (GDH) on plant growth and metabolism. The gdhA gene from Escherichia coli encoding a NADPH-GDH was expressed in tobacco plants under the control of the 35 S promoter. The specific activity of GDH in gdhA plants was 8-fold of that in E. coli. Damage caused by spray application of 1.35 mM of phosphinothricin (PPT) herbicide, a glutamine synthetase (GS) inhibitor, was less pronounced in gdhA plants as compared with the control plants which suggests that the introduced GDH can assimilate some of the excess ammonium, at least during GS inhibition. However, gdhA plants were susceptible to 2.7 mM PPT. Biomass production was consistently increased in gdhA transgenic plants grown under controlled conditions and in the field. Total free amino acids and total carbohydrates were increased in gdhA plants grown in the greenhouse suggesting that both nitrogen and carbon metabolism were altered. We conclude that the modifications in transgenic plants may result from both increased nitrogen efficiency and altered gene expression and metabolism. This revised version was published online in June 2006 with corrections to the Cover Date.     

Ammonium removal using algae–bacteria consortia: the effect of ammonium concentration,algae biomass,and light

In this study, the effects of ammonium nitrogen concentration, algae biomass concentration, and light conditions (wavelength and intensity) on the ammonium removal efficiency of algae-bacteria consortia from wastewater were investigated. The results indicated that ammonium concentration and light intensity had a significant impact on nitrification. It was found that the highest ammonia concentration (430 mg N/L) in the influent resulted in the highest ammonia removal rate of 108 ± 3.6 mg N/L/days, which was two times higher than the influent with low ammonia concentration (40 mg N/L). At the lowest light intensity of 1000 Lux, algae biomass concentration, light wavelength, and light cycle did not show a significant effect on the performance of algal–bacterial consortium. Furthermore, the ammonia removal rate was approximately 83 ± 1.0 mg N/L/days, which was up to 40% faster than at the light intensity of 2500 Lux. It was concluded that the algae-bacteria consortia can effectively remove nitrogen from wastewater and the removal performance can be stabilized and enhanced using the low light intensity of 1000 Lux that is also a cost-effective strategy.     

Robust biological nitrogen fixation in a model grass–bacterial association

Nitrogen‐fixing rhizobacteria can promote plant growth; however, it is controversial whether biological nitrogen fixation (BNF) from associative interaction contributes to growth promotion. The roots of Setaria viridis, a model C₄ grass, were effectively colonized by bacterial inoculants resulting in a significant enhancement of growth. Nitrogen‐13 tracer studies provided direct evidence for tracer uptake by the host plant and incorporation into protein. Indeed, plants showed robust growth under nitrogen‐limiting conditions when inoculated with an ammonium‐excreting strain of Azospirillum brasilense. ¹¹C‐labeling experiments showed that patterns in central carbon metabolism and resource allocation exhibited by nitrogen‐starved plants were largely reversed by bacterial inoculation, such that they resembled plants grown under nitrogen‐sufficient conditions. Adoption of S. viridis as a model should promote research into the mechanisms of associative nitrogen fixation with the ultimate goal of greater adoption of BNF for sustainable crop production.     

氮素营养对苗期菘蓝叶中硝酸还原酶活性与矿质元素吸收的影响

采用正交试验设计,研究铵态氮、硝态氮和酰胺态氮3种氮素形态及其不同浓度配比对苗期菘蓝的单株干重、叶内的硝酸还原酶活性及矿质元素吸收的影响。结果显示:(1)影响苗期菘蓝单株干重的氮素形态依次为酰胺态氮>铵态氮>硝态氮。(2)不同氮素形态对叶片硝酸还原酶活性影响有差异,铵态氮影响最大,其次是硝态氮和酰胺态氮。(3)不同形态氮素配合施用后均能促进P、K、Ca、Mg、Cd、Mn、Cr、Sr 8种元素的吸收,但不利于Ni和Fe的吸收;元素吸收受铵态氮影响最大的矿质元素有K、Ba、Se、Ni、B、Si、Fe 7种元素,受硝态氮影响最大的元素有P、Cd、Ti、Al、Cu 5种元素,受酰胺态氮影响最大的元素有Na、Ca、Mg、Zn、Mo、Mn、Cr、Sr 8种元素。研究表明,不同形态氮素对苗期菘蓝吸收矿质元素的影响存在很大的差异,应注重酰胺态氮与无机的铵态氮、硝态氮的配合施用;适宜氮素形态及其配比能提高叶中硝酸还原酶的活性并促进矿质元素的吸收,从而有效地促进菘蓝的生长。     

Evaluation of inhibitory effects of heavy metals on anaerobic ammonium oxidation (anammox) by continuous feeding tests

To facilitate the application of anaerobic ammonium oxidation (anammox) to a nitrogen removal process, the effects of heavy metals (Ni, Cu, Co, Zn, and Mo) on anammox bacteria entrapped in gel carriers were examined by conducting continuous feeding tests for each metal. The results show that all anammox activities decreased by more than 10 % when influent concentrations of Ni, Cu, Co, Zn, and Mo were 5, 5, 5, 10, and 0.2 mg/L, respectively. It was observed that the effects of Ni, Cu, Co, and Zn on anammox activity were reversible and that of Mo on anammox activity was irreversible. Anammox activity was not affected when influent containing mixed Ni, Cu, Co, and Zn (0.5 mg/L) was fed into the reactor.     

Competition for ammonium between plant roots and nitrifying and heterotrophic bacteria and the effects of protozoan grazing

The competition for limiting amounts of ammonium between the chemolithotrophic ammonium-oxidizing species Nitrosomonas europaea, the heterotrophic species Arthrobacter globiformis and roots of Plantago lanceolata (Ribwort plantain) was studied in a series of model systems of increasing complexity, i.e. energy-limited continuous cultures, non-water-saturated continuously percolated soil columns and pots with γ-sterilized soil planted with axetic P. lanceolata seedlings. The effects of bacterial grazing by the flagellate species Adriamonas peritocrescens on the competition for ammonium were also investigated in the three model systems. It was found that N. europaea was a weaker competitor for ammonium than either A. globiformis or plant roots of P. lanceolata. It is assumed that the heterotrophic bacteria have a higher affinity for ammonium than the nitrifying bacteria, whereas growing plant roots have a greater capacity to exploit the soil for ammonium than the immobile nitrifying bacteria. It is not very likely that allelochemicals were involved in suppressing the nitrification process. Four reasons are given for this assumption. Presence of the flagellates strongly stimulated the potential nitrification rate in all model systems. It is assumed that there is a more even distribution over the soil of either nitrifying bacteria or their substrate ammonium in the presence of flagellates. In addition to the distribution effect, there is a stimulation of the potential ammonium oxidation rate. The results are discussed in the light of the function of nitrate as nitrogen sink in the biogeochemical nitrogen cycle.     

Development of high density mammalian cell culture system for the production of tissue-type plasminogen activator

A high cell density culture system for the anchorage dependent CHO cells was developed based on the combination ofin situ removal of ammonium ion and microcarrier culture system, and semi-fed-batch feeding of glucose and glutamine was employed to the developed culture system. The glass bead was selected as an optimum microcarrier in terms of cell growth. An ammonium ion selective zeolite, Phillipsite-Gismondine, was packed in a dialysis membrane and equipped on the agitator of spinner reactor forin situ removal of ammonium ion. The semi-fed-batch operation was employed to the novel culture system for the high density cell culture, and the results showed the cell growth was improved by 32% and tPA productivity by 250%.     

Trials to create artificial nitrogen-fixing symbioses and associations using in vitro methods: An outlook

Summary Biological nitrogen fixation is the most important process in which some prokaryotic organisms fix N₂ into ammonium. From an agricultural standpoint, biological nitrogen fixation (BNF) is critical because industrial production of nitrogen fertilizers seldom meets agricultural demands. To increase the BNF is one of the main challenges for the future. There are different possibilities for extending biological nitrogen fixation to the economically important plants. One of the possibilities is to create new artificial systems between diazotrophic bacteria and different higher plants. This is the main topic of the present review article which discusses the establishment of new associative and/or symbiotic systems, via introduction of diazotrophic bacteria into the roots by different methods; and incorporation of nitrogen-fixing bacteria in the entire plant by in vitro methods, through the establishment of intracellular endosymbioses via induced uptake of bacteria by plant protoplasts (endocytobiosis), and establishment of intercellular associations by forced introduction of bacteria into the plant tissues (exocytobiosis). The common characteristic of the methods to create artificial plant-microbe systems for atmospheric nitrogen fixation is the use of in vitro plant systems: cells, tissues and organ cultures. The review pays particular attention to new bacterial inoculation procedures for introduction of the diazotrophic bacteria inside the plant tissues.     

Effects of light intensity and magnesium supplementation in pretreatment cycle on ammonium removal from wastewater of photobioreactor using a biofilter composed of the aerial microalga Trentepohlia aurea

Application of a laboratory-scale photobioreactor containing a biofilter composed of the aerial microalga Trentepohlia aurea to the removal of ammonium from synthetic wastewater was assessed to determine whether the system could be applied to water purification and the treatment of eutrophic water. The removal efficiency of the photobioreactor was tested after ten biofilter sheets (total dry weight cells: 50 mg) were cycled in nitrogen-free Bold’s basal (BB) medium for 72 h (pretreatment cycle). The ammonium removal ability was significantly enhanced when the photobioreactor was operated after performing the pretreatment cycle using nitrogen-free BB medium supplemented with magnesium. Moreover, the illumination conditions during the treatment were shown to affect the nitrogen removal ability, and this ability was strongly dependent on the concentrations of organic compounds (e.g., α-ketoglutarate and pyruvate) for assimilating the nitrogen source in the T. aurea biofilter.     

Polarographische Messung der Nitritreduktion von Chlorella im monochromatischen Licht

Summary In green plant cells nitrite is reduced by two systems, one dependent on photosynthesis and the other upon respiration. Using a polarographic method for continuous measurement of nitrite uptake, the relationship between light driven and respiration linked nitrite reduction of Chlorella cells was studied.Photosynthetic nitrite reduction is characterized by a pronounced increase in the velocity of nitrite uptake upon illumination. After the light is turned off the velocity immediately returns to the preillumination value. Photosynthetic nitrite reduction of Chlorella is separated from respiration linked nitrite reduction by illumination with red light under anaerobic conditions; it is stimulated by CO₂ and is inhibited by DCMU, findings which confirm earlier observations.In white light a special blue light stimulation of nitrite uptake is overlapped by photosynthetic nitrite reduction. In contrast to photosynthetic nitrite reduction this type of light stimulation is characterized by a lag period of about I min from the onset of illumination; it continues about 10 min when the light is turned off. It is separated from photosynthetic nitrite reduction by irradiation of the algae with low intensities of short wavelength light (<500 nm). Blue light stimulation of nitrite uptake of Chlorella is strongly dependent on the developmental stage of the cells. It is observed with young cells (autospores) of synchronized algae only.There is no evidence for any connection between blue light stimulation of nitrite uptake and photosynthesis. From the sensitivity of this process towards anaerobic conditions and antimycin A it is concluded to be a stimulation of respiration linked nitrite reduction.Under conditions of low exogenous nitrite concentration a temporary inhibition of steady state dark nitrite reduction appears immediately after the light is turned off. From several observations it is concluded that the inhibition already exists during the preceding illumination and decreases the rate of total nitrite uptake in the light. This process is suppressed by inhibition of respiration as well as by the inhibitor of photosynthesis, DCMU.If nitrate is the source of nitrogen an excretion of nitrite is found following illumination. The kinetics of this process agree with those observed for the light induced inhibition of steady state dark nitrite reduction immediately after illumination.     

Nodulation studies in legumes

Summary The influence of combined nitrogen (as ammonium nitrate) on the symbiotic performances of selected bacterial associations of four legumes was examined using sand culture.In barrel medic (Medicago tribuloides Desr.) and vetch (Vicia sativa L. andV. atropurpurea Desf.) bacterial partnerships of a host plant varied greatly in their nodulation responses to a range of amounts of nitrogen applied at sowing. Some bacterial strains exhibited varying degrees of stimulation of nodule number, growth and fixation by low or medium amounts of nitrogen. Higher levels of combined nitrogen depressed symbiosis. Other strain responses showed a severe restriction of symbiosis with any amount of added nitrogen.Seasonal influences conditioned symbiotic responses to combined nitrogen in an association of cowpea (Vigna sinensis End.) With a summer sowing small amounts of ammonium nitrate added at sowing benefited later symbiotic development. No such stimulation was evident in an autumn sowing and symbiotic injury from high levels of nitrogen was greater than in the summer sowing.The developing association of cowpea was found to be most sensitive to ammonium nitrate added just as the first leaves unfolded. Here damage was manifest in a permanent elevation of the top: root ratio with subnormal growth and functioning of nodules. Greatest benefit from added inorganic nitrogen followed applications made as the first nodules appeared on the primary root. In this case added combined nitrogen acted as an investment providing returns in additional fixation equivalent to 5–10 times the amount of nitrogen originally fed to the seedling and representing some 50 per cent greater total fixation than in minus-nitrogen controls.     

Nitrogen transformations under different conditions in open ponds by means of microalgae-bacteria consortium treating pig slurry

Four open ponds inoculated with microalgae-bacteria consortium treating different swine slurries (fresh and anaerobically digested) were evaluated in terms of nitrogen transformation under optimal and real conditions of temperature and illumination. Ammonium complete depletion was not achieved. Ponds operated under real conditions presented lower ammonium removal. Elimination capacities were around 26 mg N/L d and were subsequently increased with increasing inlet ammonium loading rate. Different nitrogen transformation was observed depending on substrate source. When anaerobically digested slurry was fed to the ponds, nitrification followed by biomass uptake and denitrification were the main nitrogen transformation taking place depending on inlet ammonium loading rate and operational conditions. Ponds fed with fresh slurry exhibited denitrification as the main nitrogen removal mechanism for the pond operated under real conditions while under optimal conditions stripping, denitrification and biomass uptake contributed similarly. Therefore, this study confirmed that the so-claimed nitrogen recovery by microalgae biomass is frequently overestimated.