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Biological Nitrogen Fixation - Science topic

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How we need to consider soil mineral nitrogen availability regarding this?
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You need to follow the steps from the attached document from old times but useful
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General perception is that a fast growing species are better carbon sequester if it holds than all CDM projects will have these species. What will be role of old species and other slow or moderate growth species vis a vis role of site quality with respect to fast growing species. How to determine carbon sequestration potential of and species mean ideal parameters for consideration eg.  age of tree, site factor / surrounds, tree associates/ allelopathy nature, nutrient cycle / physiology/ silvics of the species etc.
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Definitely because of high biomass, but lamina area also matters. Kindly consult this Govt. report
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N-fixation by various leguminous and non-leguminous plants is important and its quantification as function of nature of crop, crop growing condition, soil environment, soil N availability and N-addition needs to be evaluated. We wish to know from the experts colleagues on this kind of work done and like to share the publications by the group on this important aspect, as well some of the empirical technical coefficient generators in this regard.
regards.  
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Also check please the following very good link: https://ageconsearch.umn.edu/record/118041/files/11.pdf
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All plants need relatively large amounts of nitrogen (N) for proper growth and development. Biological nitrogen fixation (BNF) is the term used for a process in which nitrogen gas (N2) from the atmosphere is incorporated into the tissue of certain plants. Only a select group of plants (legumes) is able to obtain N this way, with the help of soil microorganisms. What characteristics make legumes fix nitrogen?
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Several non-leguminous plants such as Alnus, Casuarina, Ceanothus, Colletia, Comptonia, Datisca, Discaria, Eleagnu, Hippophae, Morella,, Myrica, Purshia, Shepherdia, Trevoa, etc. also fix atmospheric nitrogen by forming symbiotic association with a Gram positive bacterium Frankia.
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I am doing some literature review to better understand the processes governing the biological fixation of nitrogen by non-symbiotic micro-organisms (associative, endophytic, free-living...).
I am not interested in symbiotic relations like legumes (which have their use), but rather to find solutions to promote this fixation throughout the cultivation (perhaps through composting?).
So it could be in the field or in a compost pile on the farm.
Could you share some insights?
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following
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Diazotrophs 'fix' atmospheric nitrogen into biologically available forms. In very general, non-technical terms, an over-abundance of these organic nitrogen forms within a soil beyond what biota in the soil metabolically require has been suggested to result in the increase of nitric acid in the soil. What I would be very interested in is if anyone has done work to help establish a rate of nitric acid production in response to this over abundance of organic nitrogen.
Cheers!
Eron
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I think yes...please do experiment
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I want to assay the ACC deaminase in my isolates, and the commonly used medium is the DF medium which contains MOO3 and gluconic acid. Can I replace them by sodium molybdate and glucose? Or can sombody help me with another minimal medium?
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ACC deaminase activity on the sterile minimal DF (Dworkin and Foster) salts media (DF salts per liter: 4.0 g KH2PO4, 6.0 g Na2HPO4, 0.2 g MgSO4.7H2O, 2.0 g glucose, 2.0 g gluconic acid and 2.0 g citric acid with trace elements: 1 mg FeSO4.7H2O, 10 mg H3BO3, 11.19 mg MnSO4.H2O, 124.6 mg ZnSO4.7H2O, 78.22 mg CuSO4.5H2O, 10 mg MoO3, pH 7.2) amended with 3 mM ACC instead of (NH4)2SO4 as sole nitrogen source (Dworkin and Foster, 1958; Penrose and Glick, 2003). The inoculated plates were incubated at 28°C for 3 days and growth was monitored on a daily basis. Colonies growing on the plates were taken as ACC deaminase producers. Samar Salama Natasha Tilikj
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I am looking for a way to estimate the root biomass of a grass-clover stock using visual methods, such as counting roots in a defined area of the soil cross-section. The overall objective is to calculate the total amount of nitrogen and thus also the fixation capacity with the aid of the biomass determined in this way. So, which methods are known for this?
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I found some literature which might be useful. A method described by Newman (1966) has been referenced in the past. It was titled: A METHOD OF ESTIMATING THE TOTAL LENGTH OF ROOT IN A SAMPLE.
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I heard that some species of Rhizobia can fix nitrogen even in the absence of plant roots. If someone knows about it, please let me know what conditions are required for Rhizobia to fix atmospheric nitrogen. Thanks
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This is an interesting issue. Papermill process waters are nitrogen deficient and recent microbiome testing is showing many rhizobia genera in the waters. It could be that they are within the biofilms, protected from oxygen (which is what leghemoglobin is supposed to do). I haven't worked in N-fixation for many years and my experience was with Frankia.
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Economic and environmental costs of chemical N in agriculture is becoming a global concern. At the same time climate, change together with food insecurity is the most pressing problem for the 21st century. Indicating the demand for sustainable agricultural practices including the use of legume- rhizobium symbiosis driven biological nitrogen fixation. Is it economically visible if these N-fixing properties of legume also introduced to cereals?
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Yes, it is possible. We coordinated a project at Embrapa for this purpose "Biological Nitrogen Fixation in Rice Irrigated by Flood"
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Pulses are known to fix atmospheric nitrogen through bacterial symbiosis. But only a single molecule of fixed nitrogen consumes about 16 molecules of ATP and the carbohydrate provided to the symbionts by the plant is also used up for the growth and development of the symbionts. So it is clear that a copious amount of photosynthates are consumed in the process of nitrogen fixation.
On the other hand, pulses have the roots which can preferentially uptake soil available nitrogen inhibiting the symbiosis. So, there is a possibility that a normal nitrogen fertilization to pulses may inhibit biological fixation and may help to divert more photosynthates to the economic part.
The problem of excessive vegetative growth may be solved by the judicious splitting of the doses or with drip fertigation techniques.
Please share your views on the matter.
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Dear Prithwiraj
Whether one uses or N fertiliser or symbiotic N fixation, soil moisture & pH and P fertilisation are critical except for Mo requirements. Whilst you can obtain good yield response with increasing fertiliser-N application in theory, the price of N fertiliser has to be factored into estimating profit, without which pulse farming could become an academic exercise.
Some papers argue increase in fertiliser-N use in association with P does not necessarily result in increasing pulse yield (e.g.
Some argue biofertiliser approach provides better yield than mineral fertiliser-N application (e.g. Bangladesh J. Agril. Res. 40(3): 501-506, September 2015).
If fertiliser-N use is compelling, starter-N application may be more beneficial (e.g. Agronomy 2018, 8, 120; doi:10.3390/agronomy8070120).
There is convincing evidence on the benefit BNF (biological N fixation) in grain and forage legume production without compromising yield (e.g.
When considering crop rotation, fertiliser-N use becomes more complex (e.g.
Even if wish to ignore energy use and the above factors, there is also compelling evidence on heavy nitrate leaching from high fertiliser-N use, even by split dosing.
Kind regards
Selva
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Azotobacter is a nonsymbiotic nitrogen fixer used to fix atmospheric nitrogen, so we can save N through chemical fertilisers upto 20-25 %. PSB are added in soil to solubilise P in the soil,. Besides, they secrete  Plant Growth Promoting  hormones  thus both biofertilizers r beneficial for plant growth. Can these biofertilizers also play a role in solubilising silica in the soil, which increases the resistance of plants against diseases?
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I agree with Dr Hani Antoun as far as solubalization is concerned
however statistically significant yield enhancement are only reported with foliar application of silicon. marked enhancement in plant Si content on fresh weight basis has been poorly reported, even though the underlying mechanism of Silicic acid mediated absorption is suitably characterized
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I am working with Nitrogen fixating organisms especially cyanobacteria and Heterotrophic bacteria. For quantifying nitrogen fixation rates by these micro organisms ARA used. I need to know the about the Gas chromatography condition separate acetylene and ethylene (I am using Elite Capillary Alumina KCL column (0.53*50m)). I am waiting for your valuable comments.
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I am giving you a recent and a good alternative of what you are asking
Microbial assay of N2 fixation rate, a simple alternate for acetylene reduction assay
lSubhajitDasTarun KumarDe
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What are the most important environmental factors, including the soil factors most affecting nodule formation or biological nitrogen fixation?
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there are a lot of factors ranging from edaphic to biotic that affect BNF, however you asked only about environmental ones, following are some really important of them, whose affect is readily visible are
1. light regime and photosynthetic efficiency, , upon which biological nitrogen fixation depends. This is demonstrated by diurnal variations in nitrogenase activity. A very few plants can grow and fix N2 under shade (e.g., Flemingia congesta under plantain canopy). In alley farming if hedgerows are not weeded, or if trees are planted with food crops like cassava, their nitrogen fixation and growth will be reduced due to shading. Early growth of legume trees is slow and they cannot compete successfully for light.
2. Extreme temperatures affect N2 fixation adversely. This is easy to understand because N2 fixation is an enzymatic process. However, there are differences between symbiotic systems in their ability to tolerate high (>35°C) and low (<25°C) temperatures.
in additon soil pH, salinity, organic matter content are some of the edaphic factors affecting BNF
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Is nitrogease enzyme (nifH) are stable in extreme environments??
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Dear Mr. Thajudeen,
The premiere enzyme responsible for fixation of dinitrogen to ammonia, the nitrogenase, is extremely sensitive to the presence of oxygen because of the degradative tendencies of oxygen towards the Fe-S co-factor of the enzyme. However, hot springs provide an ideal environment for diazotrophs or symbionts as the excess temperature ensures that the partial pressure of gaseous oxygen would be higher than the ambient atmosphere and generally results in outgasing of oxygen into the atmosphere and the water becomes hypoxic. This is effect is absolutely conducive for the nitrogenase to function. However, the temperature might prove to be a hindrance for the enzyme to perform optimally for which hot spring cyanobacteria or other microbes tend to synthesize excessive extracellular mucopolypeptides and other polymers. The dissolved carbonates in the alkaline hot springs serve as buffers to any pH swing, thereby protecting the enzyme.
At or near deep sea vents, nitrogen fixers encounter environments where the element with highest electron affinity is usually sulfur. And the absolute absence of free or dissolved oxygen in there immediate environments enable the nitrogen fixers to perform optimally. Production of methane, hydrogen sulfide, sulfur di-oxides farther bolster the situation of hypoxia. There are articles indicating that gaseous nitrogen is abundant in hydrothermal fluids and the environment itself is limited by fixed nitrogen. Hence thermophilic nitrogen fixers reduce N2 to ammonia in the unsedimented deep-sea vent regions.
In very cold regions, like the polar climates, the nitrogen fixation do take place beneath the ice or permafrost, in peat bogs etc but at very slow rates. And in it very rare in arctic oceans since the cold ensures presence of excess dissolved oxygen in the water which is a big roadblock for the nitrogenase. The global nitrogen cycle is not balanced since in the arctic regions, nitrogen fixation hardly, if ever, occurs, apart from some limited coastal settings and the rate of microbial denitrification far outweighs the ammonification in high latitudes. The tropical regions are characterized by higher rates of nitrogen fixation within the euphotic depth followed by greater ammonia oxidation through Annamox in the lower reaches of the nitricline.
This is why if all the niches of the world were considered, nitrogen fixation concept would have been needed to be rewritten as a result of a paradigm shift. Hence, in terrestrial conditions nitrogen fixation may or may not occur in polar climates but in water it is truly a rarefied phenomenon and can only, theoretically/hypothetically take place in oxygen minima zones, which need research to corroborate.
Thanking you,
Dr. Abhishek Mukherjee
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I want to measure biological nitrogen fixation activity in some legumes using acetylene reduction assay. I got an acetylene cylinder with a regulator and just wondering how to take constant gas samples each time? Thank you.
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Hi Malinda,
Attach a rubber hose to the outlet of the gas regulator. Usually a 30 - 50 cm hose is enough. Clamp the end of the hose to avoid excessive gas leaking. The hose should be soft enough to allow easy perforation using an insulin syringe for example. Open the valve, put some pressure using the pressure regulator (just until you start to see that the pressure indicator moved a little bit). When you add pressure, the attached hose will be filled with gas. Using one syringe of appropriate volume, punch the hose and collect the gas. Usually 10 % acetylene is added to the flask.
I hope it helps.
Cheers
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Hi, I'm not biogeochemistry specialist. This isn't my area. So this question may seem stupid to some.
I would like to know if there may be excess biological nitrogen fixation and if this carries some negative impact. If anyone can give me references on this topic, I will be grateful.
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Efficiency of BNF could be observed optimum in tune with optimum N reauirement of crop since such processes are signal - based. No physiological N demand by crop , then no triggering in BNF...
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I want to know the importance of %nitrogen derived from atmosphere(%Ndfa) in estimating biological nitrogen fixation(BNF).
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It is the key parameter required for quantifying the amount of N biologically fixed by a plant or crop. It can be determined using a standard method that utilize the 15N isotope as a tracer (either by employing the '15N isotope dilution method' by using 15N enriched N compounds or by '15N natural abundance method'.
Detailed methodology for both options can be found in this book:
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Nitrogenase activity, Microbiology.
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Please have a look at some PDFs , hope you find them useful...
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Hello everyone :) I'd appreciate it so much if someone would explain me what negative nitrogen (N) mineralization rates mean. Do they mean that microorganisms convert more N than it is available? Thank you!
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This paper may help for better interpretation. 
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Some nitrogen fixing endophytic bacteria belong to the genera such as Klebsiella, Enterobacter, Herbaspirillum, Stenotrophomonas, Rhizobium, Bradirhizobium had been detected from plant extracted DNA.Same plant samples were stamped on Nitrogen minus MR agar plate for isolation of the mentioned bacteria. Only Rhizobium and Bradirhizobium was isolated and other not isolated.can I get information of suitable media/technique for isolation of  Klebsiella, Enterobacter, Herbaspirillum,Stenotrophomonas please?
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Vasvi,
We just isolate on YES (agar with yeast extract (1%) and sucrose (1%)) or a comparable medium then culture on Norris nitrogen free to screen for possible N fixers. The real trick is to get them out of the plant. Many are really hard to culture directly from the plant. Once you get them into culture they grow fine.
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I'm doing isolation of endophytic PGP from plant root and I've read about journal that use bromothymol blue (BTB) as indicator, is there any other option other than using BTB?
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@all, what about growing a potential nitrogen fixer on Glucose nitrogen-free medium (GNFM) and BTB as an indicator for nitrogen-fixation?
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There are several methods of measuring nitrogen fixation ability of bacterial cells (like acetylene reduction assay etc) , but is there any protocol or research paper from which we can directly find out nitrogen fixing ability of root nodule.
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Thanks for the reply
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I am working on N2 production by none denitrifying bacteria. To be sure that N2 produced by these organisms actually is from the N2O route, I need to spike the culture medium with labelled N2O and trace the yield of labelled N2
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buy labeled N2O (don't know if it is possible), or produce it yourself from labeled nitrate, using a denitrifying organism that lacks nosZ. There are several methods to produce N2O chemically (see Wikipedia), but I would not trust them, because you may get a mixture of N oxides.. Good luck!
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Share your views about quantification of nodule count in legumes.
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 Thanks very much dear Dr Bhatt and Dr Patil ji
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Particularly in the inhibition of nitrogen fixation.
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Interesting discussion. Why should we invariably consider , exogenous application of urea  will adversely affect the efficacy of inoculated rhizobium with regard to nitrogen fixation . If soil N-test value is sub-critical , any rhizobium species will respond to urea as a starter nutrient . however , this will not be the case when soil N-test value is above optimum. 
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Hi, I am interested in measuring nitrogenase activity in free living bacteria. Specifically i am interested in measuring through the 15N2 method which measures the organic nitrogen through mas spectometry. Nevertheless, I would l would like to know why it does not take into account  other forms of nitrogen in the supernatant or intracellular. For example Labelled amonnia.
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 Thanks, yes i assume that the organic nitrogen is the main product but i wonder about the inorganic nitrogen that could also be produced.
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Can nitrogen fixation occurs in non legume plants? It is well know that nitrogen fixation occurs in legume plants. But our Q Can nitrogen fixation occurs in non legume plants such as wheat, rice and corn.
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Under some restriction BNF as been extesively found in non legumes with limited flux, as in https://academic.oup.com/aob/article/111/5/743/193622/Biological-nitrogen-fixation-in-non-legume-plants
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I’m planning to investigate free-living nitrogen-fixing microorganism application as an alternative to chemical fertilization and soil restoration. In the application of nitrogen-fixing microorganisms (diazotrophs) it is very common the use those who are in symbiosis with leguminous plants. But, it is well known that a significant number of plant species that feed humanity (e.g. wheat, corn, rice) do not have the capacity to form this type of symbiosis; Therefore, other ways have been sought to supply for this lack. One of these is the use of microorganisms that make asymbiotic fixation. However, the species until now used, have been studied and obtained in pure microbial cultures, and for various causes low ability to colonize soils for agricultural use has been found, especially those highly degraded.
Recently, alternatives have been developed for the use of microbial communities in mixed cultures or consortia, but so far no relationship with biological nitrogen fixation. So, I’m really looking to find out what you’d consider to be the most limiting factors in the application of this nitrogen fixation consortia to no-leguminous crops.
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Dear Landázuri,
You can go through my published paper in this context. You have to optimize the growth conditions such as temperature, moisture, pH, required C:N ratio etc. for maximization of your biological. Regarding non-symbiotic N2 fixation, you may also go for Azotobacter sp., Azospirillum sp. etc. as these microbes have potential role in enhancing the soil nitrogen status.
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In the extreme desert, soil nutrients, especially nitrogen are always very poor. Are significant effects observed among local desert plants? I want to know your opinions about which are the key factors controlling nitrogen use efficiency of desert plants in extreme environments?
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Interesting question . There could be two types of factors , one related to desert plant itself and another related to soil, since most of the desert plants are xerophytes in nature growing naturally  . Soils are predominantly alkaline in nature coupled with salinity issue , low nitrogen level , low P level , and most of the micronutrients are also within deficient range . Plants also need to have low transpiration rate , low water requirement  ( having capacity of more crop per drop) . So , nitrogen -use -efficiency under such trying environment , needs to be dictated by both plants ability to respond to applied nutrients and  produce maximum at unit cost of nitrogen ...
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How nitrogen fixation ability of Azotobacter on solid media? please give exact composition. We tried GNFMM but it produced yellow color rather blue?
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Thank you for your answer. We need to check the nitrogen fixing ability of Azotobacter and literature says that on GNFMM medium with BTB produces blue color. We are getting yellow rather blue. Even reference Azotobacter also giving yellow color,I would like to know the composition of GNFMM medium with BTB.
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I'm having the hardest time finding this information. If anyone knows of a reference leading me to it I'd be most appreciative.
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Dear Suresh Malhotra, I think the assertion than cropping systems can or do not have ability to satisfy the cropping N requirements of competitive crop systems in a misnomer. If we have a Nitrogen demanding crop like maize or rice the use of legume cover crop or green manure can provide all the Nitrogen for top yields. In addition the yield of maize following a soybean crop for instance will produce about 18% higher crop yield based on rotation effect. When legume crop are used in hay forage mixture they not only reduce the Nitrogen required as amendment but also improve the Carbon content in soil rejuvenating the soil structure and biology in addition. When legumes are added to grass pastures the earthworm populations skyrocket 3 to 4 times the grass monoculture. Interestingly work in Australia has shown than when termites are restricted within two years up to 80 to 90% of the soil Nitrogen level is lost. The symbiotic microbial communities in the termite gut are very active sites of Nitrogen fixation. Recently in Guinea West Africa I witnessed the abundant prairie production and health where termite mounds were abundant at the concentrations of the termites I observed the rates of Biological Nitrogen fixation would be estimated at 300 to 400 kgN/ha/year enough for top tier grass production. The Nitrogen fixation concentrated on legumes is also not exhaustive as symbiotic Azolla in rice paddy can provide the total Nitrogen need for top rice production. This idea that Nitrogen in biological fixed forms is also being limited is being challenged by the association of Nitrogen fixing rhizobacterial in tropical grass in work done in Brazil. As a past research Director for Rodale Institute Farming System Trial showed through legume based cover cropping and rotation can produce all the cropping systems Nitrogen needs and increase crop yields while improving critical soil organic matter. The application of effective methodologies to do this are available but often the application has lagged from lack of knowledge desire and aptitude. 
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I know rhizobia bacteria fix nitrogen when symbiotic with leguminous plant.But when culture on nitrogen free media that's time can fix atmospheric nitrogen.
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Rhizobia bacteria can fix nitrogen and can growth on nitrogen free media.
Pham Van Toan
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By qualitative assay, we have found some bacteria showing positive to utilize  nitrogen in Nfb media. Now we  are trying to quantify nitrogen fixation by these endophytic bacteria. So if anyone can suggest which method we can apply to quantify nitrogen fixation . How is the Kjeldahl method?
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You could work with stable isotopes (15N2). Ohyama et al. did something similar in sugarcane plants.
Ohyama, T., Momose, A., Ohtake, N., Sueyoshi, K., Sato, T., Nakanishi, Y., & Ando, S. (2014). Nitrogen Fixation in Sugarcane. Advances in Biology and Ecology of Nitrogen Fixation, 47-70.
If you don't have access to a mass  spectrometer I could imagine that an acetylene essay could work, too.
Hardy, R. W., Holsten, R. D., Jackson, E. K., & Burns, R. C. (1968). The acetylene-ethylene assay for N2 fixation: laboratory and field evaluation. Plant physiology, 43(8), 1185-1207.
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We are growing Azotobacter in liquid medium and can't obtain cysts after fermentation. We tried to use minimal salt medium, tried to mix the obtained liquid with other materials such as butanol, glycerol, liquid humus, starch, gum arabicum, PVP but no formation of cysts we can observe. Furthermore the number of colony forming units are decreasing very faster, i.e. during storage of one month the number of CFU 109 decrease until 108. Maybe someone can suggest some methods how to induce cyst formation of Azotobacter vinelandii in liquid medium and how to keep the number of cells for a long storage?
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Please email me on : kuldeepjpatel@yahoo.com
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I am working on biological nitrogen fixation and I wanted to screen the microbes for nitrogen fixation attributes. Is there a method to find nitrogen fixation by microbial isolates apart from Gas chromatography method (ARA).
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The potential for biological nitrogen fixing ability can be detected initially by culturing the isolates on nitrogen free media containing bromothymol blue as an indicator. Bromothymol, a pH indicator, shows yellow colour in acidic medium, green colour in neutral medium and blue colour in basic medium. during nitrogen fixation, free atmospheric nitrogen is converted into ammonia which makes the medium basic and hence turns blue (Dobereiner, 1972).
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i need to make primary screening for biological nitrogen fixing endophytes, concerning the amount of fixed nitrogen in liquid media?
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Thank you D. Hani.
i am interested in Endophytes. i have some isolate, i need to make selection for the best isolates depending on; nitrogen fixation, Indole acetic acid production and phosphate solubilization. so i need a method for quantitative measurement of nitrogen fixation other than acetylene reduction assay.
many thanks.
Ahmed Eid.
Assistant Lecturer, Botany and microbiology dep.
faculty of science. Al azhar universty
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I am working of Nitrogen fixation and I wanted to find Nitrogenase activity. I want to know a very good method to find nitrogen fixation method apart from Gas chromatography method.
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Dear Kartik Shivappa Ganiger,
The following are some methods for the determination of nitrogenase activity:
1-Acetylene Reduction Assay (ARA): Measuring Nitrogenase Activity
The discovery that the nitrogenase enzyme responsible for N2-fixation also reduced C2H2 (acetylene) to C2H4 (ethylene) (Dilworth, 1966) provided a useful assay for the quantification of the N2-fixation process. For quantitative determinations of N2-fixation, 15N2 techniques should be used, however, the acetylene reduction assay is still used widely because it provides a highly sensitive and inexpensive way to quantify relative nitrogenase enzyme activity in N2 fixing samples.
Because the presence of acetylene blocks the conversion of N2O to N2, we are able to simultaneously measure denitrification (NO3
- → N2O → N2) by measuring N2O.
For the method details, please use the following method:
Environ Microbiol. 2001 May;3(5):343-51.
Nitrogenase activity in cyanobacteria measured by the acetylene reduction assay: a comparison between batch incubation and on-line monitoring.
Staal M1, Lintel-Hekkert ST, Harren F, Stal L.
Author information
 
Abstract
A new on-line method for measuring acetylene reduction is described. It consists of a gas-flow cell connected to an electronic gas-mixing system and an automatic sample loop in the gas chromatograph. Alternatively, ethylene can be determined by using laser-based trace gas detection. The laser-based trace gas detection technique achieves a detection limit that is three orders of magnitude better than gas chromatography. We have applied the on-line method to the measurement of nitrogen fixation in a culture of the heterocystous cyanobacterium Nodularia spumigena and compared it with conventional batch-type incubations. Incubation of N. spumigena in the gas-flow cell resulted in very short response times with a steady-state flux of ethylene obtained within 2 min. Nitrogenase was shown to respond immediately to changes in light and oxygen. Monitoring of nitrogenase activity could be continued for several hours without having a negative impact on nitrogen fixation rates in N. spumigena. This was not the case in batch incubations, in which changes in nitrogenase activities were recorded during incubations, probably as a result of varying oxygen concentrations. It was therefore concluded that the on-line method is superior to batch incubations when rates of nitrogenase activity are to be measured. The method is suitable for natural samples (water or sediment).
2-A much easier, safer and more accurate method of assaying nitrogenase activity is by measurement of H2 evolution from nodulated roots of legumes (Hunt and Layzell, 1993). Reduction of protons to H2 is an obligate part of the nitrogenase reaction, and the rate of H2 release into the soil is directly related to nitrogenase activity. By attaching a plant to a gas exchange system incorporating a H2 sensor, nitrogenase activity in vivo can be measured noninvasively, and variations in activity can be observed in real time. Recently, a simple H2 sensor designed for undergraduate teaching has been developed at Queen’s University, and students in
introductory level, and advanced level, courses in plant science are using this with great success. 
For more details on this method, please use the following link:
3-Gas Exchange of Legume Nodules and the Regulation of Nitrogenase Activity
Annual Review of Plant Physiology and Plant Molecular Biology
Vol. 44: 483-511 (Volume publication date June 1993)
DOI: 10.1146/annurev.pp.44.060193.002411
Hoping this will be helpful,
Rafik
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I want to determine the nitrogen fixation efficiency of some bacterial isolates in my lab. How can i do it with simplest lab settings? 
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ya I think u can check Klawonn et al. methods (2015)
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I want to extract growth substances such as gibberellins and  Auxins from nitrogen fixing cyanobacteria Anabaena sp..
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 we have done this work as part of M.Sc. thesis by one of my students. the procedure is to grow the cyanobacteria in pure culture and then extract   a known volume of the pure culture with known volume of 80% methanol three times., and then follow the extraction procedure with partitioning with diethyl ether after adjusting the PH of the extractto 3.5. then  reducing the volume of the extract to 1ml using rotary evaporator at 45C.then use HPLC for the determination of gibberellins and  IAA.. the thesis is written in Arabic, and it published in arabic in BASRAH J.AGRIC. SCI,1998.
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Dear researcher, I know that nitrogenase is responsible to fix atmospheric nitrogen. Which technique are used to detect the nitrogenase enzyme? Please tell me answer so I will gratitude for everybody.
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Dear Noorain,
The following publications describe the detection methods for nitrogenase from bacteria:
EPR spectroscopy is the technique often used to study the nitrogenase and its cluster. EPR experiments show that MoFe cofactor posses a 3/2 spin state.12 Information such as this spin state give a valuable spectroscopic fingerprint that can help detect what will happen to nitrogenase under different circumstances, i.e. what happens to the active site when it is purified or isolated.
2-JOURNAL OF BACTERIOLOGY, Jan. 1991, p. 365-371
Detection of Alternative Nitrogenases in Aerobic Gram-Negative
Nitrogen-Fixing Bacteria
ELAZAR FALLIK,1 YIU-KWOK CHAN,2 AND ROBERT L. ROBSON'*
Strains of aerobic, microaerobic, nonsymbiotic, and symbiotic dinitrogen-fixing bacteria were screened for the presence of alternative nitrogenase (N2ase) genes by DNA hybridization between genomic DNA and DNA encoding structural genes for components 1 of three different enzymes. A niJDK gene probe was used as a
control to test for the presence of the commonly occurring Mo-Fe N2ase, a vnfDGK gene probe was used to show the presence of V-Fe N2ase, and an anfDGK probe was used to detect Fe N2ase. Hitherto, all three enzymes have been identified in Azotobacter vinelandii OP, and all but the Fe N2ase are present in Azotobacter chroococcum ATCC 4412 (MCD1). Mo-Fe N2ase and V-Fe N2ase structural genes only were confirmed in this strain and in two other strains of A. chroococcum (ATCC 480 and ATCC 9043). A similar pattern was observed with Azotobacter beijerinckii ATCC 19360 and Azotobacter nigricans ATCC 35009. Genes for all three systems are apparently present in two strains of Azotobacter paspali (ATCC 23367 and ATCC 238331; ,nd also in Azomonas agilis ATCC 7494. There was no good evidence for the existence of any genes other than A ,o-Fe N2ase structural
genes in several Rhizobium meliloti strains, cowpea Rhizobium strain 32H1, or Bradyrhizobium japonicum. Nitrogenase and nitrogenase genes in Azorhizobium caulinodans behaved in an intermediate fashion, showing (i) the formation of ethane from acetylene under Mo starvation, a characteristic of alternative nitrogenases, and (ii) a surprising degree of cross-hybridization to the vnfDGK, but not the anfDGK, probe. vnfDGK- and anJDGK-like sequences were not detected in two saccharolytic Pseudomonas species or Azospirillum brasilense Sp7. The occurrence of alternative N2ases seems restricted to members of the family Azotobacteraceae among the aerobic and microaerobic diazotrophs tested, suggesting that an ability to cop
See attached file.
See attached paper.
Hoping this will be helpful,
Rafik
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Nitrogen fixing plant endophytes.
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Thanks to all scholars for sharing your experiences. Would you please share a reference or detailed protocol?
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Hi,
I'm just wondering whether molybdenum is absolutely necessary to isolate nitrogen fixers using nitrogen-free media. Your replies would be invaluable.
Best,
Dilantha
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But Rhizobium don´t fix N in culture medium. The molibdenum is essential in the process of nitrogen fixation, in nodule. In culture medium Mo is essential in the cultures when we grow the N fixers not simbiotics, as Azotobacter, Azospirillum etc. 
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It is possible to apply this method on root nodules and/or on Rhizobia strains (like nitrogen fixation by the free bacteria) and how for each case?
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The use of ARA for evaluate the activity of nitrogenase is only for comparative effects and not for quantify the fixed N. If we want to compare, for example, two strains or the effect of a micronutrient in the nitrogenase activity, the ARA is a good tool.
The link above gives the practice:
And can search by Boddey, R M (1981, 1987, 1995) and Boddey & Dobereiner.
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How long Azotobacter will go on multiplying continuously   in soil after adding  ? What are the factors responsible for their decrease in population in soil ? OR simply adding organic matter in the soil will help to multiply Azotobacter , which was used to treat seeds at the time of sowing   ?
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This is really a good question . We often debate on this issue. Any microbial inoculation will be effective only when there is sufficient organic matter ( especially the labile fraction )  in the soil plus other supplementary conditions like pH, Ec  etc etc....   ..However the bigger question is  , once you inoculate the seeds , those seeds germinate exploiting the microbial secretions towards germination  and onward growth , but at later stages , especially in perennial crops , need to be budded with some compatible rootstock , thereby , change the growing medium  preferably using containers , then you need another inoculation , followed by planting of those  budded plants in main  field , then you need yet another inoculation to really witness the response of any microbe(s) in the main field  in terms of real field evaluation . On the contrary , this is not the case with annual crops , since seeds continue to grow in the same piece  of land till crop harvest . In this case also , we need to  discriminate the difference between starter inoculation and growth responsive stages of crop to boost up the  further crop performance .  I am still willing to know from my learned  colleagues , which is better , inoculating seeds or inoculating growing medium ? . which of the inoculation practices will favour better survival and subsequent  utilisation of inoculated microbes?
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I will work on effect of Nitrogen levels on Brachypodium distachyon, and I want know on better of Nitrogen levels can I use it..
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81.6-78.2 kg/h
the source Conservation Agriculture: Global Prospects and Challenges
 edited by Ram A. Jat, Kanwar L. Sahrawat, Amir H. Kassam
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We can't find any nodules. Why? Are we using the wrong methodology? We dig up roots and look for nodules. This is in southern Mozambique.
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Collect soil from field in pots and germinate the seed - you should get nodules in young seedlings
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I am studying the effects of pH on Biological Nitrogen Fixation and planning to use N15 natural abundance method for determining BNF. Can anyone suggest me if there is any other method more suitable for this particular study?
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Hi Prashanta,
I agree, 15-N isotope techniques are the most reliable of the techniques available for measuring biological N2 fixation (BNF) by legumes under field conditions.  There are two 15N based methods for the determination of BNF: (a) natural abundance method, (b) 15N enrichment methods.
Of these two methods, ‘natural abundance’ method is less costly, but needs very careful preparation of samples to avoid cross contamination.  Also, soil ‘δ15N’ at your experimental site need to be significantly different from the ‘δ15N’ value of the atmospheric N (which considered as 0 ‰).  A soil δ15N value around 2 ‰ is recommended by Unkovich et al.  Both methods require a reference (non-nitrogen fixing plant) plant.  The papers published by Unkovich et al. are good source for understanding the assumptions and principles related to the selection of a suitable reference plant for your study as well as general methodology of the two 15N methods.
Good luck.
-SJ
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I am looking for some scholarly articles on the effects of increased soil pH on nitrogen fixation. Can anyone suggest the most read and cited articles on this topic please?
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'Factors influencing biological nitrogen fixation and nitrogen release in biological soil crust' __J. Belnap. Ecological studies. Vol. 150. Springer-Verlag Berlin Heidelberg 2001.
THE INFLUENCE OF SOIL ACIDITY ON SYMBIOTIC AND
NON - SYMBIOTIC NITROGEN FIXATION
Edmundas LAPINSKAS, Loreta PIAULOKAITĖ-MOTUZIENĖ
Zemdirbyste / Agriculture, vol. 93, No. 4 (2006), p. 210-220
There are many more references if you google the topic.
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In studies aimed to evaluate the soybean response to nitrogen fertilization in reproductive stages, the rationale for nitrogen application is the assignment or decrease in BNF after flowering. However, I think that if the BNF continues during the reproductive stages, such justification would not make sense.
I appreciate if someone can help me.
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Dear Vitor,
There is a review by Salvagiotti et al. (2008) on N uptake, fixation and response to fertilizer N in soybeans.  This review has examined 40 years (1966-2006) of research on this subject and will definitely provide you a comprehensive answer.  It can be found here:
Briefly, this review has indicated that:
(a) biological N fixation can provide the majority of the required N supply for soybean, unless there are soil restrictions for a normal nodule activity (e.g. moisture stress, soil temperature stress, soil pH)
(b) soybean grain yield is more likely to respond to N fertilization in high yielding (> 4.5 Mg/ha) environments, usually with deep placement of slow release fertilizer below the nodulation zone or late N application during the reproductive stages
(c) there is a negative exponential relationship between N fertilizer rate and N2 fixation when N was applied on the surface or incorporated in the topmost soil
(d) N fertilization would only be profitable where N2 fixation was not able to meet the total N demand of high yielding soybeans.
-SJ
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In drylands, rhizobial community are in low density. In your opinion, in a experiment to measure the rhizobial population in sites where you expect to be low density, what is the most appropriate serial x-fold dilution to determine the most probably number (MPN)? 10-fold, 5-fold, 2-fold?
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For MPN method, we use 5-fold dilution and inoculate the legume plant with highest dilution (5-6) to the lowest (5-1) dilution.
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I am trying to count the nitrogen fixing bacteria in interior rice roots by (MPM) method
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Dear Dr. Abdel-Gawad,
To enumerate the endophytic nitrogen fixers in the root, I do not prefer to use the MPN but the plate count using the general medium for isolation of nitrogen fixers.
First, take 10 g of the root and just use the method followed to wash and sterilize the root nodules of legumes to sterilize the roots.
Secondly, use a sterilized mortar to crash the roots carefully then transfer the crushed roots quantitatively to the dilution bottle that contains 90 ml of sterile distilled water. After that follow the procedure of the total counting.
Wishing you all success   
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While conducting experiments, I experienced that 1000 mg/l ammonium nitrogen was converted to 130 mg/l nitrate nitrogen. This did not make sense to me, although the experiments were conducted in triplicates. Additional experiments also gave similar results
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You either had denitrification going on as well. pH was higher and you stripped NH3 or due to some uknwon factor ammonium was converted in N2O. But essential there cannot be 'loss' of N
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I want to study root nodule properties to fix N and its effect on soil N2O emission in rice paddies.
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N15 Isotope
Acetylene ethylene reduction
Use of N2O Gas Monitors such as Aeroqual series 500
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B value represents the fractionation given by the nitrogenase and the 15N distribution in the plant. According to Höwberg (1997), the fractionation during N2-fixation is generally small. Besides, 15N distribution in the plant is not important when we harvest and analyze both above and below ground biomass. I think B value estimation is important when we harvest only above ground biomass, but if it is possible to harvest all biomass, can we assume B=0? What is your opinion about this?
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The B value basically refers to the fractionation that occurs for the two N isotopes. As a plant takes up nitrogen it preferentially uptakes the lighter isotope, 14N, by a small degree. This is something that would have to be determined experimentally, but there are also a range of generic values that you could use. I will paste a section below that discusses some of that. It is obviously important to know the δ15N of your soil...the larger the difference between the soil and the air then the less significant the B value is. You could do the experiment with the entire range of B values, and then say your results fall within that range of nitrogen fixation to be safe. Or to do your calculations with one "best guess" B value, and note that the B value may change. I will paste a couple sections below that may help.
Isotope fractionation constants are ex-
pressed as β in the expression 14k/15k = β, where
14k=first-order reaction rate constant of the conver-
sion of the 14N isotope substrate to product and 15k
the same constant for the 15N species. Shearer et al.
(1974) used a value of β of 1.005 for the incorpora-
tion of ammonium ion into microbial protoplasm and
1.025 for nitrification (Delwiche & Steyn, 1970). For
a similar model describing ammonification, microbial
assimilation of ammonium and nitrification, Herman
& Rundel (1989) used values of β of 1.0046, 1.0046
and 1.0176 for these three transformations, respect-
ively. Handley & Raven (1992) have cited values of
β for NH+ assimilation of between 1.012 to 1.020, 4
and for nitrification of between 1.015 and 1.035.
Good papers usmmarizing:
Use of the 15N natural abundance technique to quantify biological nitrogen fixation by woody perennials. Boddey et al 2000.
Measurement of Nitrogen Fixation by Soybean in the Field Using the Ureide and Natural 15N Abundance Methods' Herridge et al 1990.
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This is for a class at a low-budget community college.  Thanks
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Yes, It will work.
The above suggestions by the experts are best methods.
Still if you are looking for simple method,
First of all collect healthy root nodules, and follow the surface sterilization process. Then squeeze the nodules in sterile conditions into sterile broth of 1ml then streak on to YEMA medium with 0.025% Congo red will give good results.
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Which is the best fixation solution to preserve RNA in aquatic microorganisms prior to water filtering?.
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Hi Frederic
RNA Later is supposed to be fine for aquatic samples but people working with soils and sediments tend to favor LifeGuard from MoBio - it is very expensive though.
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I want information on how to calculate N2 fixation using 15-N based methods for measuring BNF .That is the Principles of the δ15N (15N natural abundance) technique to determine biological N2 fixation (BNF).
With the formula
. %Ndfa = δ15N of reference plant - δ15N of N2-fixing legume x 100
δ15N of reference plant – B 1
), expecally how to calculate for the B vale in the formula
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Go through this manuscript "Singh Bhupinder, Usha K. 2003. Nodulation and symbiotic nitrogen fixation of cowpea genotypes as affected by fertilizer nitrogen. Journal of Plant Nutrition 26 (2): 463-473".
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We succeded in culturing using Vinodgrasky media, but are unable to purify it.
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Thank you very much.
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I'm working on nitrogen cycle and I want to quantify the potential rate of nitrification and denitrification through the quantification of functional genes. I want to known how many copies of these genes (nosZ, nirS, nirK and amoA genes) there are in a bacterial genome?
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We are interested in getting rhizosphere bacteria with plant growth-promoting activities. We got hundreds of bacterial colonies from environmental samples and we must now evaluate nitrogen fixation in them. Does anyone know selective microbiological methods for these activities?
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You can try acetylene reduction assay for measuring nitrogen fixation by any microbes. Wherein depending upon nitrogenase activity certain amount of acetylene will be converted to ethylene which can be measured using the technique of gas chromatography.
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Thanks a lot. I tried looking for papers on type iii machinery  but I did not come across anything I was interested in. But now that you mentioned, I would search again. Thank you very much for your response once again.
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Suppose I have over-fertilized soil ( festival area - places with the big  urine concentration) and I want to accumulate the nitrogen in plants to recycle it  further and make it available for other plants. What kind of plant could help me to bind the nitrogen from soil (in similar way as leguminosae fix and accumulate the nitrogen from atmosphere)? Is there any solution to make a use of this natural fertilizer (urine I mean ) which has a peak only two times a year?
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Dear Natalia,
First of all  study the local flora of the place where you are planning to work. As native plants need less effort to establish and have less impact of local climate on their growth. Make list of plants then we can make out which could be best according to the suitablity of your place. If you are planning to construct wetland then Eicchornia have great potential to accumulate nitrogen and after harvesting it can be used in biogas plant as well for the production of manure. In my native place people are using it in biogas plant and organic manure formed is being used by farmers. Cyperus and Typha  are also good options.
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Symbiotic N fixation has been shown to respond to N availability and is limited by high energy requirements. Plants appear to use symbiotic N fixation as a facultative strategy to overcome N limitation. However, in many ecosystems, N fixing plants are not present, and even in ecosystem where they are present, N fixation by free-living organisms appears to be an important additional source of N to the ecosystem. But, what controls this input on an ecosystem scale? Is there any observed variation across certain gradients? And how close is the interaction between plants and free-living fixers? Is there plant exudation of labile C as energy provision to free-living heterotrophs? - Thanks for any answers and references!
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Any newer synthesis would have cited this:
Cleveland, C. C. et al. 1999. Global patterns of terrestrial biological nitrogen (N2) fixation in natural ecosystems. Global Biogeochemical Cycles 13(2): 623-645
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I am looking for the database about herbs biomass. I need some average data about biomass (C and N) sequestered in entire plant, roots and shoots.
e.g.
Achillea millefolium
Alchemilla vulgaris
Anemone nemorosa
Angelica sylvestris
Anthriscus sylvestris
Asarum europaeum
Asperula odorata
Blackcurrant
Bryophytes
Vaccinium myrtillus
etc.
I need it to evaluate "plant biomass" of different sites, according to counted "plant units".
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This publication may help you:
also check if your library has the book:
Root Demographics and Their Efficiencies in Sustainable Agriculture, Grasslands and Forest Ecosystems
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I would like to know if it would be possible to combine both measurements and measure them simultaneously. Are there any studies doing this?
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Here is the first publication on this aspect:
T. Yoshinari, R. Hynes, R. Knowles (1977). Acetylene inhibition of nitrous oxide reduction and measurement of denitrification and nitrogen fixation in soil. Soil Biology & Biochemistry, Vol 9, Pages 177-188.
This will obviously work for measuring actual denitrification rate as well as nitrogen fixation. However, an important point is that if commercial acetylene is employed, it should be passed through sulfuric acid to remove acetone (in which acetylene is dissolved and which may server as carbon source for bacteria).
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We got results of nitrogen fixation by unicellular cyanobacteria in an eutrophic estuary. In an eutrophic estuary they have enough nutrients available for their metabolism, so they do not have the need for nitrogen fixation. However, even under these circumstance they continue with nitrogen fixation. What is the reason for this?
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Well its a known fact that cyanobacteria can fix nitrogen. But in eutrophic waterbodies they have to compete with other algal species to bloom. At this stage, cyanobacteria can not risk to loose their energy in Nitrogen fixation, rather they use the available nitrogen source to increase their biomass rapidly.
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How is measurement of nitrogen fixation by the acetylene reduction assay (ARA) by gas chromatograph done?
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The method you refer to can be used as a qualitative measure for nitrogen fixation only. It has long been abandoned to arrive at quantitative estimates.
For details, I refer to an online publication you can download for free:
Unkovich M, Herridge D, Peoples M, Cadisch G, Boddey B, Giller K, Alves B, Chalk P. 2008. Measuring plant-associated nitrogen fixation in agricultural systems: Australian Centre for International Agricultural Research (ACIAR).
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Nitrogen input and output from a Capsicum annuum- soil system may be important to determine amount of nitrogen got leached from Chile soil system. We know the added amount of nitrogen to system but what about the N in output (Capsicum annuum fruits)?
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Dear Sudhir,
Data from food databases show that protein content in green pepper fruits fluctuates from about 0.8 to 0.9% and from 1.48 to 2 % in red fruits . As the protein content in foods is estimated by multiplying the determined nitrogen content by the nitrogen-to-protein conversion factor, 6.25, you can calculate the nitrogen content in green fruits : 0.128-0.144 % and in red fruits: 0.237-0.32%.
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Synechococcus sp.PCC 7002 grown in A+ media containing NaNO. Is it possible if I replace NaNO3 with N2?
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Nope. The strain does not fix atmospheric nitrogen.
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I tried to isolate about 150 Endophytic bacteria from the roots and nodules of the legume White Lupin. However, I can't identify them due to the lack of PCR or any other apparatus required for molecular identification in our laboratory.
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I dont know with what purpose you are interested to identify these root nodulating bacteria. But, I am working on root noudlating bacteria and also identify these bugs using MALDI-TOF MS and sequencing of several genes. I am working in an international culture collection (http://www.nccs.res.in/mcc), so if you are interested in their identification, we can figure out a possible way for this.
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Conventional microscopic identification of nitrogen fixing cyanobacteria and heterotrophic bacteria (growing in nitrogen-free medium) is a difficult task. Does anyone work with a diversity of nitrogen fixing cyanobacteria and heterotrophic bacteria?
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Sorry - here's the other paper. Also anything by Jon Zehr will be worth your while looking at.
Cheers
Clare
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It would be great if the bacteria were confirmed by NifH pcr screening or acetylene reduction assay. If you have one and are interested in sharing with me, kindly let me know. Need to screen my isolates
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Thanks for the kind reply. I have some isolates which are able to grow on N free medium, but my isolates from plants which is growing on the poor nutrient as well as stressed condition. So i am wondering ability of my isolates to grow on N free medium due to their N fixing capability or some other reason.
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I need help to prepare diagram similar to attached diagram.
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Hi
Do you want a different way to create pathways or you just want a more appealing diagram than what KEGG gives you? If the latter, you can use iPath2- http://pathways.embl.de/ It is same as KEGG, just more beautiful and interactive and an option to download publication quality images. And its completely free!
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There are some concerns about the use of 15N enrichment and 15N natural abundance to quantify the contribution of biological nitrogen fixation.
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Enderson, the N15 based methods are the gold standard for overall biological nitrogen fixation, simply because they work. Their main question is finding the reference species, especially here in the tropics, since so many species are showing BNF themselves (or rather some kind of associative BNF system).
If you are working under sterile conditions the good old Leonard jar (or derived) systems can give you a really useful estimate, with all the limitations derived from the different environmental conditions.
On the field, though, I don´t know of any method with more trustworthy estimations, especially for total BNF over a time frame.
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I'll try to find all chemical reactions related N - Cycle occurring in soil.
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Thank you very much Fulvio Rivano
I need more help in this area.
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Molecular tools allow us to measure the diversity and also abundance of some functional genes, such as nitrogen fixation genes(nif H), ammonia oxidation genes (amo B) etc. For the gene diversity and gene abundance, which one has more contribution to the biogeochemical processes and to the ecosystem?