report microbiology

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Nitrogen fixation by bacteria

Ahlam Ali

200700521

DR. Abdulmajeed Al-Khajah

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Nitrogen is essential for all life on this planet, but most of it is in the air, making up

about 78% of the earth's atmosphere.

Nitrogen is often referred to as a primary limiting nutrient in plant growth. Simply

put, when nitrogen is not available plants stop growing. Although lack of nitrogen isoften viewed as a problem, nature has an immense reserve of nitrogen everywhere

plants grow in the air. Air consists of approximately 80% nitrogen gas (N2),

representing about 6400kg of N2 above every hectare of land. However, N2 is a stable

gas, normally unavailable to plants. Nitrogen fixation, a process by which certain

plants "fix" or gather atmospheric N2 and make it biologically available is an

underlying pattern in nature. Bacteria are the only organisms capable of taking

gaseous nitrogen and combining it with hydrogen to make ammonia.

Biological nitrogen fixation was discovered by the Dutch microbiologist Martinus

Beijerinck

Martinus Willem Beijerinck(March 16, 1851 January 1, 1931) was a Dutch

microbiologist and botanist. He was born in Amsterdam.

Beijerinck studied at Leiden University and became a teacher in microbiology at the

Agricultural School in Wageningen (now Wageningen University) and later at the

Polytechnische Hogeschool Delft(Delft Polytechnic, currently Delft University ofTechnology) (from 1895). He established the Delft School of Microbiology. His

studies of agricultural microbiology and industrial microbiology yielded fundamental

discoveries in the field of biology. His achievements have been perhaps unfairly

overshadowed by those of his contemporaries Robert Koch and Louis Pasteur,

because unlike them, Beijerinck never studied human disease.

He is considered the founder of virology. In 1898, he used filtration experiments to

show that tobacco mosaic disease is caused by an agent smaller than a bacterium. He

named that new pathogen virus. (Dimitri Ivanovski discovered viruses in 1892, butfailed to report his findings.) Beijerinck maintained that viruses were liquid in nature,

a theory later discredited by Wendell Stanley, who proved they were particulate.

Beijerinck also discovered nitrogen fixation, the process by which diatomic nitrogen

gas is converted to ammonium and becomes available to plants. Bacteria perform

nitrogen fixation, dwelling inside root nodules of certain plants (legumes). In additionto having discovered a biochemial reaction vital to soil fertility and agriculture,

Beijerinck revealed this archetypical example of symbiosis between plants and

bacteria.

Beijerinck discovered the phenomenon of bacterial sulfate reduction, a form of

anaerobic respiration. He learned that bacteria could use sulfate as a terminal electron

acceptor, instead of oxygen. This discovery has had an important impact on our

current understanding of biogeochemical cycles. Spirillum desulfuricans, the first

known sulfate-reducing bacterium, was isolated and described by Beijerinck.

Beijerinck invented the enrichment culture, a fundamental method of studyingmicrobes from the environment. He is often incorrectly credited with framing the

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microbial ecology idea that "everything is everywhere, the environment decides,"

which was stated by Lourens Baas Becking.

Role of nitrogen in the biosphere

The growth of all organisms depends on the availability of mineral nutrients, and none

is more important than nitrogen, which is required in large amounts as an essential

component of proteins, nucleic acids and other cellular constituents. There is an

abundant supply of nitrogen in the earth's atmosphere - nearly 79% in the form of N2gas. However, N2 is unavailable for use by most organisms because there is a triple

bond between the two nitrogen atoms, making the molecule almost inert. In order for

nitrogen to be used for growth it must be "fixed" (combined) in the form of

ammonium (NH4) or nitrate (NO3) ions. The weathering of rocks releases these ions

so slowly that it has a neglible effect on the availability of fixed nitrogen. So, nitrogen

is often the limiting factor for growth and biomass production in all environments

where there is suitable climate and availability of water to support life.

Microorganisms have a central role in almost all aspects of nitrogen availability and

thus for life support on earth:

Some bacteria can convert N2 into ammonia by the process termed nitrogen

fixation; these bacteria are either free-living or form symbiotic associations

with plants or other organisms (e.g. termites, protozoa)

other bacteria bring about transformations of ammonia to nitrate, and of nitrate

to N2 or other nitrogen gases

many bacteria and fungi degrade organic matter, releasing fixed nitrogen for reuse byother organisms.

The nitrogen cycle

The diagram below shows an overview of the nitrogen cycle in soil or aquatic

environments. At any one time a large proportion of the total fixed nitrogen will be

locked up in the biomass or in the dead remains of organisms (shown collectively as

"organic matter"). So, the only nitrogen available to support new growth will be that

which is supplied by nitrogen fixation from the atmosphere (pathway 6 in thediagram) or by the release of ammonium or simple organic nitrogen compounds

through the decomposition of organic matter (pathway 2). Some of other stages in this

cycle are mediated by specialised groups of microorganisms and are explained below.

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Nitrification

The term nitrification refers to the conversion of ammonium to nitrate (pathway 3-4).

This is brought about by the nitrifying bacteria, which are specialised to gain their

energy by oxidising ammonium, while using CO2 as their source of carbon to

synthesise organic compounds. Organisms of this sort are termed chemoautotrophs -

they gain their energy by chemical oxidations (chemo-) and they are autotrophs (self-

feeders) because they do not depend on pre-formed organic matter. In principle the

oxidation of ammonium by these bacteria is no different from the way in which

humans gain energy by oxidising sugars. Their use of CO2 to produce organic matter

is no different in principle from the behaviour of plants.

The nitrifying bacteria are found in most soils and waters of moderate pH, but are not

active in highly acidic soils. They almost always are found as mixed-species

communities (termed consortia) because some of them - e.g.Nitrosomonas species -

are specialised to convert ammonium to nitrite (NO2-) while others - e.g.Nitrobacter

species - convert nitrite to nitrate (NO3-). In fact, the accumulation of nitrite inhibits

Nitrosomonas, so it depends onNitrobacterto convert this to nitrate, whereas

Nitrobacterdepends onNitrosomonas to generate nitrite.

The nitrifying bacteria have some important environmental consequences, because

they are so common that most of the ammonium in oxygenated soil or natural waters

is readily converted to nitrate. Most plants and microorganisms can take up either

nitrate or ammonium (arrows marked "1" in the diagram). However, process of

nitrification has some undesirable consequences. The ammonium ion (NH4+) has a

positive charge and so is readily adsorbed onto the negatively charged clay colloids

and soil organic matter, preventing it from being washed out of the soil by rainfall. In

contrast, the negatively charged nitrate ion is not held on soil particles and so can be

washed down the soil profile - the process termed leaching (arrow marked 7 in the

diagram). In this way, valuable nitrogen can be lost from the soil, reducing the soil

fertility. The nitrates can then accumulate in groundwater, and ultimately in drinkingwater. There are strict regulations governing the amount of nitrate that can be present

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in drinking water, because nitrates can be reduced to highly reactive nitrites by

microorganisms in the anaerobic conditions of the gut. Nitrites are absorbed from the

gut and bind to haemoglobin, reducing its oxygen-carrying capacity. In young babies

this can lead to respiratory distress - the condition known as "blue baby syndrome".

Nitrite in the gut also can react with amino compounds, forming highly carcinogenic

nitrosamines.

Denitrification

Denitrification refers to the process in which nitrate is converted to gaseous

compounds (nitric oxide, nitrous oxide and N2) by microorganisms. The sequence

usually involves the production of nitrite (NO2-) as an intermediate step is shown as

"5" in the diagram above. Several types of bacteria perform this conversion when

growing on organic matter in anaerobic conditions. Because of the lack of oxygen for

normal aerobic respiration, they use nitrate in place of oxygen as the terminal electron

acceptor. This is termed anaerobic respiration and can be illustrated as follows:

In aerobic respiration (as in humans), organic molecules are oxidised to obtain energy,

while oxygen is reduced to water:

C6H12O6 + 6 O2 = 6 CO2 + 6 H2O + energy

In the absence of oxygen, any reducible substance such as nitrate (NO3-) could serve

the same role and be reduced to nitrite, nitric oxide, nitrous oxide or N 2.

Thus, the conditions in which we find denitrifying organisms are characterised by (1)

a supply of oxidisable organic matter, and (2) absence of oxygen but availability ofreducible nitrogen sources. A mixture of gaseous nitrogen products is often produced

because of the stepwise use of nitrate, nitrite, nitric oxide and nitrous oxide as electron

acceptors in anaerobic respiration. The common denitrifying bacteria include several

species ofPseudomonas,Alkaligenes andBacillus. Their activities result in

substantial losses of nitrogen into the atmosphere, roughly balancing the amount of

nitrogen fixation that occurs each year.Biological Nitrogen Fixation

Approximately 80 percent of the atmosphere is nitrogen gas (N2). Unfortunately, N2 is

unusable by most living organisms. Plants, animals and microorganisms can die of

nitrogen deficiency, surrounded by N2 they cannot use. All organisms use the

ammonia (NH3) form of nitrogen to manufacture amino acids, proteins, nucleic acidsand other nitrogen-containing components necessary for life.

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Biological nitrogen fixation is the process that changes inert N2 to biologically useful

NH3. This process is mediated in nature only by bacteria. Other plants benefit from

nitrogen-fixing bacteria when the bacteria die and release nitrogen to the environment

or when the bacteria live in close association with the plant. In legumes and a few

other plants, the bacteria live in small growths on the roots called nodules. Within

these nodules, nitrogen fixation is done by the bacteria, and the NH3produced is

absorbed by the plant. Nitrogen fixation by legumes is a partnership between a

bacterium and a plant. Biological nitrogen fixation can take many forms in nature,

including bluegreen algae (a bacterium), lichens and free-living soil bacteria. These

types of nitrogen fixation contribute significant quantities of NH3 to natural

ecosystems but not to most cropping systems, with the exception of paddy rice. Their

contributions are less than 5 lbs of nitrogen per acre per year. However, nitrogen

fixation by legumes can be in the range of 25-75 pounds of nitrogen per acre per year

in a natural ecosystem and several hundred pounds in a cropping system.

Some ammonia also is produced industrially by the Haber-Bosch process, using an

iron-based catalyst, very high pressures and fairly high temperature.

Legume Nodules

Legume nitrogen fixation starts with the formation of a nodule. A common soil

bacterium,Rhizobium, invades the root and multiplies within the cortex cells. The

plant supplies all the necessary nutrients and energy for the bacteria. Within a weekafter infection, small nodules are visible with the naked eye. In the field, small

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nodules can be seen 2-3 weeks after planting, depending on legume species and

germination conditions.

When nodules are young and not yet fixing nitrogen, they are usually white or gray

inside. As nodules grow in size, they gradually turn pink or reddish in colour,

indicating nitrogen fixation has started. The pink or red color is caused by

leghemoglobin (similar to hemoglobin in blood) that controls oxygen flow to the

bacteria. Nodules on many perennial legumes, such as alfalfa and clover, are

fingerlike in shape. Mature nodules may actually resemble a hand with a center mass(palm) and protruding portions (fingers), although the entire nodule is generally less

than 1/2 inch in diameter. Nodules on perennials are long-lived and will fix nitrogen

through the entire growing season, as long as conditions are favorable. Most of the

nodules (10-50 per large alfalfa plant) will be centered around the tap root. Nodules

on annual legumes, such as beans, peanuts and soybeans, are round and can reach the

size of a large pea. Nodules on annuals are short-lived and will be replaced constantly

during the growing season. At the time of pod fill, nodules on annual legumes

generally lose their ability to fix nitrogen, because the plant feeds the developing seed

rather than the nodule. Beans will generally have less than 100 nodules per plant,

soybeans will have several hundred per. plant and peanuts may have 1,000 or more

nodules on a well-developed plant. Legume nodules that are no longer fixing nitrogen

usually turn green and may actually be discarded by the plant. Pink or red nodules

should predominate on a legume in the middle of the growing season. If white, grey

or green nodules predominate, little nitrogen fixation is occurring as a result of an

inefficientRhizobium strain, poor plant nutrition, pod filling or other plant stress.

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Close up view of bacteroids

The nitrogen fixed is not free. The plant must contribute a significant amount of

energy in the form of photosynthate (photosynthesis derived sugars) and other

nutritional factors for the bacteria. A soybean plant may divert 20-30 percent of its

photosynthate to the nodule instead of to other plant functions when the nodule isactively fixing nitrogen. Any stress that reduces plant activity will reduce nitrogen

fixation. Factors like temperature and water may not be under the farmer control. But

nutrition stress (especially phosphorus, potassium, zinc, iron, molybdenum and

cobalt) can be corrected with fertilizers. When a nutritional stress is corrected, the

legume responds directly to the nutrient and indirectly to the increased nitrogen

nutrition resulting from enhanced nitrogen fixation. Poor nitrogen fixation in the field

can be easily corrected by inoculation, fertilization, irrigation or other management

practices.

The nitrogen-fixing organismsAll the nitrogen-fixing organisms are prokaryotes (bacteria). Some of them live

independently of other organisms - the so-called free-living nitrogen-fixing bacteria.

Others live in intimate symbiotic associations with plants or with other organisms

(e.g. protozoa). Examples are shown in the table below.

Examples of nitrogen-fixing bacteria (* denotes a photosyntheticbacterium)

Free living Symbiotic with plants

AerobicAnaerobic (see

Winogradsky column for

details)

Legumes Other plants

Azotobacter

Beijerinckia

Klebsiella (some)

Cyanobacteria(some)*

Clostridium (some)

DesulfovibrioPurple sulphur bacteria*

Purple non-sulphur

bacteria*

Green sulphur bacteria*

RhizobiumFrankia

Azospirillum

A point of special interest is that the nitrogenase enzyme complex is highly sensitive

to oxygen. It is inactivated when exposed to oxygen, because this reacts with the iron

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component of the proteins. Although this is not a problem for anaerobic bacteria, it

could be a major problem for the aerobic species such as cyanobacteria (which

generate oxygen during photosynthesis) and the free-living aerobic bacteria of soils,

such asAzotobacterandBeijerinckia. These organisms have various methods toovercome the problem. For example,Azotobacterspecies have the highest known rate

of respiratory metabolism of any organism, so they might protect the enzyme bymaintaining a very low level of oxygen in their cells.Azotobacterspecies also

produce copious amounts of extracellular polysaccharide (as doRhizobium species in

culture - see Exopolysaccharides). By maintaining water within the polysaccharide

slime layer, these bacteria can limit the diffusion rate of oxygen to the cells. In the

symbiotic nitrogen-fixing organisms such asRhizobium, the root nodules can contain

oxygen-scavenging molecules such as leghaemoglobin, which shows as a pink colour

when the active nitrogen-fixing nodules of legume roots are cut open.

Leghaemoglobin may regulate the supply of oxygen to the nodule tissues in the same

way as haemoglobin regulates the supply of oxygen to mammalian tissues. Some of

the cyanobacteria have yet another mechanism for protecting nitrogenase: nitrogen

fixation occurs in special cells (heterocysts) which possess only photosystem I (usedto generate ATP by light-mediated reactions) whereas the other cells have both

photosystem I and photosystem II (which generates oxygen when light energy is used

to split water to supply H2 for synthesis of organic compounds).

Symbiotic nitrogen fixation1. Legume symbiosesThe most familiar examples of nitrogen-fixing symbioses are the root nodules of

legumes (peas, beans, clover, etc.).

Clover root nodules at higher magnification, showing two partly crushed nodules (arrowheads) with

pink-coloured contents. This colour is caused by the presence of the pigment leghaemoglobin - a

unique metabolite of this type of symbiosis. Leghaemoglobin is found only in the nodules and is not

produced by either the bacterium or the plant when grown alone.

In these leguminous associations the bacteria usually areRhizobium species, but the

root nodules of soybeans, chickpea and some other legumes are formed by small-

celled rhizobia termedBradyrhizobium. Nodules on some tropical leguminous plants

are formed by yet other genera. In all cases the bacteria "invade" the plant and cause

the formation of a nodule by inducing localised proliferation of the plant host cells.

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Yet the bacteria always remain separated from the host cytoplasm by being enclosed

in a membrane - a necessary feature in symbioses (see the image below).

Part of a crushed root nodule of a pea plant, showing four root cells containing colonies ofRhizobium.The nuclei (n) of two root cells are shown; cw indicates the cell wall that separates two plant cells.

Although it cannot be seen clearly in this image, the bacteria occur in clusters which are enclosed in

membranes, separating them from the cytoplasm of the plant cells.

In nodules where nitrogen-fixation is occurring, the plant tissues contain the oxygen-

scavenging molecule, leghaemoglobin (serving the same function as the oxygen-

carrying haemoglobin in blood). The function of this molecule in nodules is to reducethe amount of free oxygen, and thereby to protect the nitrogen-fixing enzyme

nitrogenase, which is irreversibly inactivated by oxygen.

Nostoc is a genus of fresh water cyanobacteria that forms spherical colonies

composed of filaments of moniliform cells in a gelatinous sheath. When on the

ground, a Nostoc colony is ordinarily not seen; but after a rain it swells up into a

conspicuous jellylike mass, which was once thought to have fallen from the sky,

hence the popular names, fallen star and star jelly. It is also called witches' butter

(not to be confused with the fungus Tremella mesenterica). Michael Quinion of the

World Wide Words newsletter says that it is known in Welsh aspwdre sr, orrot of

the stars.

Nostoc can be found on moist rocks, at the bottom of lakes and springs, and rarely in

marine habitats. It may also grow symbiotically within the tissues of plants, such as

the evolutionarily ancient (Gunnera) or hornworts, providing nitrogen to its host.

These bacteria contain photosynthetic pigments in their cytoplasm to perform

photosynthesis.

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Relationship between bacteria and plant:The relationship between the plant and the bacteria is known as a symbiotic

relationship since both the plant and the bacteria benefit from their relationship.

Legumes including peas, lentils and alfalfa can form symbiotic associations for

nitrogen fixation with a soil bacterium calledRhizobium. TheRhizobium enters into

the roots and forms nodules, which the bacteria then use as their home. In the nodules,

theRhizobium fixes N 2 into a form that the plant can use. Nitrogen is fixed by binding

it to hydrogen and making it into ammonia which the legume plant can use. Rhizobia

benefit as the plant provides carbohydrates to the rhizobia. Carbohydrates are required

by rhizobia as a source of energy. Also, the carbohydrates produced by the legume

plant are transported to the nodules where they are used by the rhizobia as a source of

hydrogen in the conversion of nitrogen to ammonia.

Nitrogen Fixation Efficiency and Nitrogen FertilizationSome legumes are better at fixing nitrogen than others. Common beans are poor fixers

(less than 50 lbs per acre) and fix less than their nitrogen needs. Maximum economic

yield for beans in New Mexico requires an additional 30-50 lbs of fertilizer nitrogen

per acre. However, if beans are not nodulated, yields often remain low, regardless of

the amount of nitrogen applied. Nodules apparently help the plant use fertilizer

nitrogen efficiently. Other grain legumes, such as peanuts, cowpeas, soybeans and

faba, beans are good nitrogen fixers and will fix all of their nitrogen needs other than

that absorbed from the soil. These legumes may fix up to 250 lbs of nitrogen per acre

and are not usually fertilized. In fact, they usually dont respond to nitrogen fertilizer

as long as they are capable of fixing nitrogen. Nitrogen fertilizer is applied at planting

to these legumes when grown on sandy or low organic matter soils to supply nitrogen

to the plant before nitrogen fixation starts. If nitrogen is applied, the rate is low, 10-15

lbs per acre. When large amounts of nitrogen are applied, the plant literally slows or

shuts down the nitrogen fixation process. It is easier and less energy consuming for

the plant to absorb nitrogen from the soil than to fix it from the air. Perennial and

forage legumes, such as alfalfa, sweetclover, true clovers and vetches, may fix 250-

500 lbs of nitrogen per acre. Like the grain legumes previously discussed, they are not

normally fertilized with nitrogen. They occasionally respond to nitrogen fertilizer at

planting or immediately after a cutting when the photosynthate supply is too low for

adequate nitrogen fixation.

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Nitrogen Return to the Soil and Other CropsThe amount of nitrogen returned to the soil during or after a legume crop can be

misleading. Almost all of the nitrogen fixed goes directly into the plant. Little leaks

into the soil for a neighboring nonlegume plant. However, nitrogen eventually returns

to the soil for a neighboring plant when vegetation (roots, leaves, fruits) of the legume

dies and decomposes. When the grain from a grain legume crop is harvested, little

nitrogen is returned for the following crop. Most of the nitrogen fixed during the

season is removed from the field. The stalks, leaves and roots of grain legumes, such

as soybeans and beans contain about the same concentration of nitrogen as found in

non-legume crop residue. In fact, the residue from a corn crop contains more nitrogen

than the residue from a bean crop, simply because the corn crop has more residue. A

perennial or forage legume crop only adds significant nitrogen for the following crop

if the entire biomass (stems, leaves, roots) is incorporated into the soil. If a forage is

cut and removed from the field, most of the nitrogen fixed by the forage is removed.

Roots and crowns add little soil nitrogen compared with the aboveground biomass.

Nitrogen Fixation Problems in the Field

Measuring nitrogen fixation in the field is difficult. However, a grower can make

some field observations that can help indicate if nitrogen fixation is adequate in some

of the common legumes. If a newly planted field is light green and slow growing, suspectinsufficient nitrogen fixation. This is often seen with beans and alfalfa. In a new field, the

poor fixation is often attributed to the lack of nativeRhizobium to nodulate the legume, butthe cause may also be poor plant nutrition or other plant stresses that inhibit nitrogen fixation.

Small nodules should be present 2-3 weeks after germination. If no nodules are present,

consider the following options. A. Replant using seed inoculated with the correctRhizobium.B. Try to inoculate the plants in the field through the irrigation system or by other means.

Caution: this technique often does not work and expert advice is needed C. Consider nitrogen

fertilization to meet all of the plants nitrogen needs. This may not be an option for aperennial legume, such as alfalfa, if the field is kept in alfalfa for several years. Also, some

legumes use soil or fertilizer nitrogen more efficiently if nodules are present. If young nodules

are present, sufficient soil nitrogen may not be available for the young plant before nitrogen

fixation starts. The plant usually grows out of this condition or a small amount of nitrogen can

be applied. Also, inefficient nativeRhizobium may result in poor nitrogen fixation. Consider

other soil stresses that may be inhibiting plant growth, especially plant nutrition and water

stress. If an established crop becomes nitrogen deficient in the middle of the growing season,

when plant growth and nitrogen demands are greatest, poor or inefficient nitrogen fixation

might be the cause. Nodules should be clearly evident, at about the size and number per plant

as previously described and be pink or red in color. If only a few nodules are present,

insufficientRhizobium numbers have limited nodulation or plant stresses may be inhibitingnitrogen fixation. At this time, the grower may be able to remove a plant stress, but it is too

late to inoculate if the nodules are mostly green, gray or white, the native may be inefficient

nitrogen fixers. The only choice may be to apply nitrogen fertilizer on the present crop and

heavily inoculate the next crop.New Mexico State University Extension Guide A-130,

Inoculation of Legumes, describes when and how to inoculate legumes.

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Reference:http://www.biology.ed.ac.uk/research/groups/jdeacon/microbes/nitrogen.htm

http://www.alexagri.com/forum/showthread.php?t=7577

http://www.britannica.com/EBchecked/topic-video/416291/83734/Some-bacteria-

convert-nitrogen-gas-into-complex-compounds-that-can

http://www.microbiologybytes.com/video/Azotobacter.htmlwww.goooglevedio.com

http://students.kennesaw.edu/~dmw5091/NitrogenCycle.ppt

cahe.nmsu.edu/pubs/_a/a-129.pdf

bioteach.snunit.k12.il/upload/.arab/ecolarab.ppt

http://en.wikipedia.org/wiki/Nitrogen_fixation

http://hcs.osu.edu/hcs300/bact.htm

book: biology of plant, third edition, university of Wisconsin, Madison, Peter

H.Raven,1981(in uaeu:QK47R25)

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