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NITROGEN FIXATION
Nitrogen Fixation
• The growth of all organisms depend on the availability of Nitrogen (e.g. amino acids)
• Nitrogen in the form of Dinitrogen (N2) makes up 80% of the air we breathe but is essentially inert due to the triple bond (NN)
• In order for nitrogen to be used for growth it must be "fixed" (combined) in the form of ammonium (NH4) or nitrate (NO3) ions.
Nitrogen Fixation
• The nitrogen molecule (N2) is quite inert. To break it apart so that its atoms can combine with other atoms requires the input of substantial amounts of energy.
• Three processes are responsible for most of the nitrogen fixation in the biosphere:
• atmospheric fixation
• biological fixation • industrial fixation
Industrial Fixation
• Under great pressure, at a temperature of 600oC, and with the use of a catalyst, atmospheric nitrogen and hydrogen (usually derived from natural gas or petroleum) can be combined to form ammonia (NH3).
• Ammonia can be used directly as fertilizer, but most of its is further processed to urea and ammonium nitrate (NH4NO3).
Haber-Bosch
3CH4 + 6H2O --> 3CO2 + 12H2
4N2+12H2 --> 8NH3 (high T,press)
Changing Nitrogen Cycle
Humans have doubled the N fixation rates
over natural levels
Nitrogen Fixation Process
Energetics• NN• Haber-Bosch (100-200 atm, 400-500°C,
8,000 kcal kg-1 N)• Nitrogenase (4,000 kcal kg-1 N)
Biological Fixation
The ability to fix nitrogen is found only in certain bacteria.
Some live in a symbiotic relationship with plants of the legume family (e.g., soybeans, alfalfa).
Some establish symbiotic relationships with plants other than legumes (e.g., alders).
Some nitrogen-fixing bacteria live free in the soil. Nitrogen-fixing cyanobacteria are essential to
maintaining the fertility of semi-aquatic environments like rice paddies.
Biological Fixation cont.
• Biological nitrogen fixation requires a complex set of enzymes and a huge expenditure of ATP.
• Although the first stable product of the process is ammonia, this is quickly incorporated into protein and other organic nitrogen compounds.
• Scientist estimate that biological fixation globally adds approximately 140 million metric tons of nitrogen to ecosystems every year.
Some nitrogen fixing organisms
• Free living aerobic bacteriao Azotobactero Beijerinckiao Klebsiellao Cyanobacteria (lichens)
• Free living anaerobic bacteriao Clostridiumo Desulfovibrioo Purple sulphur bacteriao Purple non-sulphur bacteriao Green sulphur bacteria
• Free living associative bacteriao Azospirillum
• Symbiontso Rhizobium (legumes)o Frankia (alden trees)
Some nitrogen fixing organisms
Estimated Average Rates of Biological N2 Fixation
40-3001-1501-50
50-15050
Actinorhizal plant symbioses with FrankiaAlnusHippophaëCeanothusCoriariaCasuarina
50-100100-600
Leguminous plant symbioses with rhizobiaGrain legumes (Glycine, Vigna, Lespedeza, Phaseolus)Pasture legumes (Trifolium, Medicago, Lupinus)
10-20300
40-80
Cyanobacterial associationsGunnera AzollaLichens
5-25Grass-Bacteria associative symbioses
Azospirillum
250.3
0.1-0.5
Free-living microorganismsCyanobacteria Azotobacter Clostridium pasteurianum
N2 fixed (kg ha-1 y-1)Organism or system
Rank of Biological Nitrogen Fixation
0.1 - 25Free- living
5 - 25Rhizosphere associations
10 - 300Cyanobacteria- moss
50 - 600Rhizobium-legume
Nitrogen Fixation (kg N/ha/year)
N2 fixing system
Nitrogen Fixation
• All nitrogen fixing bacteria use highly conserved enzyme complex called Nitrogenase
• Nitrogenase is composed of of two subunits: an iron-sulfur protein and a molybdenum-iron-sulfur protein
• Aerobic organisms face special challenges to nitrogen fixation because nitrogenase is inactivated when oxygen reacts with the iron component of the proteins
Nitrogenase
FeMo Cofactor
N2 + 8H+
2NH3 + H2
8e-
4C2H2 + 8H+ 4C2H2
DinitrogenaseDinitrogenasereductase
Fd(red)
Fd(ox)
nMgATP
nMgADP + nPi
N2 + 8H+ + 8e- + 16 MgATP 2NH3 + H2 + 16MgADP
Nitrogenase
Genetics of Nitrogenase
Dinitrogenase reductaseDinitrogenaseRegulatory, activator of most nif and fix genesFeMo cofactor biosynthesisFeMo cofactor biosynthesisUnknownElectron transferRegulatoryRegulatory, two-component sensor/effectorElectron transferTransmembrane complex
nifHnifDKnifAnifBnifENnifSfixABCXfixKfixLJfixNOQPfixGHIS
Properties and functionGene
Types of Biological Nitrogen Fixation
Free-living (asymbiotic)• Cyanobacteria• Azotobacter
Associative• Rhizosphere–Azospirillum• Lichens–cyanobacteria• Leaf nodules
Symbiotic• Legume-rhizobia• Actinorhizal-Frankia
Free-living N2 Fixation
Energy• 20-120 g C used to fix 1 g N
Combined Nitrogen• nif genes tightly regulated• Inhibited at low NH4
+ and NO3- (1 μg g-1 soil, 300 μM)
Oxygen• Avoidance (anaerobes)• Microaerophilly• Respiratory protection• Specialized cells (heterocysts, vesicles)• Spatial/temporal separation• Conformational protection
Heterocyst
Associative N2 Fixation
• Phyllosphere or rhizosphere (tropical grasses)• Azosprillum, Acetobacter• 1 to 10% of rhizosphere population• Some establish within root• Same energy and oxygen limitations as free-
living• Acetobacter diazotrophicus lives in internal
tissue of sugar cane, grows in 30% sucrose, can reach populations of 106 to 107 cells g-1 tissue, and fix 100 to 150 kg N ha-1 y-1
Phototrophic N2-fixing Associations
• Lichens–cyanobacteria and fungi• Mosses and liverworts–some have associated
cyanobacteria• Azolla-Anabaena (Nostoc)–cyanobacteria in
stem of water fern Gunnera-Nostoc–cyanobacteria in stem nodule of dicot Cycas-Nostoc–cyanobacteria in roots of gymnosperm
Azolla pinnata (left) 1cm. Anabaena from crushed leaves Of Azolla.
Simbiosis Anabaena-Azolla
Frankia and Actinorhizal Plants
• Actinomycetes (Gram +, filamentous); septate hyphae; spores in sporangia; thick-walled vesicles
Frankia vesicles showing thick walls that confer protection from oxygen. Bars are 100 nm.
Alder and the other woody hosts of Frankia are typical pioneer species that invade nutrient-poor soils. These plants benefit from the nitrogen-fixing association, while supplying the bacterial symbiont with photosynthetic products.
Actinorhizal Plant Hosts
DatiscaDatiscaceae
CoriariaCoriariaceae
Cercocarpus, Chamaebatia, Cowania, Dryas, Purshia
Rosaceae
Ceanothus, Colletia, Discaria, Kentrothamnus, Retanilla, Talguenea, Trevoa
Rhamnaceae
Elaeagnus, Hippophaë, ShepherdiaElaeagnaceae
Comptonia, MyricaMyricaceae
Allocasuarina, Casuarina, Ceuthostoma, Gymnostoma
Casuarinaceae
AlnusBetulaceae
GeneraFamily
Legume-Rhizobium Symbiosis
• The subfamilies of legumes (Caesalpinioideae, Mimosoideae, Papilionoideae), 700 genera, and 19,700 species of legumes
• Only about 15% of the species have been evaluated for nodulation
• Rhizobiumo Gram -, rodo Most studied symbiotic N2-fixing bacteriao Now subdivided into several generao Many genes known that are involved in nodulation (nod, nol,
noe genes)
Taxonomy of Rhizobia
Aeschynomene (stem nodule)spp.Photorhizobium
Galega (goat’s rue), Leucaenagalegae[Rhizobium]
Lotus (trefoil)Astragalus (milkvetch)Cicer (chickpea)
Cicer (chickpea)
lotihuakuiiciceritianshanensemediterraneum
‘Meso rhizobium’
Sesbania (stem nodule)caulinodansAzorhizobium
Glycine (soybean)Glycine (soybean)Glycine (soybean)
japonicumelkaniiliaoningense
Bradyrhizobium
Melilotus (sweetclover), Medicago (alfalfa), TrigonellaGlycine (soybean)Sesbania Sesbania, Acacia
melilotifrediisaheliteranga
Sinorhizobium
Trifolium (clovers)Pisum (peas), Vicia (field beans), Lens (lentils), LathyrusPhaseolus (bean)Phaseolus (bean), LeucaenaPhaseolus (bean)
leguminosarum bv. trifolii“ bv. viciae“ bv. phaseolitropicietli
Rhizobium
Host plantSpeciesGenus
Rhizobium Root Nodules
The picture above shows a clover root nodule. Available from [Internet]
Rhizobium Root Nodules
A few legumes (such as Sesbania rostrata) have stem nodules as well as root nodules. Stem nodules (arrows) are capable of photosynthesis as well as nitrogen fixation.
Formation of a Root Nodule
Nodulation in Legumes
Infection Process
• Attachment• Root hair curling• Localized cell wall
degradation• Infection thread• Cortical cell
differentiation• Rhizobia released into
cytoplasm• Bacterioid
differentiation (symbiosome formation)
• Induction of nodulins
Role of Root Exudates
General• Amino sugars, sugars
Specific• Flavones (luteolin), isoflavones
(genistein), flavanones, chalcones
• Inducers/repressors of nod genes
• Vary by plant species• Responsiveness varies by
rhizobia species
nod Gene Expression
Commonnod
genes
Nod factor–LCO(lipo-chitin
oligosaccharide)
Nodule Metabolism
Oxygen metabolism• Variable diffusion barrier• Leghemoglobin
Nitrogen metabolism• NH3 diffuses to cytosol• Assimilation by GOGAT• Conversion to organic-N
for transportCarbon metabolism• Sucrose converted to
dicarboxylic acids• Functioning TCA in
bacteroids• C stored in nodules as
starch
• Anaerobic jar
Anaerobic Culture Methods
• Anaerobic chamber
Anaerobic Culture Methods
• Encourages growth of desired microbe• Assume a soil sample contains a few
phenol-degrading bacteria and thousands of other bacteriao Inoculate phenol-containing culture medium with
the soil and incubateo Transfer 1 ml to another flask of the phenol
medium and incubateo Transfer 1 ml to another flask of the phenol
medium and incubateo Only phenol-metabolizing bacteria will be
growing
Enrichment Media
• Suppress unwanted microbes and encourage desired microbes.
Selective Media
Streak Plate
• After incubation, count colonies on plates that have 25-250 colonies (CFUs)
Plate Count