Presentation on theme: "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."— Presentation transcript:
Nitrogen Fixation The growth of all organisms depend on the availability of Nitrogen (e.g. amino acids) Nitrogen in the form of Dinitrogen (N 2 ) makes up 80% of the air we breathe but is essentially inert due to the triple bond (N N) In order for nitrogen to be used for growth it must be "fixed" (combined) in the form of ammonium (NH 4 ) or nitrate (NO 3 ) ions.
Nitrogen Fixation The nitrogen molecule (N 2 ) 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 600 o C, and with the use of a catalyst, atmospheric nitrogen and hydrogen (usually derived from natural gas or petroleum) can be combined to form ammonia (NH 3 ). Ammonia can be used directly as fertilizer, but most of its is further processed to urea and ammonium nitrate (NH 4 NO 3 ).
Haber-Bosch 3CH 4 + 6H 2 O --> 3CO 2 + 12H 2 4N 2 +12H 2 --> 8NH 3 (high T,press) Changing Nitrogen Cycle Humans have doubled the N fixation rates over natural levels
Nitrogen Fixation Process Energetics N N 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 bacteria o Azotobacter o Beijerinckia o Klebsiella o Cyanobacteria (lichens) Free living anaerobic bacteria o Clostridium o Desulfovibrio o Purple sulphur bacteria o Purple non-sulphur bacteria o Green sulphur bacteria Free living associative bacteria o Azospirillum Symbionts o Rhizobium (legumes) o Frankia (alden trees)
Some nitrogen fixing organisms
Estimated Average Rates of Biological N 2 Fixation 40-300 1-150 1-50 50-150 50 Actinorhizal plant symbioses with Frankia Alnus Hippophaë Ceanothus Coriaria Casuarina 50-100 100-600 Leguminous plant symbioses with rhizobia Grain legumes (Glycine, Vigna, Lespedeza, Phaseolus) Pasture legumes (Trifolium, Medicago, Lupinus) 10-20 300 40-80 Cyanobacterial associations Gunnera Azolla Lichens 5-25 Grass-Bacteria associative symbioses Azospirillum 25 0.3 0.1-0.5 Free-living microorganisms Cyanobacteria Azotobacter Clostridium pasteurianum N 2 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) N 2 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 N 2 + 8H + 2NH 3 + H 2 8e-8e- 4C 2 H 2 + 8H + 4C 2 H 2 Dinitrogenase reductase Fd(red) Fd(ox) nMgATP nMgADP + nP i N 2 + 8H + + 8e - + 16 MgATP 2NH 3 + H 2 + 16MgADP
Genetics of Nitrogenase Dinitrogenase reductase Dinitrogenase Regulatory, activator of most nif and fix genes FeMo cofactor biosynthesis Unknown Electron transfer Regulatory Regulatory, two-component sensor/effector Electron transfer Transmembrane complex nifH nifDK nifA nifB nifEN nifS fixABCX fixK fixLJ fixNOQP fixGHIS Properties and functionGene
Free-living N 2 Fixation Energy 20-120 g C used to fix 1 g N Combined Nitrogen nif genes tightly regulated Inhibited at low NH 4 + and NO 3 - (1 μg g -1 soil, 300 μM) Oxygen Avoidance (anaerobes) Microaerophilly Respiratory protection Specialized cells (heterocysts, vesicles) Spatial/temporal separation Conformational protection
Associative N 2 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 10 6 to 10 7 cells g -1 tissue, and fix 100 to 150 kg N ha -1 y -1
Phototrophic N 2 -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.
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.
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 Rhizobium o Gram -, rod o Most studied symbiotic N 2 -fixing bacteria o Now subdivided into several genera o Many genes known that are involved in nodulation (nod, nol, noe genes)
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
Nodule Metabolism Oxygen metabolism Variable diffusion barrier Leghemoglobin Nitrogen metabolism NH 3 diffuses to cytosol Assimilation by GOGAT Conversion to organic-N for transport Carbon metabolism Sucrose converted to dicarboxylic acids Functioning TCA in bacteroids C stored in nodules as starch
Anaerobi c jar Anaerobic Culture Methods
Anaerobi c chamber Anaerobic Culture Methods
Encourages growth of desired microbe Assume a soil sample contains a few phenol-degrading bacteria and thousands of other bacteria o Inoculate phenol-containing culture medium with the soil and incubate o Transfer 1 ml to another flask of the phenol medium and incubate o Only phenol-metabolizing bacteria will be growing Enrichment Media
Suppress unwanted microbes and encourage desired microbes. Selective Media
After incubation, count colonies on plates that have 25-250 colonies (CFUs) Plate Count