1 NITROGEN FIXATION

2 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.

3 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

4 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 ).

5 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

6 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)

7 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.

8 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.

9 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)

10 Some nitrogen fixing organisms

11 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

12 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

13 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

14 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

15 Nitrogenas e

16 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

17 Types of Biological Nitrogen Fixation Free-living (asymbiotic) Cyanobacteria Azotobacter Associative Rhizosphere–Azospirillum Lichens–cyanobacteria Leaf nodules Symbiotic Legume-rhizobia Actinorhizal-Frankia

18 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

19 Heterocyst

20 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

21 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

22 Azolla pinnata (left) 1cm. Anabaena from crushed leaves Of Azolla.

23 Simbiosis Anabaena-Azolla

24 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.

25 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.

26 Actinorhizal Plant Hosts DatiscaDatiscaceae CoriariaCoriariaceae Cercocarpus, Chamaebatia, Cowania, Dryas, Purshia Rosaceae Ceanothus, Colletia, Discaria, Kentrothamnus, Retanilla, Talguenea, Trevoa Rhamnaceae Elaeagnus, Hippophaë, ShepherdiaElaeagnacea e Comptonia, MyricaMyricaceae Allocasuarina, Casuarina, Ceuthostoma, Gymnostoma Casuarinace ae AlnusBetulaceae GeneraFamily

27 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)

28 Taxonomy of Rhizobia Aeschynomene (stem nodule)spp.Photorhizobium Galega (goat’s rue), Leucaenagalegae[Rhizobium] Lotus (trefoil) Astragalus (milkvetch) Cicer (chickpea) loti huakuii ciceri tianshanense mediterraneum ‘Meso rhizobium’ Sesbania (stem nodule)caulinodansAzorhizobium Glycine (soybean) japonicum elkanii liaoningense Bradyrhizobium Melilotus (sweetclover), Medicago (alfalfa), Trigonella Glycine (soybean) Sesbania Sesbania, Acacia meliloti fredii saheli teranga Sinorhizobium Trifolium (clovers) Pisum (peas), Vicia (field beans), Lens (lentils), Lathyrus Phaseolus (bean) Phaseolus (bean), Leucaena Phaseolus (bean) leguminosarum bv. trifolii “ bv. viciae “ bv. phaseoli tropici etli Rhizobium Host plantSpeciesGenus

29 Rhizobium Root Nodules The picture above shows a clover root nodule. Available from [Internet]

30 Rhizobium Root Nodules

31 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.

32 Formation of a Root Nodule

33 Nodulation in Legumes

34 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

35 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

36 nod Gene Expression Common nod genes Nod factor–LCO (lipo-chitin oligosaccharide)

37 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

38 Anaerobi c jar Anaerobic Culture Methods

39 Anaerobi c chamber Anaerobic Culture Methods

40 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

41 Suppress unwanted microbes and encourage desired microbes. Selective Media

42 Streak Plate

43 After incubation, count colonies on plates that have 25-250 colonies (CFUs) Plate Count