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The Nitrogen Cycle
Component
Input to soil
Loss from soil
Atmospheric
nitrogen
Atmospheric
fixation
and deposition
Industrial fixation
(commercial fertilizers)
Crop
harvest
Animal
manures
and biosolids
Volatilization
Plant
residues
Runoff and
erosion
Biological
fixation by
legume plants
Plant
uptake
Imm
Organic
nitrogen
Min
era
li
Denitrification
obi
liza
tion
zat
ion
Nitrate
(NO-3)
Ammonium
(NH4+)
cation
Nitrifi
Leaching
1. Imobilisasi; 2.Fiksasi NH4+
3
1.Pelindian (leaching), limpasan (runoff);
2.Nitrifikasi, 3.Denitrifikasi; 4.Penguapan
4
1.Penambatan N2 hayati; 2.Presipitasi NH3 dan NO3;
3.Pemupukan (Organik & anorganik)
PENYIMPANAN
2
KEHILANGAN
1.Amonifikasi; 2.Nitrifikasi
PENAMBAHAN
1
PROSES HABER-BOSCH VS
PENAMBATAN N2 HAYATI
3 H2

N2
Suhu 1200oC
Tekanan 500 atm
NH3
 1.3 TON MINYAK UNTUK MEMPRODUKSI 1 TON NITROGEN
DAMPAK : Melepaskan GRK (CO2 & NOX) serta pencemaran NO3-
N2
Suhu & tekanan normal
NH3
Terjerap mineral lempung tipe 2 : 1
(Vermikulit, Illit, Mica butir halus &
Smektit)
NH4+ lepas
lambat
N2 O
NH3
Nitrifikasi
NO
Leaching, Menurunkan kejenuhan
basa & memasamkan tanah,
Pencemaran air, Eutrofikasi,
Nitrosamin, Denitrifikasi
NH3
Methemoglobinemia (bluebaby syndrome) pada
Konsumen
FREE-LIVING (ASYMBIOTIC):
• Cyanobacteria
• Azotobacter
ASSOCIATIVE:
• Rhizosphere–Azospirillum
• Lichens–cyanobacteria
• Leaf nodules
SYMBIOTIC:
• Legume-rhizobia
• Actinorhizal-Frankia
ORGANISM OR SYSTEM
FREE-LIVING MICROORGANISMS
Cyanobacteria
Azotobacter
Clostridium pasteurianum
N2 FIXED (kg ha-1 y-1)
25
0.3
0.1-0.5
GRASS-BACTERIA ASSOCIATIVE SYMBIOSES
Azospirillum
5-25
CYANOBACTERIAL ASSOCIATIONS
Gunnera
Azolla
Lichens
10-20
300
40-80
LEGUMINOUS PLANT SYMBIOSES WITH RHIZOBIA
Grain legumes (Glycine, Vigna, Lespedeza, Phaseolus)
Pasture legumes (Trifolium, Medicago, Lupinus)
50-100
100-600
ACTINORHIZAL PLANT SYMBIOSES WITH FRANKIA
Alnus
Hippophaë
Ceanothus
Coriaria
Casuarina
40-300
1-150
1-50
50-150
50
Rhizobia are one of the groups of microorganisms living in
soil. Rhizobia are bacteria which are single cells about one
thousandth of a millimetre long.
Rhizobia belong to a specific group of bacteria that form a
close association with legume plants. This association is
called a SYMBIOSIS. The symbiosis results in visible, balllike structures being formed on roots - these structures are
called NODULES. The nodules are formed by the plant IN
RESPONSE TO the presence of the bacteria.
Rhizobia belong to a family of bacteria called
RHIZOBIACEAE. There are a number of groups (genera and
species) of bacteria in this family.
The bacteria take nitrogen from the air (which plants
cannot use) and convert it into a form of nitrogen called
ammonium nitrogen, which plants can use. The process is
called NITROGEN FIXATION and these bacteria are often
called "NITROGEN FIXERS“ = DIAZOTROPH.
Rhizobia can be EXTREMELY BENEFICIAL to plants in areas
WHERE THE SOILS ARE LOW IN NITROGEN, if they form
associations with legumes in these soils.
Rhizobia are found in soils of many natural ecosystems.
They may also be present in agricultural areas where they
are associated with both crop legumes (like soybean) and
pasture legumes (like clover). Usually, the rhizobia in
agricultural areas have been introduced when the
legumes were sown.
The subfamilies of legumes (Caesalpinioideae,
Mimosoideae, Papilionoideae), 700 genera, and
19,700 species of legumes,
COnly about 15% of the species have been
evaluated for nodulation,
CRhizobium :
CGram -, rod
CMost studied symbiotic N2-fixing bacteria
CNow subdivided into several genera
CMany genes known that are involved in nodulation
(nod, nol, noe genes)
RHIZOBIACEAE
RHIZOBIUM
(Genus I)
leguminosorum
Biovar : phaseoli - Bean,
trifolli - Clover,
viceae - Pea,
meliloti - Alfalfa,
loti - Lupines, Lotus,
galegae - Galega,
Rhizobium spp. (host plant)
BRADYRHIZOBIUM
(Genus II)
B.japonicum - Soybeans
Bradyrhizobium spp.
SINORHIZOBIUM
(Genus III)
S. fredii - Soybeans
S. xinjiangensis
AZORHIZOBIUM
(Genus IV)
A. caulinodans - Sesbania
GENUS
SPECIES
HOST PLANT (MACROSYMBION)
Rhizobium
leguminosarum bv. trifolii
“
bv. viciae
“
bv. phaseoli
tropici
etli
Trifolium (clovers)
Pisum (peas), Vicia (field beans), Lens (lentils), Lathyrus
Phaseolus (bean)
Phaseolus (bean), Leucaena
Phaseolus (bean)
Sinorhizobium
meliloti
fredii
saheli
teranga
Melilotus (sweetclover), Medicago (alfalfa), Trigonella
Glycine (soybean)
Sesbania
Sesbania, Acacia
Bradyrhizobium
japonicum
elkanii
liaoningense
Glycine (soybean)
Glycine (soybean)
Glycine (soybean)
Azorhizobium
caulinodans
Sesbania (stem nodule)
‘Meso rhizobium’
loti
huakuii
ciceri
tianshanense
mediterraneum
Lotus (trefoil)
Astragalus (milkvetch)
Cicer (chickpea)
[Rhizobium]
galegae
Galega (goat’s rue), Leucaena
Photorhizobium
spp.
Aeschynomene (stem nodule)
Cicer (chickpea)
The nodulation process is a series of events in which
rhizobia interact with the roots of legume plants to
form a specialised structure called a ROOT NODULE.
The process involves complicated signals between the
bacteria and the roots. In the first stages, the bacteria
multiply near the root and then adhere to it. Next, the
small hairs on the root's surface curl around the
bacteria and they enter the root. Alternatively, the
bacteria may enter directly through points on the root
surface. The method of entry of the bacteria into the
root depends on the type of plant.
Once inside the root, the bacteria multiply within thin
threads. Signals stimulate cell multiplication of both the
plant's cells and the bacteria and this repeated division
results in a mass of root cells containing many bacterial
cells. Some of these bacteria then change into a form
that is able to convert gaseous nitrogen into
ammonium nitrogen (that is, they can "fix" nitrogen).
These bacteria are then called BACTEROIDS.
The shape the nodules form are controlled by the plant
and nodules can vary considerably - both in size and
shape.
Most plants need very specific kinds of rhizobia to form
nodules. For example, the rhizobia that form nodules
on peas cannot form nodules on clover.
Specificity is the DEGREE TO WHICH A BACTERIA
SPECIES CAN FIX ATMOSPHERIC NITROGEN IN
ASSOCIATION WITH MORE THAN ONE HOST
SPECIES. For example, the bacteria Bradyrhizobium
japonicum can only form symbiotic associations with
soy bean plants and is said to have HIGH
SPECIFICITY. Whereas another bacteria in the genus
Bradyrhizobium can form symbiotic associations with
both lupins and serradella and so has lower specificity.
NITROGENASE is the bacterial enzyme that
allows atmospheric nitrogen to be converted to
ammonium. Enzymes are special kinds of
proteins that allow reactions to occur at faster
speeds than normal or under less extreme
conditions. Without nitrogenase, atmospheric
nitrogen can only be converted to ammonium
with HIGH PRESSURE and TEMPERATURES.
Nitrogenase requires a low oxygen environment
to function.
Fd(ox)
Fd(red)
FeMo Cofactor
N2 + 8H+
8e-
2NH3 + H2
nMgATP
nMgADP + nPi
Dinitrogenase
reductase
4C2H2 + 8H+
4C2H4
Dinitrogenase
N2 + 8H+ + 8e- + 16 MgATP  2NH3 + H2 + 16MgADP
GENE
nifH
nifDK
nifA
nifB
nifEN
nifS
fixABCX
fixK
fixLJ
fixNOQP
fixGHIS
PROPERTIES AND FUNCTION
Dinitrogenase reductase
Dinitrogenase
Regulatory, activator of most nif and fix genes
FeMo cofactor biosynthesis
FeMo cofactor biosynthesis
Unknown
Electron transfer
Regulatory
Regulatory, two-component sensor/effector
Electron transfer
Transmembrane complex
LEGHAEMOGLOBIN is an organic
molecule made up of two protein
molecules that control the flow of oxygen
to the bacteria that fix atmospheric
nitrogen. Leghaemoglobin gives the inside
of nodules their REDISH PINK
COLOURING.
Rhizobia are identified according to HOW FAST THEY GROW
AND MULTIPLY ON ARTIFICIAL FOOD SOURCES. They are
very difficult to identify just from their shape or size alone,
because all of them are very small and all are shaped like short
rods with rounded ends. SO YOU CANNOT TELL THE
DIFFERENT TYPES APART JUST BY LOOKING AT THEM,
EVEN WITH A VERY POWERFUL MICROSCOPE.
Rhizobia are usually grouped into "FAST" and "SLOW" growers,
based on HOW QUICKLY THEY GROW ON ARTIFICIAL
FOOD SOURCES.
Recently, new methods of characterisation of rhizobia have
been developed using DNA from known rhizobia.
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,





Phyllosphere or rhizosphere (tropical grasses),
Azosprillum, Acetobacter,
1 to 10% of rhizosphere population,
Some establish within root,
Same energy and oxygen limitations as freeliving,
 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
• Lichens–cyanobacteria and fungi
• Mosses and liverworts–some have
associated cyanobacteria
• Azolla-Anabaena (Nostoc)–cyanobacteria
in stem of water fern
C Gunnera-Nostoc–cyanobacteria in stem
nodule of dicot
C Cycas-Nostoc–cyanobacteria in roots of
gymnosperm
• 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.
FAMILY
Betulaceae
GENERA
Alnus
Casuarinacea Allocasuarina, Casuarina, Ceuthostoma,
e
Gymnostoma
Myricaceae
Comptonia, Myrica
Elaeagnaceae Elaeagnus, Hippophaë, Shepherdia
Rhamnaceae Ceanothus, Colletia, Discaria, Kentrothamnus,
Retanilla, Talguenea, Trevoa
Rosaceae
Cercocarpus, Chamaebatia, Cowania, Dryas,
Purshia
Coriariaceae Coriaria
Datiscaceae
Datisca
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
Common
nod genes
Nod factor–LCO
(lipo-chitin oligosaccharide)
INFECTION PROCESS
1. Attachment,
2. Root hair curling,
3. Localized cell wall
degradation,
4. Infection thread,
5. Cortical cell
differentiation,
6. Rhizobia released into
cytoplasm,
7. Bacterioid differentiation
(symbiosome formation),
8. Induction of nodulins.
Oxygen metabolism
• Variable diffusion barrier
• Leghemoglobin
Nitrogen metabolism
• NH3 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
legume
Fixed nitrogen
(ammonia)
Fixed carbon
(malate, sucrose)
rhizobia
Rhizobium bebas
Perlekatan
secara
acak
Perlekatan
Rambut Akar
PERLEKATAN
Rhizobium
Perlekatan
Rambut akar
mengeriting setelah
Dinding Sel
kontak dengan
Rhizobium
Tanaman Legum
Buluh infeksi memasuki selsel kortek dan merangsang
pembelahan sel
polar
Reseptor
Sel-sel yang sehat
Nodula akar Vakuola
Nukleus
Rhizobium yang
menginfeksi
Membelah
Bakteroid
Peribakteroid
Rhizobium
Mitokondria
Dinding sel
Sel
terinfeksi
ATMOSPHERE
N2
N2
Atmosphere
Soil
N2
Nitrogen-fixing
bacteria
Denitrifying
bacteria
H+
(From soil)
Soil
+
NH4
NH3
(ammonia)
NH4+
(ammonium)
Organic
material (humus)
Nitrate and
nitrogenous
organic
compounds
exported in
xylem to
shoot system
–
Nitrifying
bacteria
NO3
(nitrate)
Ammonifying
bacteria
Root
R. leguminosarum
nodules
Pink color is leghaemoglobin a protein
that carries oxygen to the bacteroids
Azorhizobium
caulinodans
on
Sesbania
(Secang)
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.
1. Enhancing survival of nodule forming bacterium
by improving competitiveness of inoculant strains,
2. Extend host range of crops, which can benefit
from biological nitrogen fixation,
3. Engineer microbes with high nitrogen fixing
capacity.
Root
Nodules
YEMA
Medium
Komposisi YEMA (per 1 liter)
Bahan
K2HPO4
MgSO4.7H2O
NaCl
Mannitol
Yeast Extract
Agar
Akuades
Congo red 1%
gram
0.5
0.2
0.1
10.0
1.0
20.0
1000 ml
2.5 ml
Sumber :
Subba Rao
(1995)
Media diautoklaf pada 120o C selama 15 menit. Kalsium karbonat
(CaCO3) sebanyak 3 g per liter dapat ditambahkan bila
dikehendaki senyawa untuk menetralisir asam yang terbentuk.
REAKSI-REAKSI YANG DAPAT DIKATALISIS OLEH
NITROGENASE (Zuberer, 2005).
Sekat karet
A
Sekrup
Botol plastik
tanpa pangkal
Kantong plastik
B
Lekukan
(diisi air)
Silinder metal
Tanah
dijenuhi air
30 cm
Tempat
penyuntikan
etilen dan
pengambilan
sampel udara
Kantong plastik
Ring pemberat
3 cm
CaC2 + H2O
5 - 7 cm
Kandungan N dalam tanah cukup
tinggi.
Kandungan hara P dalam tanah
rendah.
Kemasaman tanah (pH <5.0 – 5.5),
kecuali untuk cowpea.
Kandungan molibden (Mo) rendah
(nodul akar berukuran besar &
berwarna pucat kehijauan di bagian
dalam, pertanda nodul tidak
efektif).
Pemupukan N takaran rendah (urea <
25 kg ha-1) pada tanaman legum &
hanya diberikan sebagai pupuk dasar
pada saat tanam.
Pemupukan P (TSP, batuan fosfat) pada
lubang tanam bersamaan penanaman.
Pengapuran dg kaptan (CaCO3) pada
lubang tanam bersamaan dg
penanaman.
Pemupukan amonium molibdat atau
batuan mineral yg mengandung Mo.