A Note on Biofertilizers 21
viii) The farmer s themselves can grow BGA biofer tilizers andAzolla
2 Rhizobium invades roots of legumes a n d forms nodules on
A NOTE ON BIOFERTILIZERS
The canier based ,nicrobial inoculants being added to the soil to
enrich the soil fertility are called biofertilizers. Th ey ar e often kn o,vn
as microbial fertilizers or niicrobial inoculants. A biofe,tilize1 may
contain nitrogen fixing microbes or phosphate solubilizing m icrobes
or spores of VAM fungi. It is s upplied t o t h e soil eit her by seed
treatment or by sp,eading i t over the field during cu lt ivation .
Biofertilizers reduce the use of chemical fertilizers in agriculture
and cost of produ ct ion.
The nitrogen biofertilizer m ay have nitrogen fixing bacteria or
blue-green algae. The n itrogen fl.Xing bacteria include Rhizobium,
Azospirillum, Azotobacter Azotococcus, etc . Blu e green algae such as
Anabaena, Aulosira, Nostoc, Plectone,na a nd Tolypothrix are u sed
as n itrogen biofert ilizers.
Preparations of phosphate solu bilizing bacteria such Bacillus
megatherium, B, su.bti/is, Xanthornonas a nd Pseudomonas, are u sed
as phosphate biofertilizers.
T he spores of VAM fungi like Gl01nus, Gigaspora, Acaulospora,
Sclerocystis and Endogone are used as VAM biofertilizers.
Biofertilizers have the following advantages-
i) Biofertilizers r educe the use of chemical fertilizers in agricu lture.
ii) They n ever cause pollution in air , water and land .
iii) T h ey secr ete plant gro,vth hor m ones to increase the plant
growth.
iv) They r educe the a ttack by soil-borne pathogens,
v) T hey imp,ove the quality of soil for mor e productivity.
vi) They can be mass produced by using renewable ,vastes.
vii) No special care is required while using biofertilizers.
biofe,t ilizer in their own lands.
Rhizobium
Rhizobium is a gram n egative, aerobic, rod-shaped bacterium.
It contains a refractive granule. It is a soil bacterium present in large
num bers in rhizospher e of legume roots .
the roots. In side the ,oot nodules, the bacteria exist in variou s
p leomor ph ic forms called bacterioids. The bacterioids fix the
atmospheric nitrogen in to ammonia . They pr ovid e th e fixed nitrogen
for plant's use and drav, n ou rishments from th e root cells. T his
type of association is called symbiosis.
Different species of Rhizobium can fix 50-200 kg nitrogen/ha/
year in legum in ous crops. Ther efore, they have been r ecommend ed
as nitrogen biofer tilizers in agriculture.
Different species of Rhizobium sho,v a great d egr ee of h ost
specificity. Hence th ey can be used as biofertilizers only for the specific
crops-
i) R. meliloti (Medic-Rhizobiu.m ): Lucerne and Fen ugr eek.
ii) R. trifolii (Clover-Rhizbium): Egypt ian clover and clover.
iii) R. Leguminosarum (Pea-Rhizobium): Lentil, pea, khesri
(Lathyrus) and vetch.
iv) R. Phaseo'/i (Bean-Rhizobium) : Bean, kidney bean and French
bean,
v) R. lupini (Lupin-Rhizobium): Lupin es and v,hite lupines.
vi) R. Japonicum (Soybean-Rhizobium ): Soyabean.
vii) Rhizobium sps. (Chickpea-Rhizobium): C h ickpea and
s ubabul.
viii) Rhizobium sps, (Cowpea-Rhizobium) : Sunnhe1np, cluster
b ean , p eanut, jack bean, lablab, h o,segram, moth bean, gr een
gram, blackgr arn and pigeonpea.
Production of Rhizobium Inoculants
Production of Rhizobium inoculant involves-
i) Isolation of Rhizobium
ii) Identification of Rh izobium
iii) Establislunen t of starter cultur e
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22 Manufacture of Biofertilizer and Organic Farrning A Note on Biofertilizers 23
iv) Mass culture i) CRYEMA Test: A 2. 5 ml of conga r ed dye is mi..xed ,vith a lit re
of YEMA m ed ium to prepare CRYE1'vIA m ed ium. Bacterial colonies
v) l\1ixing with carrier
on the YEMA m edium are streaked on the CRYEMA medium and
vi) Pa ckaging and s t orage. the p et ri dis h es aie incubated at 28 2c for 5-7 days.
1 . Isolation of Rhizobium Rhizobial cells uptake congo r ed very weakly.so they 27-BT
form ,vhite, cir cu la,, entire, raised , convex colonies. Agrobacteriun1
Rhizobiurn occurs in the soil as well as in the root nodules of
select ive legu1nes . Rhizobium in t he r oo t nodu l es h as least colon ies, if any, look like Rhizobial colonies , but show characteristic
c ontaminants . Therefo,e, it is isolated from root nodules of a prope, colour of congo red. The white colon ies are picked up to produ ce
leguminous plant. Rhizobium inoculant.

The leguminous plan t is carefully up1ooted from the soil and ii) Microscopic Obseroation : Bacterial cells in th e CRYEMA
the root syst em is washed with running water to remove the s oil med ium are stained with carbol fuschin a nd visualized under a
paiiicles. Firm, undainaged , pink-coloured root nodules are selected com pou nd micr oscope. T h is dye st ains th e f3-polyhydr oxybutyrat e
visually and excised from th e ,oots. g,anu le in the Rhizobium . The cell s of tho s e colonies having
f3-polyhydr o:,,.-ybutyrate granule aie picked up to establish Rhi.zobium
The root nodules are kept immersed in 0.1 /c, pot assium chloride inoculant.
solution or in 0 . 1% acidified m ercuric chloride solution for 5 minutes
to sterilize the surface of the nodu les.
T he st erilized r oot n odules are t hen "'ashed 5 or 6 tin1es ,vith
distilled water. They aie once again sterilized by immersing them in
90% ethyl alcohol for 10 seconds and ,va shed repeatedly ,vith distilled
water .
The root nodules are crushed gently in a small amount of
distilled ,vater using a pestle and mortar to get a suspension.
The suspension is dilu t ed and inoculated onto YE!v!A (Yeast
eid:ract m a nnitol agar) medium in petri dishes. The cult ure plates
ar e incu bat ed at 28C for about 10 clays. Rh izobial cells form gummy
c olonies on the medium. Leguminous plai1t
Components of YEMA medium
K, HPO, -0 .5 g

NaCl
.
MgSO .7H -0 0
-0.2g
-0.l g .-:+ - - - Sterile soil
.. I

lvl a nnitol - 10.0g . .. . . '

.
.
. . ..
Yeast extract - 1.0 g
Agar -20.0 g
Distilled wat er - 1000 m l.
2. Identification of Rhizobium: YEMA mediun1 is s uitable
for the gr owth of Rhi.zobiurn as well as Agrobacterium.. Rhizobial Fig. 1: Exp erimental set u p for modulation test.
colonies are identified from th e cultur e in the following 1nethods:

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24 Manufacture of Biofertilizer and Organic Farrning
iii) Glucose Peptone Agar Test (GPA Test): Rhizobial
colonies are stieaked on YEMA m edium and a mast er plate is made.
Colonies in the master plate are transferred to C PA medium in a
petri dish by r eplica plating. Rhizobium cannot gr ow in the m ed iu m ,
but Agrobacteria, if present in t he colonies, grow into colonies. Those
colonies in the master pla te failed to grow in t he replica p late are
pur e r h izobial colonies. They are picked up and gro\vn in YEMA
medium . This test is th e confirm ative test to test th e purity of Rhizobial
colonies.
iv) Nodulation Test; The same species of legume from \vhich
Rhizobia were isolated, is gr own in s terile soil in s t er ile jars. Nutrient
solu tion th at lacks nitrogen sour ce, is supplied through a h ole at
th e base of the jars by insert ing a cotton thread imm ersed in the
solution. Each pure Rh izobial culture is then inoculated n ear t he
root of legurne growing in a ja r. After 3 or 4 weeks, the plants are
car efully upr ooted from the j ars and visualized for root nodules.
Presence of mor e root nodules indicat es t hat it is the right st,ain of
Rhizobium for th at legume.
3. Es tablishi ng the Starter Cu lture: Pur e rhizobial colony is
transferr ed t o a flask containing YEMA m ediu 1n . The flask is k ept on
a rotary s haker system in a cons t ant temper atur e room a t 282 C.
Pur e culture of rh izobium app ears within a ,veek. It is known as
starter culture or mother culture. A st art er culture is frequ en tly sub-
cultu red for maintain ing it for a long time.
4. Mass culture of Rhizobium: Rhizobiurn is mass cult ur ed
in large bioreactors (fermenter) to prepare inoculant. YE!VI medium
or su cr ose-mannitol ,n edium is used for this purpose.
A s uita ble mediu m is formulat ed and filled int o t he bioreactor
aft er proper sterilization. One litr e of starter cu lture for 100 litres of
medium is inoculated into th e biorea ctor.
The temper ature in s ide th e reactor is maintaine d at 28 2 C .
S terile air is continuously supplied to the broth with the help of a
proper device. T he b1oth is stirr ed cont inuously by a stirrer system
kept in the bioreactor .
Cell coun ting is done a regu lar in tervals to assess th e growt h
rate of Rhizobium. Having reach ed 10 8 - 109 cells/ml, the br oth is
harvest ed to u s e as inoculant. Fu rther 1na inten ance of th e b,oth in
the bioreactor rnay lead t o death of r h izobial cells due to the deficiency
of en ough nutrien ts .
S. Making the Carrier-bas ed Inoculant : A carr ier is a neu tra.l
med iu,n used to m ix with a cultured b,oth fo, hand ling it easily.
A Note on Biofe rtilizers 25
Ch arcoal, p eat, lign ite, venniculite, p ad dy husk, farm yar d manure,
etc. are used as can ier s . T h e crurier h olds t he broth and allows the
s low growth of m icrobes. Besides this, it increases the longitivity of
the n1icrobes .
The carriers is dr ied in the sunlight and gr oun d in to fine
po,vder. It is th en n eutralized by adding enough calcium carbonat e
dust. Finally , it is s t erilized in an autoclave at 15 lb for 3 hrs and
dried ,veil befor e mixing it with the br ot h.
Th e harvested broth is aseptically n1ixed ,vith the carr ier either
m anually 0 1 m echanically . B,oth is added to th e cru,ier t ill it receives
40-50% moisture, (ie. volume equal to h alf of the total water holding
capacity of t h e carrier).
After proper mixing, the carrier-inoculant is left as such for
2-7 days for the m u lt iplication of Rhizobium in the available broth.
The process is called curing. As a result of curing, t he carrier based
Rh izobium in oculant is rea dy to use.
6 . Pack ing and S torage : The carrier based inoculant is
packe d in low density polyten e bags for s torage and mark eting.
Usually, th e biofertilizer p ockets a r e kept in a constant temp er ature
room for a bout a ,veek before st orage. During this t im e, Rhizobia
grow by consuming the lit tle amount of m edium p re sent in the
earner.
Finally, the pockets are stor ed at l 5C in a dry cool place for
field use or salse. They should n ot be exposed to direct sunlight and
h eat t hat kill t h e cells . Rhizobial cells in t h e pockets s eem to be
via ble for abou t 6 rnonths.
[Note: Qu ality con tJol tests are cond ucted a t every s tage of th e
inocu lum production based on BIS m anual as recommended by
IS i to preven t cont amination, t o test t h e self-life, to test th e cell
n umber/ grn of carrier and to tes t the suitability of the in oculum for
use].
Field Application of Rhizobium Inoculant
Rhizobium inoculru1ts rue recomn1ended for va r ious legu -
minous c,ops by seed treatlnent:
1. A 50 g of cane sugar is dissolved in 500 m l of \\>a ter and boiled
for 15 m inutes.
2. 200 g of gum arabic is added to t he boiling sugar s olution and
sti1Ted well to dissolve it. The sticker solution so formed is cooled
own.
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26 Manufacture of Biofertilizer and Organic Farrning
3 Then a 200 g of Rhizobium inocula.nt is added in to the sticker
solution and ,nixed well to get a biofertilizer slur,y.
4. T he seeds of a legume are add ed into t he s lurry and mixed well
by hands. Consequen tly, aboutl0 5 -l0 6 Rh izobial cells get
ad sorbed o n each and eve1y seed.
5. The seeds are then allo,ved to d1y by spreading them on a
polytene sheet in shade .
6. The seeds are sown in the main field.
While adopting this n1et hod , t he farm ers should tak e th e
following precautions-
Aft er seed inoculat ion, the seeds s h ould n ot be stor ed for m ore
than a day.
No fu ngicide should be used within one day after sowing t he
seeds.
Use of chernical fertilizers should be avoided for more than a
,veek immed iately after so,ving.
Pest icides should be used in small a,nounts, if r equir ed.
Crop Respons
Seed in oculation of a proper Rhizobium strain increases growt h
and yield of m any legu,nes . Rhizobium u su ally incr eases the yield of
legurnes upto 10-35%.
In Cajanus oojanit in creases yields upto 4-40% in variou s stat es
of India. In Cicer aritinurn it boot s the yield upto 4-67%.
In Vigna mungo, it increases th e yield upto 4-29%.
In Lens culinaris it improves the yield upto 21 %.
T he crop rotat ion of rhizobium in ocu lated legumes wit h cereals
increases the yield of th e subsequent cereal crop. For example, crop
rotation of Cajanus cajan " 'ith ,vheat increases th e wh eat yield upt o
16.4%Sim ilarly, crop rotation ofLens C1.llinaris with rice has increased
the r ice yield upto 13.2%
Azotobacter
Azotobacteris a gram negative, .non-symbiotic, .nitrogen fixing
bacterium. It is an aer obic bacteriun1 pr esen t in large num bers in
rhizosphere soils. Azotobacter incl udes s ix species-Azotobacter
beijerin.ckii, A .paspali, A .chroococcum, A.haplophi/s, A. vin.elan.dii and
A.miscellus.
Azotobacterfixes about 20 -4 0KgN /ha/year. Further, it produces
A Note on Biofertilizers 27
plant growth p romoting substances lik e !AA, gibberellic acid and
vit amins to favour t h e p lant growth. Hence it h as been r ecom mended
as a biofertilizer for rice, wheat, rnillets, cotton , vegetables, m ustard,
s u nflo,ver , etc.
Production of Azotobacter Inoculant
A 10 g rhizosphere soil is mixed well with 100 m l of dist illed
water and left undis t urbed for so,netime t o h ave a clear suspension.
The suspension is serially diluted a11d inoculated into per tri dishes
containin g Jensen's 1nediun1. The culture plat es ar e incubated at
30C fo, a few day s. As Jensen's m edium is a nitiogen free agar
medium, Azotobacter alone grows int o soft , rrullcy, mucoid colonies
on its surface.
Azot obact er colonies are aseptically transferred t o culture flasks
cont aining liquid Jensen's m edium and th e culture flasks are kept
on a rotary shaker system for a few days. The temperature is
maint ained at 30C . Consequently, pure cultu re of Azotobacter
develops in t he flasks . It is used as a starter culture for mass
pr oduct ion.
For mass cult ure, 1 li tre of s tarter cu lture for 100 litr es of
medium is transfe1Ted t o s terilized Jensen 's medium or Ashby's
medium or Bu rk's m edium in a b ioreactor. The temperature inside
the r eactor is m aintain ed a t 30C and s ter ile air is pumped i11to the
reactor t o give prope, aeration t o the culture. T he cu lt u,e is s tirred
continuously by an electrically operated s t irrer. Azot obacter attained
the con centration of 10 8 cells/1nl, the inoculant (broth ) is harvested
to make c.anier-based inocu lan t .
A suitable carrier is g r ound into fine powder an d sieved through
a 106 micron sieve. Its pH is neutr alised by adding enough calcium
carbonate. T he carrier is sterilized in an autoclave and allowed t o
cool. The harvest ed br oth is m ixed with the s t erilized carrier in a
s terile room t ill carrier attains 4 0/4 moisture.
T h en the inoculant is allowed for c.u,~ng for a week. The cur ed
inoculant is then packed in low density polytene bags for stage and
salse.
Field Applications
1 . Seed Treatment
Azotobacter inoculant is m ixed with a s 1nall quan tity of water
to mal<e a slu ny and the seeds a ,e k ept dipped in the sluny for one
n ight. In the early morning, the seeds are tal<en out and sown in the
main field. The ren1aining sluny is directly s prayed in the field.
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