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ISOLATION, IDENTIFICATION AND SCREENING OFENDOPHYTIC NITROGEN FIXING BACTERIA

FROMSUGARCANE AND SELECTION OF EFFICIENTSTRAINS FOR THEIR MASS PRODUCTION ASLIQUID

STATE BIOINOCULANT WITHFORMULATIONS BY FERMENTATION BASEDBIOTECHNOLOGY.

Chapter 1

Introduction

Endophytic bacterial Nitrogen fixing liquid Bioinoculant is aunique agro-based product in liquid state,
formulated withgrowth boosters and cell protectants and it is a consortium ofgroup of efficient
Endophytic Nitrogen fixing bacteria in liveform. Endophytic bacterial Nitrogen fixing Bioinoculant
isspecial product with newly developed A4H medium with highcell count, zero contamination, longer
shelf life, greaterprotection against environment stresses, increased fieldefficiency with respect to
spreading and penetration andconvenience of handling are main features of the this product.(Baldani et
al., 1978 ).

Endophytic bacteria are those bacteria that fix nitrogeninternally in plant tissues; mostly they are
present in theapoplast i.e. intercellular spaces and xylem vessels. Hence theyare called as endophytic
bacteria. Endophytic bacteria such as

Acetobacter, Azoarcus, Herbaspirillum, Azosperrillum, and

Agrobacterium are present in all parts of sugarcane plant

including leaf, stem, roots and juice. These endophytic bacteria

actively participate in biological nitrogen fixation and fix more

nitrogen as compare to ectophytic bacteria. (Dobereiner, J.

1998).

“Biofertilizers” are products consisting of selected, efficient

and beneficial live or latent (resting stage- spores)microorganisms, which help to improve plant growth
andproductivity mainly through supply of plant nutrients.Biofertilizers are also known asmicrobial
inoculants or bio-

inoculants. Biofertilizers have been introduced in Indian

agriculture since last three decades in view of their costeffectiveness, contribution to crop productivity,
soilsustainability, and eco-friendly characteristics. Biofertilizersform an integral part of integrated plant
nutrient supply system(IPNS or INM) and organic farming which constitutes the present
Chapter 2

REVIEW OF LITERATURE:

Biological Nitrogen Fixation (BNF) is a vital component ofagricultural sustainability. ‘Sustainability’ is

defined as ‘successful management of resources for agriculture to satisfy

human needs while maintaining or enhancing the quality of theenvironment and conserving resources’
(TAC, CGIAR, 1988).Economists measure sustainability as the ratio of output toinput taking into account
stock depletion. Stocks in agricultureinclude soil, water, non-renewable energy resources
andenvironmental quality.

Modern agriculture is based on maximum output in theshort term with inadequate concern for input
efficiency or stockmaintenance (Odum, 1989). Nitrogen fertilizer ranks firstamong the external inputs to
maximize output in agriculture.Input efficiency of Nitrogen fertilizer is one of the lowest amongthe plant
nutrients and in turn contributes sustainability toenvironment pollution. The continued and unabated
use of Nfertilizer would further accelerate depletion of stocks of non-renewable energy resources used
in fertilizer production. Theremoval of large quantities of crop produce from the landadditionally
depletes soil of its native N reserves. On the otherhand, BNF, a microbiological process in the biosphere,
convertsatmospheric dinitrogen into a plant usable form through themicrobial enzyme nitrogenase.

This chapter gives the comprehensive review of literatureof the project work & review is summarized
under followingheadings.

2.1

N2 fixing Endophytic bacteria.

2.1.1 Acetobacter Diazotrophicus

2.1.2 Agrobacterium Diazotrophicus
2.1.3 Azoarcus
2.1.4 Azospirrillum
2.1.5 Herbaspirrillum

2.2

Importance of N2 fixing endophytic bacteria.

2.3

Endophytes as a Biofertilizer.

2.1N2 fixing Endophytic bacteria.

Nitrogen input through BNF can help maintain soilN reserves as well as substitute for N fertilizer to
attain largecrop yields. (Peoples and Croswell, 1992). Biological NitrogenFixation (BNF) can therefore be
a major source of N inagriculture when symbiotic N fixing systems are used. Theamount of N input
reported to be as high as 360 kg N ha-1. Onthe other hand, a contribution for non-symbiotic
(associativeand free-living) N2-fixation in upland agriculture is generally notSubstantial, although N2-
fixation to the order of 160kg N ha-1has been reported for sugarcane (Ladha et al., 1992).
Braziliancultivars of sugarcane rarely respond to N fertilizer applicationsduring the plant crop. Among
135 NPK fertilizer trials all over

the country, only 19% showed significant increase in cane yielddue to N application. This indicates that
some endophyticbacteria may contribute for Biological Nitrogen fixation. Initiallyreported by Azeredo et
al.,(1986). Tremendous progress inBNF has been made during the last more than 30 years and yetwe are
still hoping for breakthrough during the years to comeand as such abundant literature is already
available especiallyon the BNF except BNF in sugarcane associations and thereforethe review of
literature is especially is confined to therhizospheric associative diazotrophs in sugarcane.
JohannaDöbereiner initiated research on BNF with grasses in Brazilwhen she joined the research team
at the National Center ofEducation and Agricultural Research of the Ministry ofAgriculture, in the fifties.
The first studies were Dedicated to thememory of Dr. Johanna Döbereiner by two of her disciples
wholearned through working with her that research could be donewith simplicity, perseverance,
honesty, ethics and sagacity.

2.1.1Acetobacter:

Acetobacter is Gram negative, Micro aerophilic

bacteria motile with 1-3 lateral flagella present in high numberin roots and stems showing optimum
growth with 19% sugarand pH around 5.5 precisely this condition prevailing insugarcane. First isolation
of Acetobacter diazotrophicus strains

365 fold greater than for A. vinelandii depending upon thesubstrate. In general, A.
diazotrophicus supports respirationover a wider pH range than does A. vinelandii. In Germany
1999study was carried out for Analysis of nitrogen fixation andregulatory genes in the sugarcane
endophyteAcetobacter

diazotrophicusby

Lee-S; Sevilla-M; Meletzus-D; Texeira-K;Baldani-I; Kennedy-C; Martinez-E (ed.); Hernandez-G .The

mcpAgene product is involved in responses to extracellularchemotactic signals, which may be important
in plant-microbeinteractions. Study on the respiratory system and diazotrophicactivity of Acetobacter
diazotrophicus PAL5 carried out byFlores et al., (1999) The characteristics of the respiratorysystem
of Acetobacter diazotrophicus PAL5 were investigated.Increasing aeration (from 0.5 to 4.0 liters of
air/min per liter)had a strong positive effect on growth and on the diazotrophicactivity of cultures. Cells
obtained from well aerated anddiazotrophically active cultures possessed a highly active,membrane-
bound

electron
transport

system

withdehydrogenases for NADH, glucose, and acetaldehyde as themain electron donors. Ethanol,
succinate, and gluconate wereoxidized but to only a minor extent. Fuentes-Ramirez et al.,(1999)
reported that colonization of sugarcane byAcetoba cter

diazotrophicus is inhibited by high N-fertilization.

2.2

Endophytes as a Biofertilizer:

Herbaspirillum seropedicae the first nitrogen-fixing bacterium

with endophytic characteristics was isolated in 1984 from theRhizhosphere, washed roots and surface
sterilized roots ofmaize, sorghum and rice plant and named asAzospirillum

seropedicae(Ba lda ni et al., 1984).Biological nitrogen fixation

in non-leguminous field crops: recent advances. By Kennedy,I.R., and Y.T. Tchan. 1992. Recent advances
in BNF with non-legume plants. By Baldani et al., Nitrogen fixation in endophyticand associative
symbiosis. By James, E. K. 1999 Field Crop Res.65:197-209. Infection and colonization of sugarcane and
othergraminaceous plants by endophytic diazotrophs. By Jameset

al., Study on influence of nitrogen fertilization on the population

of diazotrophic bacteriaHerbaspiril lum spp. andAcetobacter

diazotrophicus in sugar cane (Saccharum spp.) by Reis-Junior-

FB-dos; Reis-VM; Urquiaga-S; Dobereiner-Brazil (2000).Comparison of benefit to sugarcane plant growth

and15N2incorporation following inoculation of sterile plants withAcetobacter diazotrophicus wild type
and nifmutant strains.By Sevilla et al., Bacterial endophytes: potential role indeveloping sustainable
systems of crop production. By Sturz, A.V., B. R. Christie, and J. Nowak. 2000. In15N study by Lima et al.,

(1987) showed thatafter plant analysis it was revealed that50% of the plant N in cultivars CB-47-89 had
been derived fromthe atmosphere.Gillis et al., (1989) reported that within thegenusAcetoba cter, seven
species are described, however, untilnow, Acetobacter diazotrophicus was the only one able to fixthe
atmospheric nitrogen. Certain sugar cane varieties arecapable of obtaining large contribution of
nitrogen from plantassociated N2 fixation. It was estimated that up to 60 to 80 % ofplant N, equivalent
to over 200 kg n ha-1 /year, could bederived from this source, under good conditions of water
andmineral nutrient supply. R.M Boddey, et al., Brazil (1991).
ranging from 20°C to 50°C. The effect of temperature on growth was

recorded after 10 days of incubation period.

3.2.6.2 Optimumization Hydrogen ion concentration (pH)

for growth of E ndophyte isolates.

The response of Endophytes strains were studied atdifferent pH ranging from 3.5 to 6.5 by adjusting pH
with thehelp of 1N HCl or 1NaOH of respected broth N-free medium.After sterilization of broth the pH of
broth may change whichwas again checked and readjusted aseptically. The isolateswere inoculated
separately. The growth and change in pH was

observed after 5 days of incubation at 30°C.

3.2.6.3 Response of E ndophytes to various sucrose

concentrations

The respected broth containing various concentration of

sucrose, as 5 % to 40% were inoculated with Endophytes

and recorded the growth after 5 days of incubation at 30°C.

3.2.6.4 Utilization of different carbon compounds

The carbon requirement for growth of Endophytes(Azospirillum, Agrobacterium diazotrophicus,

Azoarcus) wasestimated by growing the organisms on 10% of different carbonsource in the medium viz.,
sucrose, fructose, D-glucose,maltose, mannitol, ethanol (1%) keeping other basiccomposition of medium
(basal medium) and opt. conditions

same for growth. The growth was recorded after 5 days

incubation at 30° C (Cavalcante and Dobereiner, 1988). The testtube containing 10 % sucrose with basal
medium serves ascontrol.

3.2.7 Media Designing:

Mass production was done with three isolates and two purestrains of Endophytes viz. Acetobacter
diazotrophicus andHerbaspirrillum & their broth cultures are provided by institutefor further study.

On considering above parameters with their results and bycomparing the selective media’s of three
isolates and two purestrain appropriate medium was designed for the massproduction of all the
endophytes as a Liquid Bioinoculant, andnamed asA4H media. It is used for mass production by scale
upof Fermentation technology and finally mass production byFermentation based Biotechnology.
3.2.7. Mass Production:

After formulation and designing a media the massproduction of Endophytes were carried. Mother
cultureprepared was further inoculated in newly designed media

A4H (250ml) & its scale up of fermentation up to 5 liter

was carried out further.

Mass production was carried out with 100 liters of A4Hmedia by fermentation-based biotechnology,
using 5-10%inoculums of scale up of fermentation.

3.2.8. Growth and Chemical analysis:

3.2.8.1.CHEMICAL ANALYSIS AFTER FORMULATIONS of

Broth:

I. Estimation of reducing sugar by DNSA method:

Reducing sugar estimation was carried out by DNSA reagent methodand the dilution system is given in
table 3.1 as the dilutions werecompleted optical density measured for calculating theconcentration of
reducing sugar present in the sample. Glucose was

used as standard sample (2000µ g/ml).

Table 3.1: Dilution scheme for reducing sugar by DNSA method

COMPOSITION OF MEDIA

1. Azosperillum medium (for 1 liter.) (pH- 6.6-7)

(ForAzos perillum)

Malic acid

- 5gm

K2HPO4

-4gm

FeSo4 x 7 H20

– 0.05gm

Na2 Mo04 x 2 H20

– 0.002gm

MnSo4 x H20

- 0.01gm

MgS04 x 7 H20

-0.10gm

Nacl

- o.o2gm

CaCl2 x 2 H20

- 0.01gm

Distil water

- 1000ml

Agar-Agar

- 30gm

2. Azoa rcus medium (for 1 liter)(pH- 6.6-7)

(ForAzoa rcus )
Malic acid

- 2-5gm

KOH

- 2-5gm

KH2PO4

- 1.5gm

MgS04 x 7 H20

-1gm

Nacl

-1gm.
Sodium Molybdate– 2mg.
CaCl2

-1gm

MnSo4 x H20

- 10mg

Fe EDTA

- 66mg

Biotin

- 1 mg

NH4Cl

- 2mg

Beef Extract

- 3gm

Yeast extract

- 1gm

Agar-Agar
- 15gm

Distil water

- 1000ml

3. Agrobacterium Diazotrophicusmedium (for 1 liter) (pH-5.5-

6.0) For (Agrobacterium Diazotrophicus)

Sucrose

- 100gm

Nacl

- 0.2 gm

MgS04

- 0.02 gm

CaCo3

- 1gm

Na MoO4

- 0.005gm

Agar

- 15gm

4.

LGI Medium (for 1 liter) (pH-5.5-6.0)

for Acetobacter Diazotrophicus.

Cane Sugar

– 100 gm

KH2PO4

- 0.6 gm

K2HpO4
- 0.2 gm

MgS04

- 0.02 gm
Sodium molybdate – 0.002gm
Ferric Chloride

- 0.01gm

CaCl2

- O.O2 gm

BTB

- 5ml

Yeast Extract

- 0.5 gm

Agar agar

- 30gm

D/W

- 1000ml.

5. Herbasperrillum medium (for 1 liter) (pH-7)

For Herbasperrillum

KH2PO4

- 0.400 gm

K2HpO4

- 0.100 gm

MgS04X 7 H20

- 0. 200 gm

Nacl

- 0.100gm
Cacl2

- 0.020 gm

Fecl2 X 6 H20

- 0.010 gm
Sodium Molybdate- 0.002 gm
Yeast Extract

- 0.025gm

D/W

- 950 ml.

Agar Agar

- 15 gm

utilization efficiency of liquid Bioinoculant by stem; leaves andplantlets average standard treatment,

foliar application anddipping in plantlets.

Date:

Mrs.Chaitali Niratker

(Major Advisor)
Bibliography

BIBLIOGRAPHY

Bellone, C. H., De Bellone, S. D.C. Padrase R, and Monson, M.

A. (1997).

Cell colonization and infection thread formation insugarcane roots by Acetobacter diazotrophicus.
Soil.Biol-Biochem, 29;965-967.

Baldani, V.L.D., Baldani, J.I., Olivers, F., Doberenier, J. 1992

Identification of Herbaspirillum seriopicae and the

closely related

Pseudomonas rubrisubalbicans.

Symbiosis; 13:65-73.

Baldani J. L, Baldani, V.L.D., Doberner J. 1986.

Characterization of Herbaspirillum seropedicae gen novsp. Nov:a root associated nitrogen fixing
bacterium. Int.J. Syst. Bacteriol; 36:86-93

Boddey R. M. 1993.

‘Green’ energy from sugarcane. Chem. And Ind.; 17 May

1993. pp 355-358.

Bellone, C. H., De Bellone, S. D.C. Padrase R, and Monson, M. A.(1997). Cell colonization and infection
thread formation