Azospirilum- Isolation & Marketing

This Presentation offers the isolation of Azospirrilum and its marketing for organic cultivation.

1. WELCOME
TO
MICROBIOLOGY

2. Azospirillum : isolation and mass
multiplication –carrier -based
inoculants, associative effect of
different microorganisms ..

4. MICROBIOLOGY
Presented by
Dr. N.Sannigrahi, Associate
Professor,
Department of Botany,
Nistarini College, Purulia (
W.B) 723101, India

5. SUSTAINABLE DEVELOPMENT & BIOFERTILIZERS
•Biofertilizersaregainingimportanceinsustainableagriculture.Various
complementingcombinationsofmicrobialinoculantsformanagementof
majornutrientssuchasnitrogenandphosphorusarenecessaryfor
sustainability.Abroadcanvasofbio-fertilizersthatenhancenitrogenand
specifictolegumesandnonlegumesalongwithinoculantsthatenhance
phosphorusnutritionarediscussedfromseveralperspectives.Themodeof
actionofthesemicroorganismswithinandthetransformationofnutrientsis
elucidated.IntheIndianscenario,useofbio-fertilizersfacesvarious
constraints,suchaslongevity,etc,needtobeovercometoachieve
substantialfertilizersavings.Themostpromisingagentstoensure
sustainabilityareasfollows:
•1.Lowcosttechnologywithhighcostbenefitratio,
•2.Sustainedimprovementofsoilfertility,
•3.Incresedcropyieldthroughtheincreaseofnutrientsavailability&soil
fertility,
•4.Reducethesoilsalinityissuesduetoinorganicfertilizers,
•5.Protecxtsplantsagainstsoilbornepathogens

6. INTRODUCTION
•Therearedifferenttypesofbiologicalagentsthatcanpromisetooffera
goodnumberofecologicalbenefitswithapromisetoensurecrop
productivitylikeAzospirillum.Thefollowingfeaturesensurethebacteria
asgoodagentofbiofertlizer.
•1.ItisaGram(-),microaerophilicnitrogenfixingbacterium,
•BelongstoRhodospirillaceaefirstdescribedbyBeijernick,1925,
•BesideNitrogenfixation,italsopromotesplantgrowthbyenhancingplant
growthhormone,henceitiscalledPlantGrowthPromotingRhizobacteria(
PGPR)orPGPB.
•Ithasbeenisolatedfromtherootsofrice,sugarcaneandothercereals,
•ItcanpromoteIAAsynthesis,
•Solubilizephosphateandenhanceabsorption,
•Bypromotingsiderophoresecretion,itenablesplanttoabsorbFe,
•Itoffersalotofecologicalbenefitsforthesakeofsoilsustainability

7. Azospirillum -Introduction
•Azospirillum,aubiquitousrhizospherebacterium,representingthemain
groupofmicroaerophilicfreeliving/associativenitrogenfixingbacteria
(DobereinerandDay,1976).
•Theyareisolatedfromtherhizosphereofmanygrassesandcerealsallover
worldandtheirrolesonplantgrowthandyieldhavebeenwellestablished.
•Twospeciesviz.,AzospirillumbrasilenseandAzosprilliumlipoferumhave
beenfoundinsoilofatemperatezone(Conincketal.,1988)andevenin
thecoldclimateofFinland(Haathellaetal.,1983).
•Azospirillumoccursinabout90%oftropicalsoilandinabout60%ofsoils
inthetemperatezone.A.brasilenseisattributedtohaveaffinitywithplants
withphotosynthesistypeC3(wheatandchilli),whereasA.lipoferumwith
plantsofC4type(MaizeandSorghum).N2fixationmaybeoneofthe
minormechanismsinvolvedinplantgrowthpromotionbyAzospirillum
•ThesuccessofAzospirilluminoculantsinpromotingplantgrowthwill
largelydependonitsmovementtowardsthehostplantbothinblacksoil
andredsoilintherhizosphere.Thestimulatoryeffectexertedbythe
secretionofphytohormones,biologicalnitrogenfixation,andenhancement
ofmineraluptakebyplants.

8. •Azospirrilumbeinggram(-)bacteriaplayasignificantroleintheecological
sustainabilitybyprovidinganumberofecologicalbenefitsinthisregard
provideditisbeingusedlargescaleinthedomainofagriculturalpractices.
Theisolationofthisbacteriaalongwiththemassproductionneedsthe
followingstagesforthesameasstatedbelow:
•1.Isolation&massmultiplicationofbacteria,
•2.Supplyofpropernutrientmediaandgrowthfactorenrichedwithsuitable
microbiologicalambience,
•3.Carrierbasedinoculantssupply,
•Associateeffectofthedifferentmicroorganism.
•Alltheaboveprocedurestobefollowedandexercisedforthelargescale
productionalongwithitspackagingandtransportationtoreachtothe
desiredgroupstoensuremaximumproductivityinthisregard.
•Thestepsarefollowedasstatedbelow.

9. AzospirrilumISOLATION

10. Azospirillum -Isolation
•Thestandardisolationprocedure,asreportedbyDobereinerandDay
(1976),wasfollowedforisolationofAzospirillumfromtuberoseroot
samples.
•Freshrootsampleswerecutintobitsof0.5cmlengthandthenwere
washedthoroughlyinrunningtapwaterandsurfacesterilizedbydippingin
0.1%HgCl2solutionforthreeminutesfollowedbydippingin70percent
alcoholforoneminute.
•Therootswerefinallywashedinsixtoeightchangesofdistilledwater.
•Therootbitswerethenplacedatsubsurfacelevelinscrewcaptubes
containingsterilizedNitrogen-freesemisolidmalatemedium(Okonetal.,
1977)underasepticconditions.
•TheNitrogenfreesemi-solidmalatemediumistobepreparedwiththe
desiredmicrobiologicalfeaturesrequiredforthesametomaintainthe
contaminationfreeculturewiththeintroductionofdifferentprerequisitesin
thiscondition.

11. Azospirillum -Isolation
•Thetubeswereincubatedat37oCforaperiodof4-5daysandobservedfor
growthofAzospirillumassubsurfacewhiteundulatingpellicles.The
isolateswerepurifiedbyrepeatedsubculturing.
•Aloopfulofculturewasstreakedonmalateagarplatescontaining1per
centNH4Cl.Afteraweekofincubation,typicalsmall,whitedensesingle
colonieswerepickedandtransferredtosemisolidNfreemalatemediumin
culturetubes.
•Theisolatesthatformedcharacteristicsubsurfacewhiteundulatingpellicle
inthismediumweretentativelyconsideredasAzospirillum
•Biochemicalcharacterization
•Thebiochemicaltestsviz.,utilizationofglucose,biotinrequirement,acid
productioninglucosepeptonebrothanddenitrificationtestswerecarried
outforidentificationoftheAzospirillumisolates.
•TheisolatesofAzospirillumweregrowninnitrogenfreemalatemedium
(nfb)for48hrsat28°C(±2)overanenvironmentalshaker(100rpm).

12. Azospirillum: Dobereiner's malic acid broth with NH4Cl (1g
per liter
•Malicacid-5.0g
•Potassiumhydroxide-4.0g
•Dipotassiumhydrogenorthophosphate-0.5g
•Magnesiumsulphate-0.2g
•Sodiumchloride-0.1g
•Calciumchloride-0.2g
•Fe-EDTA(1.64%w/vaqueous)-4.0ml
•Traceelementsolution-2.0ml
•BTB(0.5%alcoholicsolution)-2.0ml
•Agar-1.75g
•Distilledwater-1000mlPh-6.8
•Traceelementsolution
•Sodiummolybdate-200mg
•Manganoussulphate-235mg
•Boricacid-280mg
•Coppersulphate-8mg
•Zincsulphate-24mg
•Distilledwater-200m

13. Mass Multiplication of AzospirilumSp.
Prepareappropriatemediaforspecifictothebacterialinoculantsin250
ml,500ml,3literand5literconicalflasksandsterilize.
Themediain250mlflaskisinoculatedwithefficientbacterialstrain
underasepticcondition
Keeptheflaskunderroomtemperatureinrotaryshaker(200rpm)for5-
7days.
Observetheflaskforgrowthofthecultureandestimatethepopulation,
whichservesasthestarterculture.
Usingthestarterculture(atlogphase)inoculatethelargerflasks(500ml,
3litreand5litre)containingthemedia,afterobtaininggrowthineach
flask.
Themediapreparationisdonewithfollowupofallsortsofdesired
protocolsinthisregardtomaintaintheasepticandcontaminationfree
cultureforthefurtheranalysisandexploration.

14. Mass Multiplication of AzospirilumSp.
The above media is prepared in large quantities in fermentor, sterilized
well, cooled and kept it ready.
The media in the fermentor is inoculated with the log phase culture grown
in 5 litreflask. Usually 1 -2 % inoculumis sufficient, however inoculation
is done up to 5% depending on the growth of the culture in the larger
flasks.
The cells are grown in fermentor by providing aeration (passing sterile air
through compressor and sterilizing agents like glass wool, cotton wool, acid
etc.) and given continuous stirring.
The broth is checked for the population of inoculated organism and
contamination if any at the growth period.
The cells are harvested with the population load of

15. Mass Multiplication of AzospirilumSp.
109cellsml-1afterincubationperiod.
Thereshouldnotbeanyfungaloranyotherbacterialcontaminationat10-
6dilutionlevel
Itisnotadvisabletostorethebrothafterfermentationforperiodslonger
than24hours.Evenat4oCnumberofviablecellsbeginstodecrease.
ProcessingofcarriermaterialTheuseofidealcarriermaterialisnecessary
intheproductionofgoodqualitybiofertilizer.Peatsoil,lignite,vermiculite,
charcoal,pressmud,farmyardmanureandsoilmixturecanbeusedas
carriermaterials.Theneutralizedpeatsoil/lignitearefoundtobebetter
carriermaterialsforbiofertilizerproduction.
Thefollowingpointsaretobeconsideredintheselectionofidealcarrier
material.
Cheaperincost
Shouldbelocallyavailable
Highorganicmattercontent
Notoxicchemicals•Waterholdingcapacityofmorethan50%•Easyto
process,friabilityandvulnerability

16. Mass Multiplication of AzospirilumSp.
Thecarriermaterial(peatorlignite)ispowderedtoafinepowdersoasto
passthrough212micronISsieve.•ThepHofthecarriermaterialis
neutralizedwiththehelpofcalciumcarbonate(1:10ratio),sincethepeat
soil/ligniteareacidicinnature(pHof4-5)•Theneutralizedcarrier
materialissterilizedinanautoclavetoeliminatethecontaminants.
PreparationofInoculantspacket•
Theneutralized,sterilizedcarriermaterialisspreadinaclean,dry,sterile
metallicorplastictray.
Thebacterialculturedrawnfromthefermentorisaddedtothesterilized
carrierandmixedwellbymanual(bywearingsterilegloves)orby
mechanicalmixer.Theculturesuspensionistobeaddedtoalevelof40–
50%waterholdingcapacitydependinguponthepopulation.
Theinoculantspacketof200gquantitiesinpolythenebags,sealedwith
electricsealerandallowedforcuringfor2-3daysatroomtemperature(
curingcanbedonebyspreadingtheinoculantsonacleanfloor/polythene
sheet/bykeepinginopenshallowtubs/trayswithpolythenecoveringfor2
-3daysatroomtemperaturebeforepackaging.

17. Marketing of AzospirilumSp.
Schematicrepresentationofmassproductionofbacterialbiofertilizers
Specificationofthepolythenebags
Thepolythenebagsshouldbeoflowdensitygrade.
Thethicknessofthebagshouldbearound50–75micron.
Eachpacketshouldbemarkedwiththenameofthemanufacturer,nameof
theproduct,strainnumber,thecroptowhichrecommended,methodof
inoculation,dateofmanufacture,batchnumber,dateofexpiry,price,full
addressofthemanufacturerandstorageinstructionsetc.,Storageof
biofertilizerpacket
Thepacketshouldbestoredinacoolplaceawayfromtheheatordirect
sunlight.
Thepacketsmaybestoredatroomtemperatureorincoldstorage
conditionsinlotsinplasticcratesorpolythene/gunnybags.•The
populationofinoculantsinthecarrierinoculantspacketmaybedetermined
at15daysinterval.Thereshouldbemorethan109cells/gofinoculantsat
thetimeofpreparationand107cells/gondryweightbasisbeforeexpiry
date.

18. Effects of Azetobacter in soil fertility & Sustainability
•Thebestalternativeofchemicalfertilizerisnecessarybecauseofits
adverseeffectsonthesoilhealth.Thereareseveralalternativesavailableto
enhancethesoilfertilityoneofthemisAzotobacter.
•Itisafree–livingN
2–fixerdiazotrophthathasseveralbeneficialeffectson
thecropgrowthandyield.
•Ithelpsinsynthesisofgrowthregulatingsubstanceslikeauxins,cytokinin
andGiberellicAcid(GA).
•Inaddition,itstimulatesrhizosphericmicrobes,protectstheplantsfrom
phyto–pathogens,improvesnutrientuptakeandultimatelyboostup
biologicalnitrogenfixation.
•Theabundanceofthesebacteriainsoilisrelatedtomanyfactors,mostly
soilpHandfertility.
•Drymatteraccumulationincrease,
•Increaseofleafareaindex,
•BiochemicaleffectsduetomoreuptakeofN,P,K,alongwithprotein
contentandchlorophyllcontentincomparsiontonon-inoculatedplantsby
Azotobacter.

19. Effects of Azetobacter in soil fertility & Sustainability
•Anti-pathogenicResponse:Azotobacterspp.arecapabletoproduce
siderophore,theybindtotheavailableformofironFe+3inthe
rhizosphere,thusmakingitunavailabletothephyto-pathogensand
protectingtheplanthealth;similarlyHydroCyanine(HCN)productionwas
higherintraitsofAzotobacter(77.00%).Azotobactersecretesanantibiotic
withastructuresimilartoanisomycin,whichisadocumentedfungicidal
antibiotic.Azotobacter,insufficientnumbers,willout-competepathogens
forfood.SomeofthepathogensthathavebeencontrolledbyAzotobacterin
thesoilandontheleafinclude:Alternaria,Fusarium,Collectotrichum,
Rhyzoctonia,Microfomina,Diplodia,Batryiodiplodia,Cephalosporium,
Curvularia,HelminthosporiumandAspergillus.
•EffectsofchemicalfertilizerinAzotobacter
•Combinedapplicationofbio-fertilizerwith50%ofchemicalfertilizers(N
andP)hassignificanteffectinplantgrowth,plantheight,numberof
branches,freshanddryweightofsafflowerincomparisonwithchemical
fertilizersalone.Similarly,applicationofAzotobacterbio-phosphateand
organicfertilizers,withhalfdoseofchemicalfertilizersincreasethe
economicyieldofsafflower.

20. Azetobacter-Isolation & Mass Multiplication
•Thesoilsampleswerecollectedfromvariousfieldsandserialdilutions
weredone.Theorganismwereisolatedbytheanalysisofthe
characteristicsaccordingtotheMorphologicalandBiochemical
characteristics.Thevariousbio-chemicaltestsconductedwerecitrate
utilization,catalase,urease,indole,methylred,voguesprokauer,H2Sand
nitratereductiontestwereperformedandconfirmed.Thenusingthe
specificmediumPikovskayaísmediumforphosphobacterandAshbinagar
forAzotobacterwereusedtogrowtheorganismforthemassproduction.
•FormassproductionofAzotobacter,bacterialstrainisisolatedfrom
variousregionsandgrownonslantsforpreservationasperneedculture
fromslantweretransferredtoliquidbrothofselectiveaswellasoptimized
mediumintherotaryshakerfor4daystopreparestarterculture.Lateron
thestarterculturesistransferredtothefermenterinbatchculturemode
withpropermaintenanceof300Candcontinuousagitationfor4-9days

21. Azetobacter-Isolation & Mass Multiplication
•whencellcountreachedto108-109cells/ml,thebrothusedasinoculants.
Foreasyhandling,packing,storingandtransportingbrothismixedwithan
inertcarriermaterialwhichcontainssufficientamountofcells.Inpresent
studybrothismixedwithunsterilesoil:Activatedcharcoal,A.R.(RM
1332):CaCo3inaratioof1:2:1whereasothersetpreparedbyusing
unsterilesoil:crudecoalpowder:CaCo3insameratiooverthecarrierin
suchawaythat40%moistureismaintained.Afterpropermixingcarrier
containinginoculantswasleftfor7daysandaboveformulatedmicrobial
inoculantsusedasbiofertilizer.

22. ASSOCIAED EFFECT OF DIFFERENT MICROORGANISMS
•Azospirillumextendsthefollowingbenefitsinthedomainofsustainable
agricultureaswellasorganicfarming:
•1.Ithelpsactasfreelivingbacteriatofixatmosphericnitrogen,
•2.A.brasilensereducednitrateandfreenitrogentofacilitatenitrogenfixing,
•3.Itextendstherootsystemelongationandenlargement,
•4.Itfacilitatestheabsorptionofmineralsalts,
•5.Itincreasethetoleranceingeneralandstresstoleranceinparticular
especiallyofsomecerealslikemaize,sorghumetc.
•6.Itextendsthemultipleactionwiththecombinationofothermicrobial
activitiesasstatedbelow:
•a.Azotobacterdiazotrophicus,HerbaspirillumseropedicaeandAzocarusalong
withAzoteotobacterincombinationwithAzospirrilumfoxmoreatmospheric
nitrogeninthesoilincumulativeamountthantheAzotobactoralone.
•b.Aspergillusniger,AawamorialongwithAzospirillumlipoferumjointly
increasestheweightofthewheatgrains.Theincreaseofthehuskproduction
andtheincreaseofnitrogencontentintherhizosphere.

23. THANKS FOR YOUR VISIT
•References:
•1. Fundamental Botany-Sen & Giri
•2. A text of Fungi-Vasistha,
•3. A Textbook of Microbiology-R.P. Singh,
•4.Textbook of Microbiology-Dubey & Maheswari
•5. Soil Microbiology-N.S. Subba Rao
•6. Agricultural Microbiology-G. Rangaswami
•7. Google for images
•8. Different WebPages for information.
•Disclaimer:ThisPPThasbeenmadetoenrichfreeonlinestudy
resourceswithoutanypleasureoffinancialinterest.