CHAPTERONE

1.0 INTRODUCTION
Cassava(ManihotesculentaCrantz,synonymouswithManihotutilissimaRhol)belongsto
thefamilyEuphorbiaceae.Itismainlyafoodcropwhosetubersareharvestedbetween713months
basedonthecultivarsplanted(Cook,1985Taye,1994).Cassava(ManihotesculentaCrantz)is
primarilygrownforitsstarchcontainingtuberousroots,whicharethemajorsourceofdietaryenergyfor
morethan500millionpeopleinthetropics(Lyman,1993).Theabilityofcassavatogrowandproduce
relativelywellinmarginalenvironmentunderlowmanagementlevelsmakesitanattractivecropforpoor
resource(Bencini,1991).Asafoodcrop,cassavafitswellintothefarmingsystemsofthesmallholder
farmersinNigeriabecauseitisavailableyearround,thusprovidinghouseholdfoodsecurity.Cassava
tuberscanbekeptinthegroundpriortoharvestingforuptotwoyears,butonceharvested,theybegin
todeteriorate.Toforestallearlydeterioration,andalsoduetoitsbulkynature,cassavaisusuallytraded
insomeprocessedform.Thebulkyrootscontainmuchmoisture(6065%),makingtheir
transportationfromruralareasdifficultandexpensive.Processingthetubersintoadryformreduces
themoisturecontentandconvertsitintoamoredurableandstableproductwithlessvolume,which
makesitmoretransportable(IITA,1990Ugwu,1996).Overtheyears,cassavahasbeentransformed
intoanumberofproductsbothfordomestic(dependingonlocalcustomsandpreferences)and
industrialuses.
Cassavainthefreshformcontainscyanide,whichisextremelytoxictohumansandanimalsthereis
thereforeaneedtoprocessittoreducethecyanidecontenttosafelevels(Eggelstonetal.,1992).

1

Thepoorpostharveststoragelifeoffreshcassavatubersisamajoreconomicconstraintinitsutilization
(Kehinde,2006).
Cassavaprocessinggeneratessolidandliquidresiduesthatarehazardousintheenvironment
(Cumbanaetal.,2007Jyothietal.,2005).Ontheaverage,2.62m
3
ton1ofresiduesfromwashing
and3.68m
3
ton1fromthewaterresiduesofflourproduction(Horsfalletal.,2006andIsabiryeet
al.,2007).Therearetwoimportantbiologicalwastesderivedfromcassavaprocessingwhicharethe
cassavapeelsandtheliquidsqueezedoutofthefermentedparenchymamash(Oboh,2006).
Cassavaeffluentsareliquidwastesfromthecassavamillwhichareusuallydischargedonlandorwater
inanunplannedmanner.Thecassavapeelsderivedfromitsprocessingarenormallydischargedas
wastesandallowedtorotintheopenwithasmallportionusedasanimalfeed,thusresultinginhealth
andenvironmentalhazards.
(DesseandTaye,2001AderiyeandLaleye,2003)theedibletubersareprocessedinto
variousformswhichincludechips,pellets,cakesandflour.Theflourcouldbefriedtoproducegarior
steepedinwatertofermenttoproducefufuwhencooked(OyewoleandOdunfa,1992).The
productionandconsequentconsumptionofcassavahaveincreasedextensivelyinrecenttimes.This
increasedutilizationofprocessedcassavaproductshasequallyincreasedtheenvironmentalpollution
associatedwiththedisposaloftheeffluents(Akanietal.,2006Adewoyeetal.,2005)
Inmostareas,cassavamillsaremainlyonsmallscalebasis,ownedandmanagedbyindividuals
whohavenobasicknowledgeofenvironmentalprotection.Thoughonsmallscalebasis,thereare
manyofthem,whichwhenputtogether,createenormousimpactontheenvironment.
2

Oboh,2005identifiedtwoimportantwastesthataregeneratedduringtheprocessingofcassava
tuberstoincludecassavapeelsandliquidsqueezedoutofthemash.Thebioconversionofthecassava
wasteshavebeendocumented(AntiaandMbongo,1994Okafor,1998Raimbault,1998
TwenyongyereandKatongole,2002Oboh,2005).Thewastewatercontainsheavyloadsof
microorganisms,lacticacid,lysine,amylasecapableofhydrolyzingtheglycosides(Raimbault,1998
Akindahunsietal.,1999).
Duringtheprocessingofcassavatubersinvariousproducts,liquidwastewatersgeneratedwas
reportedtocauseserioushavoctovegetation,housesandbringaboutinfection.Theliquidsqueezedout
canbedriedandusedasanimalfeeds(Okafor,1998ObohandAkindahunsi,2003a).
Microorganismsareveryimportantmembersofthesoilecosystem.Theyplaysignificantroles
inthevarioustransformationsthatgooninthesoil.Animportantfunctionofsoilorganismsisthe
decompositionoforganicresidues.Thisdecompositionprocessisdrivenbydecomposerorganisms
whichconsistofacommunityofsoilbiotaincludingmicrofloraandsoilfauna(Swiftetal.,1979Tianet
al.,1995).Fungiandbacteriaareresponsibleforthebiochemicalprocessesinthedecompositionof
organicresidues(AndersonandIneson,1983Dinda,1978).
Despitetheirimportanceinsoil,therelativeabundanceanddistributionofthesesoilorganismsis
determinedbyseveralenvironmentalfactors.Thesoilisthefinalrecipientofallformsofenvironmental
pollutantsandofrecentsuchpollutantshavehadsignificanteffectsonsoilmicrobialpopulations
(Ogboghodoetal.,2001).Variousstudiesofthemicrobiologyofhydrocationdegradationinsoil
indicatethepresenceofmicroflorainthesoilthatisabletodegradeawidevarietyofhydrocarbons
(Niessen,1970).
3

Soonafterthewidespreaduseofantibioticsbeganintheearly1950sitbecameapparentthat
strainsofbacteriawerebecomingresistanttospecificantibiotics,itwaslaterdiscoveredinEuropethat
enterobacteriaceaecantransfermultipleresistancesfromoneorganismtoanotherandevenfromone
specietoanotherbymeansofanextrachromosomalhereditaryfactors.Theproblemofantibiotic
resistancecouldbeattributedtolongtimeuseofaparticularantibioticbyhumans,thisthenmakes
bacteriatoadaptverywellwithwhateverchallengestheantibioticsmightposeandsubsequentlyitwill
becomeresistanttosuchantibiotics.
1.1 STATEMENTOFTHEPROBLEM
Cassava is important to human because they serve as a source of staple food for him and his
animals, it could be employed to produce chips, gari, fufu etc. it is also a source of viable income for
farmers who plant it and also to people involved in turning it into finished product e.g. the gari
producers, the transport drivers. But as beneficial as cassava is to humans, its effluent has always
constituted a source of nuisance as well as the odour emanating from gari processing plants are always
offensive.
Duetoeffluentsproducedintheenvironment,studieshavebeendonetofindoutthe
microorganismthatcanbeeitherpathogenicornonpathogenicthatarepresentwithinthecassavamill
factories

4

1.2 AIMSANDOBJECTIVES
Toisolate,characterizeandcarryoutantimicrobialsusceptibilitytestonsoilmicroorganisms
presentwithinthecassavamillindustry.

CHAPTERTWO
2.1 LITERATUREREVIEW
Cassava(ManihotesculentaCrantz,synonymouswithManihotutilissimaRhol)belongsto
thefamilyEuphorbiaceae.Itismainlyafoodcropwhosetubersareharvestedbetween713months
basedonthecultivarsplanted(Cook,1985Taye,1994).Thetubersarequiterichincarbohydrates
(8590%)withverysmallamountofprotein(1.3%)inadditiontocyanogenicgloucoside(Linamarin
andLotaustiallin).(NwabuezeandOdunsi,2007OyewoleandAfolami,2001).Thishighcarbohydrate
contentmakescassavaamajorfooditemespeciallyforthelowincomeearnersinmosttropical
countriesespeciallyAfricaandAsia(DesseandTaye,2001AderiyeandLaleye,2003).
Theedibletubersareprocessedintovariousformswhichincludechips,pellets,cakesandflour.
Theflourcouldbefriedtoproducegariorsteepedinwatertofermenttoproducefufuwhencooked
(OyewoleandOdunfa,1992).
Fermentationisoneoftheoldestandmostimportanttraditionalfoodprocessingand
preservationtechniques.Foodfermentationsinvolvetheuseofmicroorganismsandenzymesforthe
productionoffoodswithdistinctqualityattributesthatarequitedifferentfromtheoriginal
agriculturalrawmaterial.Theconversionofcassava(Manihotesculenta,Crantzsyn.Manihot
utilissimaPohl)togariillustratestheimportanceoftraditionalfermentations.
CassavaisnativetoSouthAmericabutwasintroducedtoWestAfricainthelate16thcentury
whereitisnowanimportantstapleinNigeria,Ghana,IvoryCoast,SierraLeone,Liberia,Guinea,
SenegalandCameroon.Nigeriaisoneoftheleadingproducersofcassavaintheworldwithanannual
6

productionof3540millionmetrictons.Over40varietiesofcassavaaregrowninNigeriaandcassava
isthemostimportantdietarystapleinthecountryaccountingforover20%ofallfoodcropsconsumed
inNigeria.Cassavatubersarerichinstarch(2030%)and,withthepossibleexceptionofsugarcane
cassavaisconsideredthehighestproducerofcarbohydratesamongcropplants.
Despiteitsvastpotential,thepresenceoftwocyanogenicglucosides,Linamarin(accountingfor93%of
thetotalcontent)andlotaustralinormethylLinamarin,thatonhydrolysisbytheenzymelinamarase
releasetoxicHCN,isthemostimportantproblemlimitingcassavautilization.Generallycassava
contains10500mgHCN/kgofrootdependingonthevariety,althoughmuchhigherlevels,exceeding
1000mgHCN/kgmaybepresentinunusualcases.Cassavavarietiesarefrequentlydescribedassweet
orbitter.Sweetcassavavarietiesarelowincyanogenswithmostofthecyanogenspresentinthepeels.
Bittercassavavarietiesarehighincyanogensthattendtobeevenlydistributedthroughouttheroots.
Environmental(soil,moisture,temperature)andotherfactorsalsoinfluencethecyanidecontent
ofcassava(Bokangaetal1994).Lowrainfallordroughtincreasescyanidelevelsincassavarootsdue
towaterstressontheplant.Apartfromacutetoxicitythatmayresultindeath,consumptionofsublethal
dosesofcyanidefromcassavaproductsoverlongperiodsoftimeresultsinchroniccyanidetoxicitythat
increasestheprevalenceofgoiterandcretinisminiodinedeficientareas.Symptomsofcyanide
poisoningfromconsumptionofcassavawithhighlevelsofcyanogensincludevomiting,stomachpains,
dizziness,headache,weaknessanddiarrhea(Akintonwaetal1994).
Chroniccyanidetoxicityisalsoassociatedwithseveralpathologicalconditionsincludingkonzo,
anirreversibleparalysisofthelegsreportedineastern,centralandsouthernAfrica(Howlettand
Konzo,1994),andtropicalataxicneuropathy,reportedinWestAfrica,characterizedbylesionsofthe
7

skin,mucousmembranes,opticandauditorynerves,spinalcordandperipheralnervesandother
symptoms(Oshuntokun,1994).Withoutthebenefitsofmodernscience,aprocessfordetoxifying
cassavarootsbyconvertingpotentiallytoxicrootsintogariwasdeveloped,presumablyempirically,in
WestAfrica.Theprocessinvolvesfermentingcassavapulpfrompeeled,gratedrootsinclothbagsand
afterdewatering,themashissiftedandfried.
Microbialfermentationshavetraditionallyplayedimportantrolesinfoodprocessingfor
thousandsofyears.Mostmarketedcassavaproductslikegari,fufu,pupuru,apuetc.,inAfrica
areobtainedthroughfermentation.Theimportanceoffermentationincassavaprocessingisbasedonits
abilitytoreducethecyanogenicglucosidestorelativelyinsignificantlevels.Unlikealcoholicfermentation,
thebiochemistryandmicrobiologyisonlysuperficiallyunderstood,butitisbelievedthatsome
cyanidrophilic/cyanidetolerantmicroorganismseffectbreakdownofthecyanogenicglucoside.Ithas
beenshownthatthehighertheretentionofstarchingratedcassavathebetterthedetoxificationprocess.
Thiscouldbeattributedtothefermentativesubstrateprovidedbythestarch.Also,thelongerthe
fermentationprocessthelowertheresidualcyanidecontent.
Generally,fermentedcassavaproductsstorebetterandoftenarelowinresidualcyanide
content.(Onabowale,1988)developedacombinedacidhydrolysisandfermentationprocessatFIIRO
(FederalInstituteforIndustrialResearch,Oshodi,Nigeria)andachieveda98%(approx.)reductionin
totalcyanideafterdehydrationofthecassavaflourforuseinthefeedingofchickens.
Cassavarootscanbeindustriallyappliedforobtainingstarchandflour.However,cassavaindustries
generatesomeundesirablesubproducts,suchassolidresiduesandaliquideffluentnamedmanipueira,
whichmayrepresentamajordisposalproblemduetothehighorganicchargeandtoxicpotential,
8

resultingfromthepresenceofcyanoglucosides.Manipueiraisrichinpotassium,nitrogen,
magnesium,phosphorous,calcium,sulfurandiron,presentingagreatpotentialasanagronomic
fertilizer.Itcontainscyanoglucosides,whichexplainstheapplicationasnematicideandinsecticide
(Palmisanoetal.,2001).
Cyanoglucosidesaresecondarymetabolitesproducedbyseveralplantspecies(Conn,1994)
usedinanimalandhumandiets,suchas:apple,bambooshoot,cassava,cherry,limabean,
maize,oat,peach,papaya,sorghumandwheat(Muro,1989).Thesecompoundsaredispersed
throughouttheplantorgans,mostlyinnonedibleparts(Jones,1998),butmaybecomeconcentratedin
ediblerootsandleaves,asinthecaseofcassava.Cassava(ManihotesculentaCrantz)rootsand
leavescontainhighconcentrationsofLinamarin(alphahydroxyisobutyronitrilebetaDglucopyranoside)
andLotaustiallin(methylLinamarin).Linamarinisthemostabundantcyanoglucosidespresentincassava
cells(Conn,1973)andmaygeneratetheequivalentto0.2100mgofHCNper100goffreshcassava
followingcellularlyses(Bradburyetal.,1991).Thecassavaeffluenthasbeenfoundtoincreasethe
numberoforganismsinthesoilecosystemwhichmaybeassociatedwithincreaseinthesoilpH,
organiccarbonandtotalnitrogen(Ogboghodoetal.,2001).

WASTEMANAGEMENTINCASSAVASTARCHFACTORIES
Wastefromcassavaprocessingmaybesolidorliquid.Thebrownpeelofcassavaroots,
knownasperiderm,variesbetween25%oftheroottotal.Thesolidwasteismadeupoffibrousroot
materialsandcontainsstarchthatphysicallycouldnotbeextracted.Theprocessofstarchextraction
fromcassavarequireslargequantityofwaterresultinginthereleaseofasignificantquantityofeffluents
9

(BalagopalanandRajalakshmy,1998).Itiscommonforfactoriestodischargetheeffluentsintothe
nearbyrivers,drainagechannels,cropfieldsortothelandadjacentthefactories.Theseeffluentsposea
seriousthreattotheenvironmentandqualityoflifeinruralareas.
Widevariationswereobservedinphysicalandchemicalconstituentsofprimaryandsecondary
effluentsfromcassavastarchfactories.(Manilaletal.,1991)observedthatthechemicaloxidation
demand(COD)rangedbetween33,600&38,223mgl
1
intheprimaryeffluents,whereasinthe
secondaryeffluents,therangewasonly38009050mgl
1
.
Thebiologicaloxidationdemand(BOD)wasintherangeof13,20014,300mgl
1
intheprimary
effluents.Thecorrespondingfiguresforthesecondaryeffluentswere3,6007,050mgl
1
.Theacidityof
theeffluentrangedbetweenpH4.5&4.7.Nitrogenandphosphorusarethemainnutrientscontributing
tothestabilityoforganicwasteandtheanalysisrevealedlownitrogencontentindicatingnecessityfor
theenrichmentoftheeffluenttoreducetheBODandCOD(Manilaletal.,1991).
BalagopalanandRajalakshmy,1998observedthattheconcentrationoftotalcyanoglucosidesinthe
effluentsrangedbetween12.9mgl
1
&16.6mgl
1
inthecaseofinitialsamples,whereasinthecaseof
finalwastesamples,theconcentrationrangedbetween10.4mgl
1
&27.4mgl
1.
Ahighconcentrationof
cyanidewasobservedinthegroundwatersourceneartheprocessingfactoriesrangingbetween
1.2mgl
1
&1.6mgl
1
.Initialsettling,anaerobiosis,filtrationthroughsand&charcoalandaerationcan
reducethepollutionloadtothedesiredlevel(Balagopalanetal.,1994)

10

Microorganismscangrowonsubstratescontainingcyanidesbyanaerobicmetabolism,orby
usinganaerobicrespirationchainasanalternativepathway(Ceredaetal.,1991).Inbothpathways,
HCNiseliminatedfromthesubstrate,andconvertedintoanontoxicproduct(Jensenetal.,1979).
Thisenzymaticcyanideremovingpropertycanbeexploitedforthedetoxificationofcyaniderich
cassavawastewaterandindustrialresidues.Theseresiduescurrentlycauseseriousenvironmental
problemsinmanycassavaflourproducingplantsinBrazil,thelargestproducerworldwide,andinmany
African,LatinAmericanandAsiancountries(Romeroetal.,2002),wherecassavaproductsare
animportantinputforhumandiet.
BACTERIA
Bacteriaaresinglecellorganismsandthemostnumerousdenizensofagriculture,with
populationsrangingfrom100millionto3billioninagram.Theyarecapableofveryrapidreproduction
bybinaryfission(dividingintotwo)infavorableconditions.Onebacteriumiscapableofproducing16
millionmoreinjust24hours.Mostsoilbacterialiveclosetoplantrootsandareoftenreferredtoas
rhizobacteria.Bacterialiveinsoilwater,includingthefilmofmoisturesurroundingsoilparticles,and
someareabletoswimbymeansofflagella.Themajorityofthebeneficialsoildwellingbacterianeed
oxygen(andarethustermedaerobicbacteria),whilstthosethatdonotrequireairarereferredtoas
anaerobic,andtendtocauseputrefactionofdeadorganicmatter.
Aerobicbacteriaaremostactiveinasoilthatismoist(butnotsaturated,asthiswilldepriveaerobic
bacteriaoftheairthattheyrequire),andneutralsoilpH,andwherethereisplentyoffood
(carbohydratesandmicronutrientsfromorganicmatter)available.Hostileconditionswillnotcompletely
11

killbacteriarather,thebacteriawillstopgrowingandgetintoadormantstage,andthoseindividuals
withproadaptivemutationsmaycompetebetterinthenewconditions.
FUNGI
Fungiaremicroscopiccellsthatusuallygrowaslongthreadsorstrandscalledhyphae,which
pushtheirwaybetweensoilparticles,roots,androcks.Hyphaeareusuallyonlyseveralthousandthsof
aninch(afewmicrometers)indiameter.Singlehyphaecanspaninlengthfromafewcellstomany
yards.Hyphaesometimesgroupintomassescalledmyceliumorthick,cordlikerhizomorphsthat
looklikeroots.
Fungiperformimportantservicesrelatedtowaterdynamics,nutrientcycling,anddiseasesuppression.
Alongwithbacteria,fungiareimportantasdecomposersinthesoilfoodweb.Theyconvert
hardtodigestorganicmaterialintoformsthatotherorganismscanuse.Fungalhyphaephysicallybind
soilparticlestogether,creatingstableaggregatesthathelpincreasewaterinfiltrationandsoilwater
holdingcapacity.
Soilfungicanbegroupedintothreegeneralfunctionalgroupsbasedonhowtheygettheirenergy.
Decomposerssaprophyticfungiconvertdeadorganicmaterialintofungalbiomass,carbon
dioxide(CO
2
),andsmallmolecules,suchasorganicacids.Thesefungigenerallyusecomplex
substrates,suchasthecelluloseandlignin,inwood,andareessentialindecomposingthe
carbonringstructuresinsomepollutants.Afewfungiarecalledsugarfungibecausetheyuse
thesamesimplesubstratesasdomanybacteria.Likebacteria,fungiareimportantfor
immobilizing,orretaining,nutrientsinthesoil.Inaddition,manyofthesecondarymetabolitesof
12

fungiareorganicacids,sotheyhelpincreasetheaccumulationofhumicacidrichorganicmatter
thatisresistanttodegradationandmaystayinthesoilforhundredsofyears.
Mutualiststhemycorrhizalfungicolonizeplantroots.Inexchangeforcarbonfromthe
plant,mycorrhizalfungihelpsolubolizephosphorusandbringsoilnutrients(phosphorus,
nitrogen,micronutrients,andperhapswater)totheplant.Onemajorgroupofmycorrhizae,the
ectomycorrhizae(seethirdphotobelow),growsonthesurfacelayersoftherootsandare
commonlyassociatedwithtrees.Thesecondmajorgroupofmycorrhizaeisthe
endomycorrhizaethatgrowwithintherootcellsandarecommonlyassociatedwithgrasses,
rowcrops,vegetables,andshrubs.Arbuscularmycorrhizal(AM)fungiareatypeof
endomycorrhizalfungi(seefourthphotobelow).Ericoidmycorrhizalfungicanbyeitherectoor
endomycorrhizal.
Thethirdgroupoffungi,pathogensorparasites,causereducedproductionordeathwhen
theycolonizerootsandotherorganisms.Rootpathogenicfungi,suchasVerticillium,Pythium,
andRhizoctonia,causemajoreconomiclossesinagricultureeachyear.Manyfungihelpcontrol
diseases.Forexample,nematodetrappingfungithatparasitizediseasecausingnematodes,and
fungithatfeedoninsectsmaybeusefulasbiocontrolagents.

2.2 ANTIBIOTICS
Thecontrolofmicroorganismiscriticalforthepreventionandtreatmentofdiseases.Modern
medicineisdependentonchemotherapeuticagents,chemicalagentsthatareusedtotreatinfections.
13

Mostoftheseagentsareantibiotics,microbialproductsortheirderivativethatcankillsusceptible
microorganismorinhibittheirgrowth.Somebacteriaandfungiareabletonaturallyproducemanyofthe
commonlyemployedantibiotics.Incontrast,severalimportantchemotherapeuticagentssuchas
sulfonamides,trimethoprim,chloramphenicol,ciprofloxacinanddapsonesaresyntheticwhileincreasing
numberofantibioticsaresemisynthetic.
Antibioticsvaryintheireffectiveness,manyarenarrowspectrumdrugsthatistheyare
effectiveonlyagainstalimitedvarietyofpathogens.Othersarebroadspectrumdrugstheyareable
toattackmanydifferentkindsofpathogens.
Drugsmayalsobeclassifiedbasedonthegeneralmicrobialgrouptheyactagainst:
antibacterial,antifungal,antiprotozoan,andantiviral.Someantibioticscanbecidalorstaticinaction.
Staticagentsreversiblyinhibitgrowth,iftheagentisremoved,themicroorganismwillrecoverandgrow
again.Althoughacidalagentkillsthetargetpathogen,itsactivityisconcentrationdependentandthe
agentmayonlybestaticatlowlevels.
2.2.1 CLASSIFICATIONOFANTIBIOTICS
Therearemanyclassesofantibioticsavailabletomodernmedicinetoday,classificationmaybe
basedonrouteofadministration,andmodeofaction(staticorcidal)etc.mostcommonlyusedgroups
ofantibioticsisthe:Penicillins,Cephalosporins,Aminoglycosides,Macrolides,Quinolonesand
fluoroquinolonesetc.
Penicillin
14

Penicilliniscidalinitsmodeofaction,anarrowspectrumantibioticthatfunctionstoinhibit
transpeptidizationenzymeinvolvedincrosslinkingthepolysaccharidechainsofthebacterialcellwall
peptidoglycan.Penicillinisusedtotreatskininfections,urinarytractinfectionsgonorrheaetc.examples
includePenicillinG,V,methicillin.
Cephalosporin
Cephalosporinsareafamilyofantibioticsoriginallyisolatedin1948fromthefungus
Cephalosporium.Theycontainalactanstructurethatissimilartothatofpenicillin.
Cephalosporinisalsocidalinaction,itisabroadspectrumantibioticthatfunctionstoinhibit
transpeptidizationenzymeinvolvedincrosslinkingthepolysaccharidechainsofthebacterialcellwall
peptidoglycan.Cephalosporinisusedtotreatpneumonia,strepthroat,staphylococcusinfectionvarious
skininfectionetc.examplesincludeCephalothin,Cefoxitin,Ceftriaxone.

Aminoglycosides
Theyarealsocidalinaction,abroadspectrumantibioticthatactsbybindingtosmallribosomal
subunits(30S)andinterferewithproteinsynthesisbydirectlyinhibitingsynthesisandcausingmisreading
ofmRNA.Aminoglycosidesaregivenforashorttimeperiodsandareinjectedintravenouslyratherthan
orallybecausetheyareeasilybrokendowninthestomach.ExamplesincludeNeomycin,Gentamicin,
andStreptomycin.
Macrolides
15

TheseantibioticsarederivedfromStreptomycinbacteria.Theyarebacteriostaticanda
broadspectrumantibiotic,bindingto23SrRNAoflargeribosomalsubunit(50S)toinhibitpeptide
chainelongationduringproteinsynthesis.Theyareusedtotreatgastrointestinalupset,respiratorytract
infectionetc.examplesincludeErythromycin,Clindamycin.
Erythromycinisarelativelybroadspectrumantibioticeffectiveagainstgrampositivebacteria,
mycoplasmasandafewgramnegativebacteria.
Trimethoprim
Trimethoprimisasyntheticantibioticthatalsointerfereswiththeproductionoffolicacid.Itdoessoby
bindingtodihydrofolatereductase(DHFR),theenzymeresponsibleforconvertingdihydrofolicacid
totetrahydrofolicacid,competingagainstdihydrofolicacidsubstrate.Itisabroadspectrumantibiotic
oftenusedtotreatrespiratoryandmiddleearinfections,urinarytractinfections,andtravelersdiarrhea

16

2.3 ANTIBIOTICSRESISTANCEBYMICROORGANISMS
Antibioticsareveryimportanttomedicinebutitisquiteunfortunatethatmicroorganismshave
beenabletoadaptthemselvestocohabitingwithantibioticsandsubsequentlydevelopingresistanceto
them(Walshetal.,2004).Whenmicroorganismsarecontinuallyexposedtothesameantibiotics,they
findwaysofadaptingthemselvestosuchantibioticandthisrendersthedrugineffectiveagainstthem.
Transferofresistancegenecanbetransferredbyconjugation,transductionortransformation(Walsh,
2003).
Widespreaduseofantibioticsbothinsideandoutsidemedicineisplayingasignificantroleinthe
emergenceofresistantorganism(FurayaandLowy,2006).Drugsfrequentlyhavebeenoverusedinthe
past.Ithasbeenestimatedthatover50%oftheantibioticprescriptionsinthehospitalaregivenwithout
clearevidenceofinfectionoradequatemedicalindication(Payneetal.,2004).Manyphysicianshave
administeredantibacterialdrugstopatientswithcolds,influenza,viralpneumonia,andotherviral
diseases.
Arecentstudyshowedthatover50%ofpatientsdiagnosedwithcoldsandupperrespiratoryinfections
and66%ofthosewithchestcolds(bronchitis)aregivenantibiotics,eventhoughover90%ofthose
casesarecausedbyviruses(FurayaandLowy,2006).
Frequentlyantibioticsareprescribedwithoutculturingandidentifyingthepathogenorwithout
determiningbacterialsensitivitytothedrug(Harbathetal.,2005).Toxic,broadspectrumdrugsare
sometimesgiveninplaceofnarrowspectrumdrugsasasubstituteforcultureandsensitivitytesting,with
theconsequentriskofdangeroussideeffects,opportunisticinfections,andtheselectionof
drugresistantmutants(Payneetal.,2005).Thesituationismadeworsebypatientsnotcompleting
17

theircourseofmedication.Whenantibiotictreatmentisendedtooearly,drugresistantmutantsmay
survive.Antibioticsareusedofteninrearinganimalsforfoodandthisuseamongothersleadsto
creationofresistantstrains.Insupposedlywellregulatedhumanmedicine,themajorproblemof
emergenceofresistantstrainsisduetomisuseandoveruseofantibioticsbydoctorsaswellaspatients
andithasbeendiscoveredthatinfectionscausedbyresistantmicroorganismoftenfailtorespondto
standardtreatmentresultinginprolongedillnessandgreaterriskofdeath(Walshetal.,2004).

18

CHAPTERTHREE

3.0 MATERIALSANDMETHODS
3.1 MATERIALS
Variousmaterialswereinvolvedintheanalysis,thesematerialsinclude:
Petridishescommonlyusedfortheholdingtheagarmediumonwhichtheorganismsare
tobegrown,
Syringesandneedleswhichareemployedindispensingaccuratemeasuresofliquids
suchasdistilledwaterinvolvedintheanalysis,
Measuringcylinderusedtomeasureapreciseamountofliquids,
Conicalflasksusedforholdingthepreparedmedium,
Ethanolwhichiscommonlyusedtoswabtheworkingenvironmentandalsotosupply
fueltospiritlamps,
Testtubesforholdingdistilledwaterneededforserialdilution,
Cottonwool,aluminiumfoil,papertapewhosefunctionsrangesfromswabbingand
pluggingofflasksmouth,wrappingofobjectsairtightlytolabeling,
Wireloopfortransferringoforganism,
Weighingbalanceusedfortakingaccuratemeasurementsofmediumtobeused.
19

Agarsusedinclude:nutrientagarforculturingbacteria,PDA(potatodextroseagar)for
culturingyeastsandmoulds.

3.2 COLLECTIONOFSAMPLES
Soilsamplesfromthree(3)differentspotsfromLautechgariprocessingindustrywerecollected:
Thefirstpointorlocationwasthepointofdischargeofthecassavaeffluentorwastewateri.e.
wherethecassavawastewaterdrainsintoandthiswaslabeledSampleA.Nextlocationwasone
hundredmeters(100m)awayfromthepointofdischargeofthecassavaeffluentorwastewateri.e.
100malongthepartofflowofcassavawastewaterandthiswaslabeledasSampleBwhilethelast
locationwassoilsamplefromaneutralsourcethathasnotwitnessedanyformofcassavaeffluent
dischargeorcontaminationandthiswaslabeledSampleC.
SampleAwhichisthesoilsamplefromthecassavaeffluentwascollectedusingasterilizedspoon,it
wascollectedintoasterileglasscontainer,andthesameprocedurewasusedforSamplesBandC
butwithdifferentsterilizedglasscontainersandspoonsinvolved.Thesamplesaftercollectionwere
transportedintothelaboratoryinasterileblackpolythenebagforsubsequentmicrobialanalysis.
3.3 CULTURINGOFMICROORGANISMS
Mediawerepreparedaccordingtomanufacturersinstructionsprintedonthecontainer,and
weresterilizedinanautoclaveat121
0
Cfor1hour.Thecollectedsoilsampleswerehomogenizedusing
distilledwater.9mlsofsteriledistilledwaterwasdispensedintoeachtesttubeandsubsequentplugging
20

withcottonwoolandaluminiumfoilthesetubeswerethentakentotheautoclaveforsterilizationat
121
o
cfor1hourandaftersterilization,coolingwasdone.
Serialdilutionwasdonetothinoutmicrobialpopulationsothatnumbersofcoloniesthatwillbe
formedwouldnotoverpopulatetheplateuntowhichtheywillbegrown.
Serialdilutionwascarriedoutforeachsampleusingsteriledistilledwaterasthediluentandthiswas
doneunderasepticcondition.Theasepticconditionwasachievedbythethoroughswabbingofthe
workingenvironmentwithethanolandcottonwool,theworktablewasalsoswabbedcleanandlitwith
spiritlampstopreventcontaminationfromatmosphere.
Threedilutionfactorsforeachsoilsamplewerepickedandusedasinoculumfortheculturingofthe
organismsusingpourplatemethod.Incubationwasdoneat37
0
cfor24hoursforbacteriaand48hours
forfungi.
Observationandrecordingwasdoneafterthecompletionoftheincubationperiod.
ISOLATIONOFORGANISMS
Fromthemixedculture,distinctcolonieswerepickedandstreakeduntoafreshlypreparedmediaunder
asepticcondition.Subculturingwasdonerepeatedlytoobtainpureisolateswhichwerestoredonslants.
IDENTIFICATIONOFISOLATES
Theisolatesweresubjectedtovariousbiochemicaltestsforidentificationaccordingto
specificationof(Starretal.,1981)inthelaboratory

21

Figure showing arrays of plates in a lamina flow chamber and an isolate growing on the
plate

3.7.2 BIOCHEMICALTESTS
Thefollowingarethebiochemicaltestsperformedontheisolates
3.7.2.1 CATALASETEST
Aslidetestwasemployed,asmallquantityofthecultureswereputonaglassslide,a
droportwoofhydrogenperoxideisthenaddedtotheslide.Thepresenceofbubblesrepresentsa
positive(+ve)testwhileanegativetestissignaledbytheabsenceofbubbles.
22

3.7.2.2 HYDROGENSULPHIDETEST
ExamineeachSIMtubeforthepresenceofablackcolor(nothingneedstobeadded).
Ablackcolorindicatesthepresenceofhydrogensulphide(H
2
S)whichcombineswiththepeptonized
ironintheSIMmedium.TheresultisFeSironsulphidewhichcausesablackeningofthemediumand
thisrepresentsapositivetesttheabsenceofablackcolorisanegativetest.

Fig.showinghydrogensulphidetest

3.7.2.3 INDOLETEST
Useadroppertoplace5dropsofKovacsreagentontothetopoftheSIMagarin
eachtube.Iftheaminoacidtryptophanhasbeenbrokendownbytheenzymetryptophanasetoform
23

indole,thekovacsreagentwillcombinewiththeindoletoformaredcoloratthetopoftheagarand
thisrepresentsapositivetest.Nocolorchangeinthekovacsreagentrepresentisanegativetest.

Fig.showingindoletest(noticetheredcoloronthetopoftheagar)

3.7.2.4 METHYLREDTEST
UsingaPasteurspipette,add10dropsofmethylredpHindicatortoeachtube,swirl
thetubegentlytomixthedropsintothebroth.Examineeachtubeforcolorchange.Bacteriathat
producemanyacidsfromthebreakdownofdextrose(glucose)intheMRVPmediumcausethepHto
dropto4.2.AtthispH,methylredisred.Aredcolorrepresentsapositivetest.Bacteriathatproduce
feweracidsfromthebreakdownofglucosedropthepHto6.0.AtthispHmethylredisyellowandthis
representsanegativetest.

24

3.7.2.5 OXIDASETEST
Drop12dropsofoxidasereagentontocoloniesofbrothculture,watchoutforgradual
colorchangefrompinktolightpurpleandthentodarkpurplewithin1030seconds.Suchacolor
changeindicatesthepresenceoftherespiratoryenzymecytochromeCoxidaseandthisrepresentsa
positivetest.Nocolorchangeisanegativetest.

3.7.2.6 OXIDATIONFERMENTATION(OF)GLUCOSETEST
OFglucosemediumcontainsthesugarglucoseandpHindicatorbromthymolblue.
ThisindicatorisgreenattheinitialpHof6.8,butturnstoyellowatapHof6.0.ifglucoseisutilized,
acidsareproducedandthepHdrops,causingthebromthymolbluetoturnfromgreentoyellow.Ifboth
tubes(withorwithoutoil)turnyellow,thetestorganismissaidtobeafacultativeanaerobeabletouse
glucoseinthepresenceorabsenceofoxygen.Ifonlythetubewithoutoilturnsyellow,thetestorganism
isconsideredanaerobeabletouseglucoseonlywhenoxygenispresent.Nocolorchangeineithertube
indicatesthatthetestorganismisunabletoutilizeglucose.

3.8 ANTIBIOTICSENSITIVITYTESTING
Coloniesfromtheslantswerepickedandusedtoinoculateappropriatebrothculture(Nutrient
brothforbacteriaandPotatodextrosebrothforfungi)andthenincubatedforlessthan18hours.Fresh
mediawerepreparedandleftovernightforsurfacemoisturetodryup.Pickingofcoloniesfromthe
25

brothcultureswasdoneusingsterileapplicatorstickandproperswabbinguntothesurfaceofthe
preparedplateswasdone.Thiswasleftfor1hourafterwhichantimicrobialdiscswereappliedusinga
sterileforcepsthediscswerepresseddownfirmlytopreventfallingoffofthediscsfromtheplates
duringincubation.
Forfungi,threeconcentrationsofantifungalstocksolutionwerepreparedandsterileperforated
filterpapersweredippedintoeachstocksolutionusingasterileforceps.Pickingandapplicationofthe
discsuntotheplateswasdoneusingasterileforceps.
Incubationwasdoneandsensitivitieswereobservedat24hoursand48hoursforbacteriawhile
fungiwereincubatedfor48hours.
Afterincubation,thezonesofinhibitionformedweremeasuredintwoperpendicular,planes
withtheaveragesdetermined.Afterthistheresultswasinterpretedusingstandardtablestodetermineif
thebacteriaareSensitive(S),Intermediate(I)orResistant(R)totheantimicrobialdrugs.

26

CHAPTERFOUR

4.0 RESULTSANDDISCUSSION
4.1 ISOLATEDORGANISMS
Atotalnumberoftwentyfivestrainswereisolatedfromallthesamplessome
ofthemare(Bacilluscereus,Bacillussubtilis,Pseudomonasaeruginosa,Listeria
monocytogenes,E.colietc),whilefungalstrainsinclude(Aspergillusniger,Aspergillus
flavusandRhizopusspetc)

PLATECOUNTRESULT
Table1showstheresultfromthecolonycountofeachsamplefromdifferentmedia.
MacConkeyAgar
SAMPLE
S
10
1
10
2
10
4
A TNC 67 36
B TNC 70 39
C TNC 80 19

27

PDA
SAMPLES 10
1
10
2
10
4
A TNC 49 17
B TNC 52 36
C 133 93 42

NutrientAgar
SAMPLES 10
1
10
3
10
5
A TNC TNC 96
B TNC 94 85
C TNC TNC 86

4.2 IDENTIFICATIONOFORGANISMS
28

Table2showstheidentifiedorganismsobtainedinthesamples(bacteriaandfungi).
S/
N
Codes Isolates
1 NAA
1
Pseudomonasaeruginosa
2 NAA
21
Bacilluscereus
3 NAA
22
Bacillussubtilis
4 NAA
31
Bacillussubtilis
5 NAA
32
Bacillussubtilis
6 NAA
53
Pseudomonasaeruginosa
7 NAA
54
Bacillussubtilis
8 NAB
11
Listeriamonocytogenes
9 NAB
12
E.coli
10 NAB
31
Listeriamonocytogenes
11 NAB
32
E.coli
12 NAB
52
E.coli
13 NAB
53
E.coli
14 NAC
31
E.coli
15 NAC
42
Bacilluscereus
16 NAC
42
Bacilluscereus
17 NAC
54
Bacillussubtilis
18 NAC
54
E.coli
29

A
11
Aspergillusniger
A
12
Aspergillusflavus
A
21
Aspergillusniger
B
11
Rhizopussp
B
12
Aspergillusniger
C
2
Aspergillusniger

MycoteneResultinmm
Fungi 50g/ml 100g/ml 200g/ml
A
11
R 18 20
A
12
13 15 19
A
21
15 30 35

Fungi 50g/ml 100g/ml 200g/ml

B
11
R R R
B
12
12.5 20 22

30

C
2
14.5 19 21

4.3 ANTIBIOTICSENSITIVITYTESTING
Table3showsthesensitivityresultfortheisolatedorganismsfromsamplesA,B,andCat24hoursand
48hoursrespectively.

SENSITIVITYTESTINGRESULT(24HOURS)
SAMPLEA(CASSAVAEFFLUENT)
SAMPLE
S
CAZ CRX GEN CPR OFL AUG NIT AMP ER
Y
CTR CX
C
veA
31
R R 21.0 24.5 21.5 18.0 18.5 11.5
veA
21
R R 19.0 24.0 23.5 13.5 17.0 10.5
+veA
32
R R 15.5 20.0 R 11.5 R R
veA
22
R R 25.0 21.0 13.0 15.0 24.5 11.0
veA
1
R R 17.0 20.5 18.0 13.0 16.5 12.0
+veA
53
R R 16.0 20.0 R 11.5 R R
veA
54
R R 18.0 23.0 20.5 12.0 15.0 11.0

31

SAMPLEB(100mfromCassavaeffluent)
SAMPLES CAZ CRX GEN CPR OFL AUG NIT AMP ERY CT
R
CX
C
veB
11
R R 16.5 23.5 21.5 12.5 13.5 R
+veB
12
R R 21.5 28.0 R 16.0 R R
+veB
53
R R 16.0 17.0 R 15.5 R R
+veB
31
R R 13.0 20.0 R R R R
veB
32
11.0 16.0 19.0 25.0 20.0 20.0 15.0 10.0
veB
52
R R 18.5 16.0 16.0 12.0 14.0 7.5

SAMPLEC(Normalsoil)
32

SAMPLE
S
CAZ CRX GEN CPR OFL AUG NIT AMP ERY CT
R
CX
C
veC
42
20.0 15.5 16.0 17.0 22.0 6.0 13.0 R
+veC
31
R R 14.5 14.5 R 9.0 R R
veC
42
20.0 17.0 17.0 25.0 25.0 8.0 9.0 R
+veC
54
R R 14.0 24.0 R 10.0 R R
veC
54
R R 17.0 20.5 20.0 10.0 14.0 10.5

SENSITIVITYTESTINGRESULTAT(48HOURS)
SAMPLEA(CASSAVAEFFLUENT)
SAMPLE
S
CAZ CRX GEN CPR OFL AUG NIT AMP ER
Y
CTR CX
C
veA
31
R R 23.0 24.5 21.5 18.0 19.0 12.0
veA
21
R R 19.0 24.0 24.0 14.5 17.0 13.0
+veA
32
R R 17.5 20.0 R 12.5 R R
veA
22
R R 25.0 23.0 18.5 15.0 24.5 17.0
veA
1
R R 19.0 21.0 19.0 13.5 18.5 12.0
+veA
53
R R 17.0 21.0 R 12.0 R R
33

veA
54
R R 20.0 27.5 25.0 13.0 17.0 12.0

SAMPLEB(100mfromCassavaeffluent)
SAMPLES CAZ CRX GEN CPR OFL AUG NIT AMP ERY CT
R
CX
C
veB
11
R R 17.5 24.5 23.0 13.0 14.0 R
+veB
12
R R 28.0 32.0 R 20.5 R R
+veB
53
R R 16.5 20.0 R 18.0 R R
+veB
31
R R 15.5 21.0 R R R R
veB
32
12.5 17.0 21.0 27.0 23.0 21.0 15.0 15.5
veB
52
R R 18.5 16.0 16.0 12.0 14.0 9.0

SAMPLEC(Normalsoil)
SAMPLE
S
CAZ CRX GEN CPR OFL AUG NIT AMP ERY CT
R
CX
C
veC
42
22.0 19.0 19.0 20.0 25.0 7.5 15.0 R
+veC
31
R R 16.0 21.0 R 11.0 R R
veC
42
22.5 17.0 19.0 25.0 25.5 8.0 14.0 R
+veC
54
R R 15.0 27.0 R 11.0 R R
veC
54
R R 19.0 26.0 26.0 13.5 16.0 11.0
34

Negative Positive
Caz Ceftazidine 30g Caz Ceftazidine 30g
Crx Cefuroxime 30g Crx Cefuroxime 30g
Gen Gentamycin 10g Gen Gentamycin 10g
Cpr Ciprofloxacin 5g Ctr Ceftriaxone 300g
Ofl Ofloxacin 5g Cxc Cloxacilin 5g
Aug Augumentin 30g Aug Augumentin 30g
Nit Nitrofurantoin 300g Ofl Ofloxacin 5g
Amp Ampicillin 10g Ery Erythromycin 5g

35

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