ASustainableApproachtoIndustry:IsolationandCharacterizationofNew

AmylaseProducingBacillusSpeciesfromAntHillSoilinHouston
By:KadriyeSudeAlmus
JohnFosterDullesHighSchool

TableofContents:
Abstract
ResearchPaper
:
I.

Introduction
a. Problem

II.

MaterialsandMethodology
III.

Results

a. Analysis
b. Discussion
IV.

Conclusion

V.

References

Abstract

Amylasesareapopularclassofenzymes,withabroadspectrumofindustrial
applications,particularlyinthosethatinvolvestarchprocessingtheyreplacemanyhazardous
chemicalprocessesasanenvironmentallyandeconomicallybeneficialalternative,asapartof
thelargerGreenChemistrymovementthathasledtosignificantenvironmentalbenefits.60%
ofcommerciallyavailableenzymesarefrombacteriaofthegenusBacillus,whichcanbeusedas
aprobioticintheanimalfeedindustry,leadingtoasignificantreductioninanimalwaste
pollutionasabiofertilizerandinthemanyindustriesthatbenefitfromtheuseofamylaseasan
enzyme.Thus,thisstudyaimedtoidentifythemostefficientamylaseproducingBacillusspecies
specifictotheHoustonareaforuseinlocalindustries.SoilsampleswerecollectedinHouston,
with728Bacilluscoloniesisolatedandtestedforamylaseproductionbystarchhydrolysis
testing.Thespeciesofamylaseproducingcolonysampleswereidentified,andmeasurements
concerningefficiencyofamylaseproductionwereobtained.11specieswereidentifiedamong
the96amylaseproducingcolonysamples.Themeanefficienciesofthespecieswereanalyzed
throughaonewayANOVA,whichshowedthatdifferencesamongidentifiedBacillusspecies
meanefficiencieswerenotstatisticallysignificantstatisticallyspeaking,noneofthespecies
standoutassignificantlymoreefficientthanothers.Thetwospeciesthatweremostabundantby
farwereBacillussiamensis(43.8%)andBacilluscereus(25%),buthadrelativelylowefficiency
meansof1.248and1.580,respectively.Ofthethreespecieswiththelargestmeanefficiencies,
Bacillussubtilishadboththelargestefficiencyof1.786andthelargestabundanceof7.3%
(thoughitwasnotthemostabundantoverall).Thus,Bacillussubtiliswasconsideredthemost
efficientandviableamylaseproducerinthisstudyhowever,theotherBacillusspeciesthatthis
studyidentifiedandanalyzedareallviableasamylaseproducingBacillusspecifictothe
Houstonarea.Asallthespeciesidentifiedareefficient,thesespeciescanandshouldbeusedin
andaroundHoustoninindustrialprocessesasanenvironmentallyviable,andoftencheaper,
alternative.

I.

Introduction

Enzymesarebiocatalyststhatacceleratebiologicalreactionsenzymessourcedfrom
fungiandbacteriahavebecomeincreasinglydesirableintheindustry.Inparticular,amylases,
knowntobeproducedbyavarietyofbacteriaandfungi,havebeenapopularclassofindustrial
enzymes,constituting25%oftheenzymemarket(Vaseekaran1).Theirhighdesirabilityresults
fromtheirstability,geneticdiversity,highenzymaticactivityinawiderangeofconditions,
simpleproductionandeasymanipulation.Thiscausesthemtohaveabroadspectrumof
industrialapplicationstheyhavepushedmanycommercialbiotechnologicalprocessesinthe
fieldsofrenewableenergy,pharmaceuticals,textileandpaperindustries,andanimalprobiotics
intonewfrontiers.Amylases,andenzymeusageingeneral,alsocontributetosaferworking
conditionsthrougheliminationofchemicaltreatmentsduringproductionprocesses,produceless
waste,andreduceenergyconsumption(Environmental).Furthermore,enzymesarehighly
efficient,andcanincreasereactionratesbyafactorof100millionto10billion(Gurung).Usage
ofsuchenzymesarealargepartoftheGreenChemistrymovementtoreducegenerationof
hazardoussubstances,whichhasledtosignificantenvironmentalbenefits(Green).

Oneofthemostsignificantfunctionsofamylaseisasacatalystinthesaccharificationof
starch.Starch,acomplexcarbohydrate,iscomposedofamyloseandamylopectin.Amylases,as
enzymes,hydrolyzestarchmoleculesintomaltose,anequivalenttotwojoinedglucose
molecules.Thesaccharificationprocessofstarchhasuseinmanyindustriesinthetextile
industriesitisusedasadesizingagentfortheremovalofstarchfromclothingbeforefurther
processinginthepaperindustryforhydrolyzingrawstarchusedforsizingandcoatingpaper
andinthedetergent,buildingproduct,andfeedindustriesitfiguresasanimportantprocess
(Singh2064).Amylaseshavelargelyreplacedchemicalhydrolysis,whichoftentookplacewith
theuseofchemicallyhazardousacids,asaneconomicallyandenvironmentallyviablealternative
(Environmental).

Theproductionofmicrobialamylasescanbesourcedfromavarietyofbacteria.
However,60%ofcommerciallyavailableenzymesarefrombacteriaofthegenusBacillus,

whichexhibitssafetyofhandling,rapidgrowthrates,andanabilitytosecreteproteinsinthe
extracellularmedium(Vijayalakshmi336).DerivationofamylasefromBacillus,then,isnot
onlyeconomicallypreferable,buthasenvironmentalbenefitsitissignificantlylesslikelytobe
hazardousinanyway.AmylaseproducingBacillushavemanyuses:oneasaprobiotic,
particularlyinthefeedindustrycattlechangingrapidlyfromaforagebaseddiettoa
grainbaseddietcausesmillionsoffiberdigestingmicrobesintheirstomachstodieoffdueto
theirinabilitytodigestthestarchingrain,andthegrainsoursintherumenduetothelackof
starchdigestingmicrobes.Asaresult,rumenpHdecreasesandcausesittostopsworkingthe
animalbecomesillandevendiesinseverecases(Hall).Moreover,animalsaccustomedtofeed
alsohavetroubledigestingstarchandthereforeproduceasignificantamountofwaste,whichhas
greatpotentialtoactasapollutant,particularlyofriversandundergrounddrinkingwater
supplies.However,waterqualityisnottheonlythingitaffectsanimalwastecanalsocontribute
significantlytoairqualityproblemsintheformofdust,smog,andgreenhousegases
(Problem).Ifusedasaprobiotic,then,amylaseproducingBacilluscanaidgreatlyindigestion
ofstarchfromthegraininanenvironmentallyfriendlymanner,andreduceanimalwaste
pollutiontoasignificantextent.Otherusesareasabiofertilizer,nonhazardousdraincleaningin
ahouseholdsetting,and,ofcourse,inthemanyindustriesthatbenefitfromtheuseofamylaseas
anenzyme.

Problem

Thus,consideringthesignificantenvironmentalandeconomicbenefitsdiscussed,this
studyaimstoidentifythemostefficientamylaseproducingBacillusspeciesspecifictothe
Houstonarea.ThisspecificitytoHoustonisimportant,asitmeansthatitwillbeattunedtoand
thriveintheHoustonenvironment,thusleadingtogreaterbenefitsofuseasanenzymesource
fortheindustriesinandaroundHouston,asaprobioticforuseonfarmsinthearea,anditsmany
otherusesdescribedabove.

II.

MaterialsandMethodology

SampleCollection

Soilsampleswerecollectedusingsterilefalcontubes.Soilfromfireanthillswiththe
coordinatesof29.667461N,95.438712WinHouston,Texaswascollectedspecialcarewas
takentoensurethatthesitewasdryandhadnotpreviouslybeensprayedwithinsecticides.

IsolationofBacterialCultures

Soilsuspensionswerecreatedbypreparing10mLofNutrientBrothMedium(MM244)
fromHiMediaLaboratoriesina50mLfalcontubeandplacingapproximately1grameachof
o
soilcollected.Thiswasthenplacedinarotaryshakerfor4hoursat30
Cand275rpm.
o
Followingincubation,1mLofeachsamplewasheatedat80
Cfor20minutes.

Themixtureswerespreadonto6.8pHand25CnutrientAgar(fromHardyDiagnostics)
platesundertheBiologicalSafetyCabinetA2.Followingthis,theplateswereincubated
o
overnightat30
C.

Purecultureswereobtainedfromvariousindividualcoloniesproducedontheplatesand
putinto15mLfalcontubescontaining3mLofnutrientbroth,formingastocklibraryofover
o
150cultures,whichwerethenincubatedovernightat30
Conrotaryshaker.

Isolationofamylaseproducingbacteria

Beforeuse,thecultures(containedinfalcontubes)fromthestocklibrarywerevortexed
onaVortexGeniemixerforapproximately2minutes.Usingapipettor,coloniesof3ulof
bacteriasamplesfromthesecultureswereinoculatedontostarchagarplateswith32gridplate
stickers(acolonysampleoneachportionofthesticker).Approximately700colonieswere
inoculatedthisway.Theseplateswerethenincubatedat30Cfor24hours,andfollowing
incubation,starchhydrolysistestingwascarriedout.

StarchHydrolysisTest:
Hydrolasesareenzymesthatcatalyzethesplittingoforganicmoleculesintosmaller
moleculesinthepresenceofwater.Twoconstituentsofthestarchmoleculearerapidly
hydrolyzedbycertainbacteriausingamylasestoyielddextrins,maltose,andglucose.
Gramsiodineisusedtoindicatepresenceofstarch.Uponcontactwithstarch,theiodine
formsadarkbluecomplex,whereashydrolyzedstarchdoesnotexhibitacolorchange.Thus,a
clearareaaroundbacteriaindicatesthehydrolysisofstarchandthereforetheproductionof
amylases.Thetestiscarriedoutthroughtheprocessofstaininganddestainingagarplateswith
Gramsiodinesolution.

Images1&2
.Colonysamplesinstarchagarplatesafterstarchhydrolysistestingbacteria
diameter(thedarkercirclethatappearstobeplacedontheplate)andhalodiameter(theclear
areaaroundthebacteria)aremeasuredwitharuler.

Coloniesthatproducedclearhalozonesinthestarchhydrolysistestwereconsidered
positiveforamylaseproducingbacteriastrains,andthesepositiveculturesweresenttoOhio
StateUniversitysBacterialGenomeSequenceCenterforbacterialstrainidentificationafterthe
followingmeasurementsweretaken.

Thehaloandcolonydiametersweremeasuredusingarulertocalculateefficiencyof
amylaseproduction:


Efficiency= HalodiameterColonydiameter

Colonydiameter

Thisefficiencycalculationisusedbecauseitaccountsforstarchhydrolyzedperamount
ofbacteriapresentusingonlythehalodiametersasabasisformeasuringstarchhydrolysis
effectivitywouldproduceinaccurateresultsassizesofbacteriaontheplatevarygreatly,andthe
halodiameterincludesthebacteriaitselfandnotonlythehydrolyzedregionsurroundingthe
bacteria.

BacterialStrainIdentification

ThefollowingproceduretookplaceattheOhioStateUniversitysBacterialGenome
SequenceCenter.Firstly,cellextractswereobtainedandstoredat4Cforuseasatemplatefor
PCR(polymerasechainreaction)amplification.

ObtainingCellExtractsfromCultures:
Cellsuspensionsfromfreshcultureswereagitatedwithvolumesof0.1mmGlassbeads
fromScientificIndustries,Inc.for1minuteusingastandardbeatbeaterdevice.Throughthe
processofcentrifugationatmaximumspeedfor5minutes,thebeadsandcelldebriswere
excised.Thesupernatant,nowconsistingonlyofthecellextracts,wasthenremovedandstored.

Thesameprimers(strandsofshortnucleicacidsequencesrequiredforDNAreplication,
usedtodeterminetheDNAfragmenttobeamplifiedbythePCRprocess)wereusedfor
amplificationandsequencing.Thesampleswereamplifiedwiththe25F(alsocalledpA)/1492r
primerpairhowever,iftheamplificationfailed,the25F/765rprimerpairwasused.

SequencesofPrimersUsed:
27FAGAGTTTGATCCTGGCTCAG
1492rCGGTTACCTTGTTACGACTT

765rCTGTTCGCTCCCCACGCTTTC

Amplification:
TheFastStartPCRMasterMixfromRocheDiagnosticswasused.A25lamplification
reactionforeachstrainwassetup,using12.5lofmastermix,12.05lofwater,0.1lofeach
primer,and0.25lofcellextractsthiswascarriedoutinaBioRadMJMinithermocycler.
Cyclingparameterswereaninitialdenaturation(2minutesat95C)35cyclesofdenaturation
(30secondsat95C),annealing(30secondsat57C),anextension(60secondsperkilobaseat
72C),andafinalextension(7minutesat72C).

TheproductswerepurifiedusingtheDNAClean&Concentrator25fromZymo
Researchwithelutionbufferatafinalvolumeof25l.Sequencingtookplaceateitherat
EurofinsOperonoratthePlantMicrobeGenomicsFacilityattheOhioStateUniversity,with
standardSangersequencingbeingperformedwiththeBigDyeTerminatorv3.1Cycle
SequencingKitfromAppliedBiosystems.Afterendtrimmingtoremovelowquality
nucleotides,andthefinalsequenceswasusedinaBLASTsearchoftheNCBIdatabasewith
typematerialchecked.Usingtheresultingdata,specieswereassignedtoeachsamplecolony.

III.

Results

Atotalof96colonysampleswerefoundtobepositiveoutofthe700testedforamylase
productionbystarchhydrolysistesting(seeImage3).Figure1liststhesesamples,alongwiththe
Bacillusspeciestheywereidentifiedasandthemeasurementsobtainedforeach.Figure3and
Figure4showthefrequenciesofeachBacillusspeciesthatwasfoundwithinthesetofcolony
samplesThemostabundantBacillusspeciesamongthesampleswasBacillussiamensisamong
the96,42weredeterminedtobeBacillussiamensis(43.2%),followedbyBacilluscereus(25%),
Bacillusamyloliquefaciens(9.4%),Bacillussubtilis(7.3%),BacilluspumilusorBacillus
safensis(4.2%),Bacillusmethylotrophicus(3.1%),BacillusthuringiensisorBacillustoyonensis

(3.1%),BacillusatrophaeusorBacillussiamensis(1%),Bacillussimplex(1%),Bacillus
siamensisorB.methylotrophicus(1%),andBacillustequilensis(1%).

Image3.
Astarchagarplatewithpositivesamplecoloniesafterthestarchhydrolysistest.

Name

Species

DataNumber Halo
diameter

Colony
diameter

Efficiency

26Za2

Bacillus
1
atrophaeusor
Bacillus
siamensis

17

0.888889

26Za1

Bacillus
amyloliquefa
ciens

14

11El8

Bacillus
amyloliquefa
ciens

15

1.5

99Cm4

Bacillus
amyloliquefa
ciens

0.4

99Cm5

Bacillus
amyloliquefa

18

2.6

ciens
99Cm7

Bacillus
amyloliquefa
ciens

0.6

99Cm8

Bacillus
amyloliquefa
ciens

19

2.166667

99Cm11

Bacillus
amyloliquefa
ciens

0.8

99Cm12

Bacillus
amyloliquefa
ciens

0.4

99Cm14

Bacillus
amyloliquefa
ciens

10

0.4

38Is2

Bacillus
cereus

11

13

0.625

38Is10

Bacillus
cereus

12

14

0.75

27Sy3

Bacillus
cereus

13

14

0.75

27Sy5

Bacillus
cereus

14

14

0.75

27Sy6

Bacillus
cereus

15

16

1.666667

33Sa8

Bacillus
cereus

16

14

1.8

33Sa10

Bacillus
cereus

17

0.8

23Jc1

Bacillus
cereus

18

10

0.25

23Jc2

Bacillus

19

19

5.333333

cereus
24Oz1

Bacillus
cereus

20

20

1.5

24Oz2

Bacillus
cereus

21

18

1.25

24Oz5

Bacillus
cereus

22

16

32Sm1

Bacillus
cereus

23

12

0.714286

32Sm2

Bacillus
cereus

24

13

1.6

4Sar4

Bacillus
cereus

25

0.75

35Mk3

Bacillus
cereus

26

17

2.4

1Ja7

Bacillus
cereus

27

15

37Ro3

Bacillus
cereus

28

13

1.166667

37Ro4

Bacillus
cereus

29

12

1.4

24Hl1

Bacillus
cereus

30

18

2.6

24Hl2

Bacillus
cereus

31

0.4

21Lo1

Bacillus
cereus

32

17

1.833333

21Lo2

Bacillus
cereus

33

13

2.25

38Mu5

Bacillus
cereus

34

20

2.333333

36Pe3

Bacillus

35

13

2.25

methylotroph
icus
11El10

Bacillus
methylotroph
icus

36

16

11El14

Bacillus
methylotroph
icus

37

15

36Pe2

Bacillus
pumilusor
Bacillus
safensis

38

14

1.333333

36Om2

Bacillus
pumilusor
Bacillus
safensis

39

15

2.75

37Ro7

Bacillus
pumilusor
Bacillus
safensis

40

15

1.5

37Ro9

Bacillus
pumilusor
Bacillus
safensis

41

15

1.5

38Is1

Bacillus
siamensis

42

13

0.857143

38Is3

Bacillus
siamensis

43

13

1.6

38Is4

Bacillus
siamensis

44

13

0.625

38Is5

Bacillus
siamensis

45

12

38Is6

Bacillus
siamensis

46

12

1.4

38Is7

Bacillus

47

13

1.166667

siamensis
38Is8

Bacillus
siamensis

48

14

1.8

38Is9

Bacillus
siamensis

49

13

0.857143

5Ju2

Bacillus
siamensis

50

16

2.2

36Pe4

Bacillus
siamensis

51

16

26Za3

Bacillus
siamensis

52

0.6

26Za4

Bacillus
siamensis

53

16

1.285714

26Za5

Bacillus
siamensis

54

13

2.25

8Rd2

Bacillus
siamensis

55

16

1.285714

8Rd3

Bacillus
siamensis

56

12

8Zn1

Bacillus
siamensis

57

0.4

8Zn2

Bacillus
siamensis

58

16

1.285714

8Zn3

Bacillus
siamensis

59

17

0.888889

8Zn4

Bacillus
siamensis

60

15

0.875

8Zn5

Bacillus
siamensis

61

15

1.142857

26Sel6

Bacillus
siamensis

62

15

11El1

Bacillus

63

15

0.875

siamensis
11El2

Bacillus
siamensis

64

14

0.75

11El3

Bacillus
siamensis

65

14

11El4

Bacillus
siamensis

66

16

1.666667

11El5

Bacillus
siamensis

67

14

1.333333

11El6

Bacillus
siamensis

68

14

1.333333

11El7

Bacillus
siamensis

69

14

1.333333

11El9

Bacillus
siamensis

70

14

1.8

11El11

Bacillus
siamensis

71

14

0.75

11El12

Bacillus
siamensis

72

14

2.5

11El13

Bacillus
siamensis

73

13

1.6

3Hm1

Bacillus
siamensis

74

12

0.5

3Hm2

Bacillus
siamensis

75

0.4

3Hm3

Bacillus
siamensis

76

13

1.166667

3Hm4

Bacillus
siamensis

77

13

2.25

38Mu1

Bacillus
siamensis

78

12

1.4

38Mu2

Bacillus

79

14

siamensis
38Mu3

Bacillus
siamensis

80

14

0.75

38Mu4

Bacillus
siamensis

81

14

1.333333

38Mu8

Bacillus
siamensis

82

16

1.666667

38Mu9

Bacillus
siamensis

83

15

1.5

7Al3

Bacillus
siamensisor
B.
methylotroph
icus

84

15

1.5

36Om17

Bacillus
simplex

85

1.25

33Sal5

Bacillus
subtilis

86

12

1.4

25Bi3

Bacillus
subtilis

87

13

1.166667

36Om4

Bacillus
subtilis

88

16

26Sel3

Bacillus
subtilis

89

14

1.8

26Sel4

Bacillus
subtilis

90

15

1Ja9

Bacillus
subtilis

91

14

1.8

37Ro6

Bacillus
subtilis

92

14

1.333333

33Sa1

Bacillus
tequilensis

93

0.6

7Al8

Bacillus
thuringiensis
orBacillus
toyonensis

94

13

0.857143

26Sel5

Bacillus
thuringiensis
orBacillus
toyonensis

95

19

2.166667

12Tr10

Bacillus
thuringiensis
orBacillus
toyonensis

96

13

1.6

Figure1.
Atableindicatingthespeciesofeachcolonysampleobtainedfromthebacterialstrain
identificationproceduredescribedpreviously,andthedataobtainedfromthemeasurements
describedinthemethodology.Thesamplesaregroupedbyalphabeticallyorderedspecies.

Figure2.
Abargraphillustratingefficiencydataofeachcolonysample.

BacillusSpecies

Frequency

Percent

CumulativePercent

BaciilusatrophaeusorBacillussiamensis

10.4

Bacillusamyloliquefaciens

9.4

35.4

Bacilluscereus

24

25

38.5

Bacillusmethylotrophicus

3.1

42.7

BacilluspumilusorBacillussafensis

4.2

86.5

Bacillussiamensis

42

43.8

87.5

BacillussiamensisorB.methylotrophicus

88.5

Bacillussimplex

95.8

Bacillussubtilis

7.3

96.9

Bacillustequilensis

100

BacillusthuringiensisorBacillustoyonensis

3.1

10.4

96

100

Total

Figure3.
AfrequencytableoftheBacillusspeciesidentifiedinthestudy,withfrequencyin
numberofsamples,percentageofthetotalamountofsamples,andcumulativepercent
illustrated.


Figure4.
ThepiechartrepresentingfrequencyofeachBacillusspeciesasapercentageofall
samples.TheBacillusspeciesarerepresentedbytypenumberthisdatacanbefoundinFigure
5below.

Analysis
AonewayAnalysisofVariance(ANOVA)testwasconductedtocompareefficiencies
ofcoloniesandexaminedifferencesamongeachtypeofBacillusthatprovedtobeamylase
producing.SPSS22.0.0wasusedtoconducttheonewayANOVA,whichisusedtoexamine
meandifferencesbetweentwoormoregroups.Itisabivariatetestwithoneindependentvariable
(IV)thatiscategoricalandonedependentvariable(DV)thatiscontinuous.

Discussion
BacillusSpecieswith3ormoresampleswereincludedintheanalysis.TheOneway
ANOVAtestresultsshowedthatmeandifferencesamongBacillusspeciesamylaseproduction
efficiencyscoreswerenotstatisticallysignificant(SeeFigure7).Thisimpliesthat,statistically
speaking,noneofthespeciesstandoutassignificantlymoreefficientthanothers.However,
BacillusspeciesBacillusmethylotrophicus(type4)withameanefficiencyof1.750,Bacillus

pumilusorBacillussafensis(type5)withameanof1.771,andBacillussubtilis(type9)witha
meanefficiencyof1.786appeartobetheoneshavingthehighestmeans(SeeFigure8),with
Bacillussubtilishavingthehighestmeanefficiency.Themostabundantofthethreespecieswith
thehighestmeanswasBacillussubtilis,with7.3%ofthesamplesintotal.Asmentionedabove,
thetwospeciesthatweremostabundantbyfarwereBacillussiamensis(43.8%)andBacillus
cereus(25%),buthadrelativelylowefficiencymeansof1.248and1.580,respectively.
However,asshownthroughtheANOVAanalysis,thedifferenceinmeanswasnotstatistically
significantthesespeciesareefficientenoughforuse.Someexperimentalerrorscouldhave
influencedresultsalso,astherewerenotsimilaramountsofeachBacillusspeciesidentified,the
analysiswaslikelysomewhatskewed.Ifmorecolonysamplesaretestedandidentified,with
largenumbersofsamplesforeachspecies,theanalysiscanbemoreaccurate.Inafuture
investigation,alargeramountofcolonysamplesshouldbeused,andsamplesourceshouldbe
diversifiedthroughouttheHoustonarea,insteadofsourcingallthesamplesfromoneanthill.

Species

AssignedNumber

MeanEfficiency

Bacillusatrophaeusor
Bacillussiamensis

0.888888889

Bacillusamyloliquefaciens

1.096296296

Bacilluscereus

1.580109127

Bacillusmethylotrophicus

1.75

BacilluspumilusorBacillus
safensis

1.770833333

Bacillussiamensis

1.248289872

BacillussiamensisorB.
methylotrophicus

1.5

Bacillussimplex

1.25

Bacillussubtilis

1.785714286

Bacillustequilensis

10

0.6

Bacillusthuringiensisor
Bacillustoyonensis

11

1.541269841

Figure5.
Atableindicatingtheassignednumberusedintheanalysisandthemeanefficiencyof
eachspecies.

Figure6.
AbargraphindicatingthemeanefficiencyofeachBacillusspeciesidentifiedinthe
investigation,listedbyassignednumber.

SourceofVariation

SS

df

MS

Pvalue

BetweenGroups

4.859

0.764

1.318

WithinGroups

49.287

85

0.580

Total

53,873

91

Figure7.
AtableillustratingtheOneWayAnalysisofVariance(ANOVA)ofAmylase
ProductionEfficiencyScoresofBacillusSpecies.

0.258


Figure8.
ThelinegraphrepresentingBacillusSpeciesefficiencymeanscores.

IV.

Conclusion

Inthisinvestigation,alloftheBacillusspeciesthatthecolonysamplesconsidered
positiveforamylaseproductionusedwereidentifiedaswerefoundtohavestatistically
insignificantefficienciesfromoneanotherthus,alloftheidentifiedspecieswerevirtually
equallygoodatamylaseproduction.However,whilenotconsideredstatisticallysignificantbyan
ANOVAanalysis,differencesdidexistinmeanefficiencies.Ofthethreespecieswiththelargest
meanefficiencies(allwithinapproximately2%orlessofeachother),Bacillussubtilishadboth
thelargestefficiencyof1.786andthelargestabundanceof7.3%.Thus,Bacillussubtilisis
consideredthemostefficientandviableamylaseproducerinthisstudyhowever,theother

Bacillusspeciesthatthisstudyidentifiedandanalyzedareallviableasamylaseproducing
bacteria.

V.

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n.d.Web.2February2016.
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February
2016.
Gurung,Neelam,SumantaRay,SutapaBose,andVivekRai.ABroaderView:Microbial
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