Immobilization of ammonia-oxidizing bacteria by polyvinyl alcohol and sodium alginate

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Environmental

Microbiology

Immobilization

of

ammonia-oxidizing

bacteria

by

polyvinyl

alcohol

and

sodium

alginate

Yuwei

Dong

_,

_Yanqiu

_Zhang

_,

_Baojun

_Tu

a_{XuzhouUniversityofTechnology,Xuzhou,PRChina}

b_{ChinaUniversityofMiningandTechnology,Xuzhou,PRChina}

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Articlehistory:

Received7March2014 Accepted5December2016 Availableonline15February2017 AssociateEditor:LaraDurãesSette

Keywords: Ammonia-oxidizingbacteria Immobilization Polyvinylalcohol Sodiumalginate Ammoniaoxidation

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Ammonia-oxidizingbacteriawereimmobilizedbypolyvinylalcohol(PVA)andsodium algi-nate. Theimmobilizationconditionsandammoniaoxidationabilityoftheimmobilized bacteriawereinvestigated.Thefollowingimmobilizationconditionswereobservedtobe optimal:PVA,12%;sodiumalginate,1.1%;calciumchloride,1.0%;inoculumconcentration, 1.3immobilized balls/mLofimmobilized medium;pH,10;andtemperature,30◦C.The immobilizedammonia-oxidizingbacteriaexhibitedstrongammoniaoxidationabilityeven afterbeingrecycledfourtimes.Theammonianitrogenremovalrateoftheimmobilized ammonia-oxidizingbacteriareached90.30%undertheoptimalimmobilizationconditions. Whencomparedwithammonia-oxidizingbacteriaimmobilizedbysodiumalginatealone, thebacteriaimmobilizedbyPVAandsodiumalginateweresuperiorwithrespecttopH resistance,thenumberofreuses,materialcost,heatresistance,andammoniaoxidation ability.

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

Introduction

Ammonia-oxidizingbacteriabelongtooneofthe physiologi-calsubsetsofthenitrifyingbacteriafamily.1_{Thesebacteria} play a role in the first rate-limiting step of nitrification2,3 and in the oxidation of amines to nitrites and are widely used in the denitrification of industrial wastewater and soil.4,5 _{However, because ammonia-oxidizing bacteria are} autotrophic,theyhavealonggenerationtime,slowgrowth

∗ _{Correspondingauthorat:XuzhouUniversityofTechnology,Xuzhou,PRChina.}

E-mail:258210902@qq.com(Y.Dong).

rate,andhighsensitivity,andtheycaneasilybeeliminated.6 To address these limitations, cell immobilization technol-ogyhasbeendeveloped.7_{Immobilizedcelltechnologyoffers} distinct advantagesand combinesliquid fermentation and immobilized enzymes. Immobilized cells exhibit improved catalytic activity, reduced productiontime, low production cost,highyield,andextensiveapplicationprospects.8,9_Many rawmaterialsand syntheticpolymerssuchassodium algi-nate,polyacrylamide,agar,andpolyvinylalcohol(PVA)have been extensively appliedincell immobilization.10–12 _{In the}

http://dx.doi.org/10.1016/j.bjm.2017.02.001

1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

presentstudy,ammonia-oxidizingbacteriawereimmobilized byPVAandsodiumalginatetoimprovetheircharacteristics andapplications.

Materials

and

methods

The ammonia-oxidizing bacteria used in this study were screenedfromactivatedsludgecollectedfromasewage treat-ment plant atChina University of Mining and Technology (Xuzhou, China) and exhibited 98% homology with Nitro-somonassp.GH22.

Culturemedia

Thesimulationsewagemedium(pH8.0–8.2)comprised4g/L (NH4)2SO4, 1g/L K2HPO4, 0.5g/L MgSO4, 2g/L NaCl, 0.4g/L

FeSO4and10g/LCaCO3.Theimmobilizedmedium(pH7.0–7.2)

contained 0.8g/L (NH4)2SO4, 1g/L K2HPO4, 0.5g/L MgSO4,

0.4g/LFeSO4and10g/LCaCO3.13,14

Preparationofimmobilizedammonia-oxidizingbacteria

Theammonia-oxidizingbacteriawereculturedinsimulated sewagemedium.Aftertheculturewasallowedtostandfora certainperiod,thesupernatantwasdiscarded.Subsequently, PVA and sodium alginate were added to the culture at a PVA/sodiumalginatecultureratioof3:1(v/v).Theculture mix-ture was addedto calcium chloride using aninjector, and thebacteriawereimmobilizedat4◦Cfor6h.Subsequently, theimmobilizedbacteriawerewashedwithdeionizedwater andinoculatedinto250–1000mLofimmobilizedmediumat aconcentrationof1–2immobilizedballs/mLofimmobilized mediumat30◦Cand100r/minfor16–20days.

Determinationofnitratenitrogenandammonianitrogen contents

The nitrite nitrogen content was determined by ␣-naphthylamine spectrophotometry and phenol disulfonic acid spectrophotometry, whereas the ammonia nitrogen contentwasdeterminedusingphenoldisulfonicacid.

Results

EffectofPVAconcentrationontheformationof immobilizedballs

TodeterminetheeffectofPVAconcentrationontheformation ofimmobilizedballs,thefollowingconditionswereapplied: 1%sodiumalginate;2.0%calcium chloride;and 6.0%,8.0%, 10.0%,12.0%,and14.0%PVA.

TheresultsshowedthatatPVAconcentrationslessthan 10%,theimmobilizedballsweretransparentandsoft,whereas atconcentrationshigherthan 14.0%,theimmobilizedballs wereopaque,hard,andtrailing.APVAconcentrationof12% producedimmobilizedballsthatshowedbettertransparency andhardness.

Effectofsodiumalginateconcentrationontheformation ofimmobilizedballs

Todeterminetheeffectofsodiumalginateconcentrationon theformationofimmobilizedballs,thefollowingconditions were employed:2.0%calcium chloride; 12%PVA;and 0.8%, 0.9%,1.0%,1.1%,and1.2%sodiumalginate.

The results showed that at sodium alginate concentra-tionslessthan1.0%,theimmobilizedballsweretransparent and soft, whereas at concentrations higher than 1.2%, the immobilizedballswereopaque,hard,andtrailing.Asodium alginate concentration of 1.1%produced immobilized balls thatshowedbettertransparencyandhardness.

Effectofcalciumchlorideconcentrationontheformation ofimmobilizedballs

Todeterminetheeffectofcalciumchlorideconcentrationon theformationofimmobilizedballs,thefollowing immobiliza-tion conditions were employed: 1.1%sodium alginate, 12% PVA,and1.0%,2.0%,3.0%,4.0%,and5.0%calciumchloride.

Theresultsshowedthatatcalciumchlorideconcentrations higherthan2%,theimmobilizedballswereopaque,hard,and trailing,whereasataconcentrationof1.0%,theimmobilized ballsexhibitedbettertransparencyandhardness.

Effectofsodiumalginateconcentrationontheammonia nitrogenremovalabilityofimmobilized

ammonia-oxidizingbacteria

To determine the effect of sodium alginate concentration on the ammonia nitrogen removal ability of immobilized ammonia-oxidizing bacteria, the following immobilization conditionswereemployed:1.0%calciumchloride;12%PVA; 0.8%, 0.9%, 1.0%, 1.1%, and 1.2% sodium alginate; and 1.3 immobilizedballs/mLofimmobilizedmedium.Thereaction was performed ina 250-mL shakeflask containing250mL ofimmobilizedmediumofpH8.0maintained at30◦C and 120r/min for16 days.Theammonianitrogenremoval abil-ityoftheimmobilizedammonia-oxidizingbacteriaunderthe above-mentionedconditionsisshowninFig.1.

The concentration of sodium alginate was observed to affectthehardnessofimmobilizedcells.Thehigherthe con-centrationofsodiumalginate was,thegreaterthe strength ofimmobilizedcellsbecame.However,if theconcentration wastoohigh,immobilizedcellswouldnotgrow,and ifthe concentration wastoo low,immobilizedballs would break. Furthermore,thenitritenitrogenconcentrationwasobserved to increase with increasing cultivation time. The optimal sodiumalginateconcentrationwas1.1%,whichproducedthe highestnitritenitrogenconcentration.

EffectofPVAconcentrationontheammonianitrogen removalabilityofimmobilizedammonia-oxidizing bacteria

The higher the concentration of polyvinyl alcohol was, the greater the strength of the immobilized cells became. However,increasedviscosity madethe operationmore dif-ficult and detrimental to matrix transfer and the growth

0.8% sodium alginate 0.9% sodium alginate 1.0% sodium alginate 1.1% sodium alginate 1.2% sodium alginate 160 140 120 100 80 60 40 20 0 Concentr ation of nitr ite nitrogen (mg/L) 0 2 4 6 8 Culture time (d) 10 12 14 16

Fig.1–Effectofsodiumalginateconcentrationontheammonianitrogenremovalabilityofimmobilized ammonia-oxidizingbacteria. 160 6% polyvinyl alcohol 8% polyvinyl alcohol 10% polyvinyl alcohol 12% polyvinyl alcohol 14% polyvinyl alcohol 140 120 100 80 60 40 20 0 0 2 4 6 8 Culture time (d) 10 12 14 16 Concentr ation of nitr ite nitrogen (mg/L)

Fig.2–EffectofPVAconcentrationontheammonianitrogenremovalabilityofimmobilizedammonia-oxidizingbacteria.

ofammonia-oxidizing bacteria. To determine the effect of PVAconcentrationontheammonianitrogenremovalability of immobilized ammonium-oxidizing bacteria, the follow-ingimmobilizationconditionswereemployed:1.1%sodium alginate; 1.0% calcium chloride; 6.0%, 8.0%, 10.0%, 12.0%, and 14.0% PVA; and 1.3 immobilized balls/mL of immobi-lized medium. The reaction was performed in a 250-mL shake flask containing 250mL of immobilized medium of pH 8.0 maintained at 30◦C and 120 r/min for 16 days. Theammonia nitrogenremoval abilityof the immobilized ammonia-oxidizingbacteriaundertheabove-mentioned con-ditionsisshowninFig.2.

Thenitritenitrogenconcentrationincreasedwith increas-ing cultivation time. The optimal PVA concentration was 12.0%,whichproducedthe highestnitritenitrogen concen-tration.

Effectofcalciumchlorideconcentrationontheammonia nitrogenremovalabilityofimmobilized

ammonia-oxidizingbacteria

To determine the effect of calcium chloride concentration on the ammonia nitrogen removal ability of immobilized ammonia-oxidizing bacteria, the following immobilization conditions were employed: 12% PVA; 1.1% sodium algi-nate; 1%, 2%, 3%, 4%, and 5% calcium chloride; and

1.3immobilizedballs/mLofimmobilizedmedium.The reac-tionwasperformedina250-mLshakeflaskcontaining250mL ofimmobilizedmediumatpH8.0,30◦C,and120r/minfor16 days.Theammonianitrogenremovalabilityofthe immobi-lizedammonia-oxidizingbacteriaundertheabove-mentioned conditionsispresentedinFig.3.

Whentheconcentrationofcalciumchloridewaslow,the immobilized balls were soft and easilybroken. Asthe cal-ciumchlorideconcentrationwasincreased,themicrobialcell activity decreasedduetothehighosmoticpressureofsalt, whichcauses celldehydrationand reducesmicrobial activ-ity. Moreover, the nitrite nitrogen concentration increased withincreasingcultivationtime.Theoptimalcalciumchloride concentrationwas1.0%,whichproducedthehighestnitrite nitrogenconcentration.

EffectofpHontheammonianitrogenremovalabilityof immobilizedammonia-oxidizingbacteria

To determine the effect of pH on the ammonia nitrogen removalabilityofimmobilizedammonia-oxidizingbacteria, thefollowingimmobilizationconditionswereemployed:12% PVA; 1.1%sodium alginate; 1.0%calcium chloride; and 1.3 immobilizedballs/mLofimmobilizedmedium.Thereaction was conducted ina 500-mL shakeflask containing500mL of immobilized medium atdifferent pH values, 30◦C, and

1% calcium chloride 2% calcium chloride 3% calcium chloride 4% calcium chloride 5% calcium chloride 180 160 140 120 100 80 60 40 20 0 Concentr ation of nitr ite nitrogen (mg/L) 0 2 4 6 8 Culture time (d) 10 12 14 16

Fig.3–Effectofcalciumchlorideconcentrationontheammonianitrogenremovalabilityofimmobilized ammonia-oxidizingbacteria. 0 180 pH 6 pH 8 pH 10 pH 12 pH 14 160 140 120 100 80 60 40 20 0 2 4 6 Culture time (d) 10 12 14 16 18 8 Concentr ation of nitr ite nitrogen (mg/L)

Fig.4–EffectofpHontheammonianitrogenremovalabilityofimmobilizedammonia-oxidizingbacteria.

110r/minfor18days.Theammonia nitrogenremoval abil-ityoftheimmobilizedammonia-oxidizingbacteriaunderthe above-mentionedconditionsispresentedinFig.4.

Strongacidicandstrongalkalineconditionsdestroyedall oftheimmobilizedcells.Hydrogenionsaffectedthecharge balance at the cell surface and the membrane permeabil-ity.Strongacidicconditionscausedthenutrientstobecome unavailabletoimmobilizedcellsinthecultureliquid.Strong alkaline conditions caused nucleic acid and protein dena-turation and cell death. Furthermore, the nitrite nitrogen concentrationwasobservedtoincreasewithincreasing culti-vationtime.TheoptimalpHwas10,atwhichpointthehighest nitritenitrogenconcentrationwasobtained.

Effectoftemperatureontheammonianitrogenremoval abilityofimmobilizedammonia-oxidizingbacteria

To determine the effect of temperature on the ammonia nitrogenremovalabilityofimmobilizedammonia-oxidizing bacteria, the following immobilization conditions were employed:12%PVA;1.1%sodiumalginate;1.0%calcium chlo-ride;and1.3immobilizedballs/mLofimmobilizedmedium. Thereaction was performed in a500-mL shake flask con-taining500mLof immobilizedmediumat pH10,different temperatures,and120r/minfor20days.Theammonia nitro-gen removalabilityofthe immobilizedammonia-oxidizing bacteriaundertheabove-mentionedconditionsispresented inFig.5.

Ifthetemperaturewastoohighortoolow,themetabolism of the immobilized ammonia-oxidizing bacteria was slow or waseveninactivated.Thenitritenitrogenconcentration increasedwithincreasingcultivationtime. Thegrowth and metabolismoftheimmobilizedammonia-oxidizingbacteria wereslowatlowtemperatureandincreasedwithincreasing temperature and the concentrationofnitritenitrogen.The enzymatic activity of the immobilized ammonia-oxidizing bacteria was disrupted and even nullifiedat high temper-atures. Theoptimaltemperature was30◦C,atwhich point thehighestnitritenitrogenconcentrationwasobtained.The immobilizedammonia-oxidizingbacteriacouldgrowathigh andlowtemperatures,indicatingthatimmobilizationbyPVA coupled with sodium alginate improved the temperature resistanceoftheammonia-oxidizingbacteria.

Effectofinoculumconcentrationontheammonianitrogen removalabilityofimmobilizedammonia-oxidizing bacteria

To determine the effect of inoculum concentration on the ammonianitrogenremovalabilityofimmobilized ammonia-oxidizing bacteria, the followingimmobilization conditions wereemployed:12%PVA;1.1%sodiumalginate;1.0%calcium chloride; and 0.6, 1.3,2.6, and 3.9immobilizedballs/mL of immobilizedmedium.Thereactionwasconductedina 500-mL shakeflaskcontaining 500mLofimmobilizedmedium at pH 10, 30◦C, and 110r/min for 18 days. The ammonia

0 180 _20ºC 25ºC 30ºC 35ºC 40ºC 160 140 120 100 80 60 40 20 0 2 4 6 Culture time (d) 10 12 14 16 18 20 8 Concentr ation of nitr ite nitrogen (mg/L)

Fig.5–Effectoftemperatureontheammonianitrogenremovalabilityofimmobilizedammonia-oxidizingbacteria.

0 2 4 6 8 Culture time (d) 10 12 14 16 18 250 200 150 100 50 0 Concentr ation of nitr ite nitrogen (mg/L) 6 balls/ 10mL 13 balls/ 10mL 26 balls/ 10mL 39 balls/ 10mL

Fig.6–Effectofinoculumconcentrationontheammonianitrogenremovalabilityofimmobilizedammonia-oxidizing bacteria.

nitrogen removal ability of the immobilized ammonia-oxidizingbacteriaundertheabove-mentionedconditionsis showninFig.6.

Thedevelopmentofammonia-oxidizingbacteria ispoor when the inoculum concentration istoo high because the bacteriawillcompetefornutrients intheculturemedium. Development is also poor when the inoculum concentra-tionistoolowbecausethenutrientsintheculturemedium arenotsufficientforimmobilizedammonia-oxidizing bacte-riametabolism.Therefore, appropriateinoculum levels are required forimmobilized ammonia-oxidizing bacteria. The nitritenitrogenconcentrationwasobservedtoincreasewith increasingcultivationtime.Whenthe inoculum concentra-tion was minimal, the growth of the ammonia-oxidizing bacteriawasslow,whereasatveryhighinoculum concentra-tions,thebacteriacompetedfornutrientsandoxygen.Atan inoculumconcentrationof1.3immobilizedballs/mLof immo-bilizedmedium,thehighestnitritenitrogenconcentrationof 143.41mg/Lwasachieved;hence,thisinoculumconcentration wasconsideredtobeoptimal.

Effectofthenumberofrecyclingtimesontheammonia nitrogenremovalabilityofimmobilized

ammonia-oxidizingbacteria

To determine the effect of the number of recycling times on the ammonia nitrogen removal ability of immobilized

ammonia-oxidizing bacteria, the following immobilization conditionswere employed:12% PVA;1.1%sodiumalginate; 1.0%calciumchloride;and1.3immobilizedballs/mLof immo-bilized medium. The reaction was conducted ina 500-mL shake flask containing 500mL of immobilized medium at pH 10,30◦C,and 120r/min.Theimmobilizedmediumwas replacedwithfreshmediumperiodically.Theeffectsofthe numberofrecyclingtimesontheammonianitrogenremoval ability of the immobilized ammonia-oxidizing bacteria are presentedinFig.7.

As the number of recycling times increased, the cells gradually aged, and their metabolisms slowed. When the immobilizedcellsgrew,thecalciumalginatewallsexpanded and were easily broken. In cases in which these protec-tivebarrierswerelost,immobilizedcellsdied.Afterthefifth recovery,thedenitrifyingcapacityandactivityofthe immo-bilized ammonia-oxidizing bacteria were better preserved, and the volume and mechanical strength of the immobi-lizedballs weresteady. However,althoughtheimmobilized balls did not break, they were soft and approximately 1.5 times larger than the original balls and showed reduced mechanical strength. Thus, we concluded that the immo-bilization process using acombination ofPVA and sodium alginateexertedaprotectiveeffectontheammonia-oxidizing bacteria and presented advantages such as the ability to recycle and save the raw materials used and the bacte-ria. The immobilized ammonia-oxidizing bacteria retained

0 2 4 6 8 Culture time (d) 10 12 14 16 18 180 One time Two times Three times Four times Five times 160 140 120 100 80 60 40 20 0 Concentr ation of nitr ite nitrogen (mg/L)

Fig.7–Effectofthenumberofrecyclingtimesontheammonianitrogenremovalabilityofimmobilizedammonia-oxidizing bacteria. 220 200 180 160 140 120 100 80 60 40 20 0 Concentr ation (mg/L) 0 1 2 3 4 5 6 7 8 9 10 11 Culture time (d)

Nitrate nitrogen concentration (mg/L) Ammonia nitrogen concentration (mg/L) Removal rate of ammonia nitrogen (%) Nitrite nitrogen concentration (mg/L)

12 13 14 15 16 17 18 220 200 180 160 140 120 100 80 60 Remo v al r

ate of ammonia nitrogen, %

40 20 0

Fig.8–Ammonianitrogenremovalabilityofimmobilized ammonia-oxidizingbacteriaunderoptimalconditions.

theirammoniaoxidationcapacityevenafterthefifth recov-ery.

Optimalimmobilizationconditions

Theoptimalimmobilizationconditionswereasfollows:12% PVA,1.1% sodium alginate, 1.0% calcium chloride,and 1.3 immobilizedballs/mLofimmobilizedmediumatpH10and 30◦C.Thereactionwasperformedina1000-mLshakeflask containing 1000mL of immobilized medium at 120r/min. Theammonia nitrogenremoval abilityof the immobilized ammonia-oxidizingbacteriaundertheoptimalconditionsis presentedinFig.8.

Clearly,theammonia-oxidizingbacteriathatwere immo-bilizedunderoptimalconditionsexhibitedbetterammonia nitrogenremovalthandidthenon-immobilizedbacteria.The ammonianitrogenremovalrateoftheimmobilized ammonia-oxidizingbacteriareached90.30%.

Discussion

Characteristicsoftheprocessofimmobilizationof ammonia-oxidizingbacteriabysodiumalginateandPVA

The experimentalresults reportedabove demonstrate that immobilizedammonia-oxidizingbacteriahaveastrong resis-tancetoadverseenvironments,areinsensitivetochangesin temperatureandpH,andexhibitincreasedstability, reusabil-ity,shortreactiontimes,lowcostandanimprovedammonia nitrogen removal ability. The ammonia-oxidizing bacteria were immobilized in sodium alginate and PVA as follows. First, forthePVA preparation,thePVA wasswelledincold waterandthendissolvedinboilingwater.Completeswelling of PVA made the immobilized balls firmer, and the PVA did not dissolve in water after crosslinking itself. Second, agglomerationofthe immobilizedcarrier wasimprovedby adding sodium alginate to defend the carrier from boric acid toxicity.ThePVAcontent(12%)wasmuchhigherthan the sodium alginate content (1.1%) to ensure the stabil-ity of the immobilizedballs. Third, althoughthe optimum pHfortheimmobilizedammonia-oxidizingbacteriawas10, experiments revealed that apH of8was optimalfor non-immobilizedammonia-oxidizingbacteria,indicatingthatthe immobilizedcarrierprotectedtheammonia-oxidizing bacte-riafromalkalineconditions.Lastly,highandlowtemperatures affected theimmobilizedammonia-oxidizing bacteria,with theoptimumtemperaturebeing30◦C.

Comparisonofthetwoimmobilizationmethods

Theammonia-oxidizingbacteriaimmobilizedbysodium algi-nateandPVAweresuperiortothoseimmobilizedbysodium alginatealone15_{forthefollowingreasons.First,the} ammonia-oxidizingbacteriaimmobilizedbysodiumalginateandPVA were toleranttoawiderrange ofpHlevels, whereas those immobilizedbysodiumalginatealonewereeasilydamagedby highlyalkalineconditions.Second,althoughtheimmobilized balls obtainedusingacombinationofsodiumalginate and PVAexhibitedaslightchangeindiameterandcolorafterthe

fifthrecovery,theydidnotbreak.Incontrast,theimmobilized ballsobtainedusingsodiumalginatealonehadalarger diam-eter,weremoretransparent,andwereseverelybrokenafter thefifthrecovery.Thisfindingsuggeststhatahigher concen-trationofsodiumalginatewasneeded,whichcouldincrease theamountofrawmaterialsrequiredandthecost.Finally,the ammonia-oxidizingbacteriaimmobilizedbysodiumalginate andPVAexhibitedabetterammoniaoxidationabilityunder optimumconditionsthanthatofthebacteriaimmobilizedby sodiumalginatealone.

Outlookoffutureresearch

Immobilizedcarriersandmethodsofimmobilized ammonia-oxidizingbacteriamustbefurtherresearched.Forexample, todeterminethebestimmobilizedcarriersandmethods,PVA ormicrocapsulescouldbeevaluatedascarriers,and immobi-lizedcellscould bepreparedusingcovalentorcrosslinking methods.Quickpreparation methodsforimmobilizedcells shouldalsobedevelopedforcontinuousandindustrialized operation.Moreover,immobilizedammonia-oxidizing bacte-riaapplicationsshouldbeextendedtosmallindustrialscales.

Conclusions

Thefollowingconclusionsweredrawnbasedonthefindings ofthepresentstudy:

1. The optimal conditions for the immobilization of ammonia-oxidizing bacteria were as follows: PVA, 12%; sodium alginate, 1.1%;calcium chloride, 1.0%;inoculum concentration, 1.3immobilized balls/mL of immobilized medium;pH,10;andtemperature,30◦C.Theimmobilized ammonia-oxidizing bacteria exhibited strong ammonia oxidation abilities even after being recycled four times. Theammonia nitrogenremovalrateofthe immobilized ammonia-oxidizing bacteria reached 90.30% under the optimalconditions.

2. Comparedwiththeammonia-oxidizingbacteria immobi-lizedbysodiumalginatealone,thoseimmobilizedbyPVA andsodiumalginateweresuperiorwithrespecttopH resis-tance,thenumberofreuses,materialcost,heatresistance, andammoniaoxidationability.

Conflict

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

Thisresearch wassupported bygrantsfrom the Collegiate NaturalScienceFoundofJiangsuProvince(14KJD180003).

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