Cost-effective production of bacterial cellulose using acidic food industry by-products

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h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Biotechnology

and

Industrial

Microbiology

Cost-effective

production

of

bacterial

cellulose

using

acidic

food

industry

by-products

Victor

Revin,

Elena

Liyaskina

∗

,

Maria

Nazarkina,

Alena

Bogatyreva,

Mikhail

Shchankin

NationalResearchMordoviaStateUniversity,FacultyofBiotechnologyandBiology,DepartmentofBiotechnology,Bioengineeringand Biochemistry,Saransk,RussianFederation

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received10February2017 Accepted14December2017 Availableonline13March2018 AssociateEditor:AdalbertoPessoa

Keywords: Bacterialcellulose Stillage Whey Gluconacetobactersucrofermentans

a

b

s

t

r

a

c

t

Toreducethecostofobtainingbacterialcellulose,acidicby-productsofthealcoholand dairyindustrieswereusedwithoutanypretreatmentoradditionofothernitrogensources. Studieshaveshownthatthegreatestaccumulationofbacterialcellulose(6.19g/L)occurs onwheatthinstillagefor3daysofcultivationunderdynamicconditions,whichisalmost 3timeshigherthanonstandardHestrinandSchrammmedium(2.14g/L).Theuseofwhey asanutrientmediummakesitpossibletoobtain5.45g/Lbacterialcelluloseundersimilar conditionsofcultivation.ItisestablishedthatthepHofthemediumduringthegrowthof GluconacetobactersucrofermentansB-11267dependsonthefeedstockusedanditsinitialvalue. Byculturingthebacteriumonthinstillageandwhey,thereisadecreaseintheacidityof thewaste.Itisshownthattheinfraredspectraofbacterialcelluloseobtainedinavarietyof environmentshaveasimilarcharacter,butwefounddifferencesinthemicromorphology andcrystallinityoftheresultingbiopolymer.

licenses/by-nc-nd/4.0/).

Introduction

Bacterialcellulose(BC)attractsextensiveattentionduetoits uniqueproperties,suchasitshighdegreeofpolymerization, high purity, good biocompatibility, biodegradability, high crystallinityandexcellentmechanicalproperties.1–5_Its_ﬁber hasahighaspectratio,withaﬁberdiameterof20–100nm.6

∗ _{Corresponding}_author_at:_National_Research_Mordovia_State_University,_Faculty_of_{Biotechnology}_and_Biology,_Department_of Biotechnol-ogy,BioengineeringandBiochemistry,Saransk,RussianFederation.

E-mail:liyaskina@yandex.ru(E.Liyaskina).

Asaresult,ithasaveryhighsurfaceareapermassunit.This property ofBC,inadditiontoitshighlyhydrophilicnature, resultsinveryhighliquidloadingcapacity.Therefore,BCis usedextensivelyinmanyﬁelds,includingbiomedical materi-als,drugdelivery,tissueengineering,foodindustry,acoustic diaphragm,functionalpaper,opticaldisplays,nanostructured biomaterialsandbiocomposites.7–16

https://doi.org/10.1016/j.bjm.2017.12.012

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Despiteall theadvantagesofBCoverpolymersofplant origin,its productionisarelativelyexpensiveprocess, due primarilytothelowproductivity ofknownstrains andthe use ofexpensive culturemedia.During BCproduction,the culturemediumrepresents approximately 30%of thetotal cost.7_Therefore,_one_important_and_challenging_aspect_of_the fermentationprocessisidentiﬁcationofanewcost-effective culture mediumthat can facilitatethe productionof high yieldswithinshortperiodsoftime.

Traditionally,glucose,fructoseandglycerolwere preferen-tialcarbonsourcesforcelluloseproduction.17,18_To_reduce_the costofBCbiosynthesis,many researchersnow suggestthe useofthefollowingmediathatincludevariouswaste prod-ucts: wheatstraw,19 _spruce _hydrolysate,20 _wood _hot _water extracts,21_pineapple_{agroindustrial}_residues,22_fruit_juices,23 rotten fruit,24 _cotton-based _waste _textiles,25 _molasses,26 waste from the dairy industry,24,27,28 _wine _fermentation waste broth,29–31 _waste _water_of_candied_{jujube-processing} industry,32_waste_and_by-product_streams_from_biodiesel_and confectioneryindustries33 _and_{acetone-butanol-ethanol} fer-mentationwastewater.34

Thinstillage(TS)isaliquidbyproductthatremainsafter microbialethanolfermentationofcarbohydratesbyyeastand subsequentdistillationofthefermentedmash.TScontains salts,carbohydrates,proteins,andorganiccompounds.Wheat thin stillage (W-TS) is a by-product of ethanol production generatedinlarge amountsin Russia. W-TSisrich in car-bonsourcessuchasglycerol,maltose andorganic acids.It contains suchsourcesofnitrogennutritionasbetaineand glycerophosphocholine,31_but_its_high_acidity_(pH_3.76–3.97)31 andhighbiologicaloxygendemand(upto70g/L)makeTSa disadvantagetodistilleryindustries.

Researchersarecurrentlystudyingthepossibilityofusing stillagetoincreasetheyieldofBCinstaticconditions.29,30_Rice thinstillage(R-TS) –awastewaterfromricewinedistillery thatisrichinorganicacids–wasusedtosupplementthe tra-ditionalHSmediumforBCproduction.Inthe50/50R-TS–HS medium,aBCconcentrationof6.26g/Lcouldbeobtainedafter 7daysofstaticcultivation.30

Whey is a by-product of the manufacture of cheese or curd. It contains a large quantity of nutritionally rich components.35_BC_formation_on_whey_was_also_studied_under staticconditions.24,27,28_Carreira_observed_a_low_level_of_BC pro-duction(0.08g/L)oncheesewhey.27_A_low-cost_medium_based ontheagriculturalby-productsoyabeanwheywasprepared andoptimizedforbiocelluloseproductionbyKomagataeibacter sp.PAP1.28_According_to_their_results,_the_optimal_conditions forBCproductionwereasfollows:pHof6.21andethanol con-centration1.61%(v/v).AmaximumBCproductionof4.10g/L wasobtainedontheseventhdayofcultivationunderstatic conditions.

DataforthestudyofBCproductiononthinstillageand whey without treatment and additional power sources in dynamicconditionsare notavailable.Theliteratureonthe cultivationofstrainsofthespeciesG.sucrofermentansonthese wastesisalsolimited.

Theaimof this study,therefore, was toinvestigate the production of bacterial cellulose by the strain Gluconaceto-bactersucrofermentansB-11267inagitatedcultureconditions using highly acidic by-products of the alcohol and dairy

industries without any pretreatment or addition of other nitrogensources.

Materials

and

methods

Bacterialstrainandcultureconditions

The bacterialculture ofG. sucrofermentansB-11267 usedin thisstudywasisolatedfromKombuchatea.1mLofthe sus-pension was withdrawn from the tea and added toa test tube with 9mL of 0.9%sodium chloride (w/v). Serial dilu-tionsfrom 100_to₁₀−6_were_prepared_using_sterilized_saline solution. Analiquot of 0.1mL ofeach dilution was spread plated onto agar containingglucose (100g/L), yeastextract (10g/L),calciumcarbonate(20g/L),andagar(15g/L).Theplates were incubated at28◦Cfor 72h. Thecolonieswithaclear zonearoundwereselectedandtransferredtotube contain-ing10mLofHestrinandSchramm(HS)medium.Acellulose positivestrainwasonethatproducedcelluloseontheliquid medium.

For amoreaccurateidentification oftheisolated strain, basesequenceswereanalyzedusingthe16SrRNAcomplete sequencingmethodandtheirsimilaritieswereexamined.16S rRNAfragmentwasamplifiedbyPCRfromgenomicDNAusing 16SrRNAuniversalprimers.Thesequencingofamplified16s rDNA fragments were performed in Research Institute for GeneticsandSelectionofIndustrialMicroorganisms(Moscow, Russia).AstrainwasdepositedintheRussianNational Col-lection ofIndustrial Microorganisms(VKPM) (AccessionNo. VKPM:B-11267).

For BCproduction,thefollowingmediawere used: Hes-trin andSchrammmedium(HS)(g/L):glucose(20),peptone (5),yeastextract(5),citricacid(1.15),anddisodiumhydrogen phosphate(2.7),pH6.0;cheesewheywithoutpHadjustment, pH4.96;thinstillage(TS)withoutpHadjustment,pH3.95;TS withpHadjustmenttopH5.0andpH6.0.Allculturemedia wereautoclavedfor20minat120◦C.

Themediawere inoculatedwith10% (v/v)inoculum.To preparetheinoculum,G.sucrofermentansB-11267fromanagar plate wastransferred asepticallyinto a 250mLErlenmeyer ﬂask containing 100mL of culture medium and incubated on a shaker incubator (Model ES-20/60, BIOSAN, Latvia) at 28◦Cfor24hat250rpm.BCwasproducedin250mL Erlen-meyerﬂaskscontaining100mLofculturemediumonashaker incubator at28◦C for 3days at 250rpm. Theexperiments wereconductedintriplicate,andtheaverageoftheresultsis reported.

PuriﬁcationofbacterialcelluloseandanalysisofBCyield

Afterincubation,BCwascollected,washedthoroughlywith distilledwatertoremovemediumcomponents,andtreated with1%sodiumhydroxide(w/v)solutionat80◦Cfor1hto eliminatebacterialcells.Further,BCwasrinsedextensively with6%aceticacid(v/v)andthenwithdistilledwateruntil the pHof the water became neutral. Thepuriﬁed BC was driedtoconstantweightat105◦C.BCproductionhasbeen reported as gram dry weight of cellulose per liter of the medium(g/L).

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AFMmicrographsofBC

The surface morphologies of BC were studied by contact atomicforcemicroscopy(AFM)usinganSPM9600(Shimadzu, Japan).BCsampleswereairdriedonaglassslideintheform ofathinﬁlm.Asiliconnitridecantileverwithnominalradius ofpyramidaltip2nmwasused.Scanratesrangedfrom0.6to 1.0Hz/s.Imageresolution256×256pointswereset.

HPLC

Theamountsofsugarsandmonocarboxylicacidswere quan-tiﬁed by high-performance liquid chromatography (HPLC) duringcultivationofG. sucrofermentansB-11267inthin stil-lage(TS)andwhey.Thecontrolwastheinitialconcentration ofsugars and monocarboxylic acids inthe media. Prior to HPLCanalysis,all samplesand standardswere mixedwith acetonitrile (ACN) (3:1, v/v), then placed into sealed tubes andcentrifugedat13,000×gfor5min.Monosaccharidesand monocarboxylic acids were analyzed on Shimadzu LC-20A ProminencewithrefractometricdetectionusingRID10Aand acomputercontroller.TheSUPELCOSILTM_LC-NH2_HPLC col-umn(4.6×150mm)wasused,andthemobilephasewasACN: DH2O(distilledwater)(3:1,v/v)withaﬂowrateof0.5mL/min. Theinjectionvolumewas20␮L,andthecolumntemperature was40◦C.

FTIRspectraofBC

BCwasfreeze-dried andcrushed into powderform,mixed with potassium bromide, and pressed into a small tablet thatwassubjectedtoFouriertransforminfrared(FT-IR) spec-troscopy using a Fourier transform infrared spectrometry (FT-IR)IRPrestige-21 (Shimadzu,Japan)inabsorptionmode. Foreachsample,32scansata4cm−1resolutionatwave num-bersrangingfrom4000to400cm−1werecollected.

X-raydiffraction

X-raydiffraction(XRD)measurementwascarriedoutto ana-lyzethe change in crystallinity of the produced BC by an X-raydiffractometerEmpyrean(PANalytical,Netherlands)in theﬁlteredradiationofthecopperanode(=0.15418nm,40kV b30mA)withinanangularrange(2) from 10to60◦. Two-coordinatedetectorPixcel3Dworkinginthemodeoflinear scanning(255pixelsonastrip)withtheresolutionof0.013 degree/strip wasalso used. Thesampleswere freeze-dried usingFreeZoneFreezeDrySystem(Labconco,USA).The crys-tallinityindex(CrI)wascalculatedfromtheratiooftheheight ofthe002peak(I002,2=22.5◦)andtheheightofminimum (Iam)betweenthe002andthe110peaks(Iam,2=18◦)(Eq.(1)). CrI (%)=

I002_I002−Iam

×100 (1)

Statisticalanalysis

All presented data are averages of at least three runs of experiments, performedwith threeto sixreplicates ofthe mean.Standarddeviationsofthemeanwerecalculatedusing

7 6 5 4 3 2 1 0 0 1 2

Culture time (days) Ts Hs Whey

3

BC (g/L)

Fig.1–BCproductionbyG.sucrofermentansB-11267in agitatedcultureconditionsusingHSmedium(HS),thin stillage(TS)andwhey.

MicrosoftExcel2013(MicrosoftCorporation,Redmond,USA). TheobtaineddatawerestatisticallyanalyzedbyStudent t-test:two-sampleassumingequalvariances.Thedifferences wereconsideredsigniﬁcantatthelevelofp<0.05.

Results

Productionofbacterialcellulose

BCproductionbyG.sucrofermentansB-11267wasinvestigated inagitatedcultureusingthinstillage(TS)andwheywithout anypretreatmentoradditionofnutrientsourcestoreducethe manufacturingcosts.HSmediumwasusedforcomparison.

AsshowninFig.1,themaximumBCyield(6.19±0.12g/L) wasobtainedafter3daysofcultivationonTS.Thisvaluewas approximately3timeshigherthantheyieldonHSmedium (2.14±0.02g/L).Inthewheymedium,aBCconcentrationof 5.45±0.09g/Lcouldbeobtainedafter3daysofcultivation.The maximumrateofproductformationonthewasteoccurson theﬁrstdayofgrowthofthebacterium.

ForadeeperunderstandingofthegrowthofG. sucrofermen-tansB-11267andBCproductiononthethinstillageandwhey, weinvestigatedthe consumptionoftheprimarysourcesof carbonthatarepartoftheseenvironments.

Todetecttheconsumptionoforganicacidsandsugars dur-ingcultivationofG.sucrofermentansB-11267inTSandwhey, HPLCanalysisofsampleswasperformed(Fig.2A).

According tothe data obtainedafter threedays of bac-teriumcultivationinTSmedium,therewasintakeofpentoses from 1.3±0.15mg/mL to 0.15±0.11mg/mL, hexoses from 0.6±0.13mg/mL to 0.15±0.10mg/mL, and dextrins from 0.84±0.12mg/mLto0.4±0.10mg/mL.Therewasalsoasteady decreaseintheconcentrationofmonocarboxylicacidsin cul-turefrom1.55±0.15mg/mLto0.11±0.10mg/mL.Therefore, withinthreedaysofcultivationofG.sucrofermentansB-11267 ontheTS,almostfullconsumptionofsugarsandorganicacids occurred.

We also investigated the consumption of sugars and organic acidsinthe processofcultivationofthebacterium onwhey(Fig.2BandC).

According tothe presenteddata, the amount oflactose decreasedfrom42.12±1.91mg/mLto22.4±1.33mg/mL dur-ingtheﬁrstdayofcultivation.Betweenoneandthreedays, its concentrationwas almoststable. Inaddition, therewas adecrease intheconcentrationofhexoseswithincreasing cultivationtimeofthebacterium.Accordingtothepresented

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2 1,5 1 0,5 0 7 6 5 4 3 2 1 0 50 45 40 35 30 25 20 15 10 5 0 0 1 2 3 0 1 2 3 0 1 2

Culture time (days)

Hexoses (including glucose) Glucose Lactose Monocarboxylic acids 3 Monocarboxylic acids Pentoses Dextrins Hexoses Concentr ation, mg/mL Concentr ation, mg/mL Concentr ation, mg/mL

A

B

C

Fig.2–Sugar,monocarboxylicacidanddextrincontentin TSmedium(A)andwhey(BandC)during3daysdynamic cultivationofG.sucrofermentansB-11267(byHPLC).

data,afterthreedaysofbacteriumcultivation,theamountof glucosedecreasedfrom3.2±0.29mg/mLto0.3±0.03mg/mL. Duetothepresenceoflargeamountsofavailable carbohy-drates,theconsumptionoforganicacidsoccurredtoalesser extentthaninthepreviousexamplewithstillage.Duringthe ﬁrst dayofcultivation, their concentrationdecreased from 2.21±0.17mg/mLto1.51±0.13mg/mL.Theconcentrationof monocarboxylicacidswasalmostthesameatalaterstage.

Asbiochemicalreactionsthatoccurinaculturemedium areassociatedtoalargeextentwithpH,wealsostudiedthe dynamicsofchangesintheacidityofthemediumduringthe cultivationofG.sucrofermentansB-11267.

After2daysofcultivatingthebacteriumonTSandwhey, apHincreasewasobserved–from3.95±0.02to6.45±0.01 andfrom4.96±0.02to8.02±0.03,respectively(Fig.3),whichis mostlikelyduetotheconsumptionoforganicacidsofstillage andwhey.Theglucoseculture(HSmedium)gavethelowest pHof4.0onthe2nddayofcultivation,astheglucose dehydro-genase(GDH)ofGluconacetobactercouldconverttheglucoseof

3 4 5 6 7 8 9 10 0 1 2 3 pH

Culture time (days)

TS HS Whey

Fig.3–Time-courseofpHduringcultivationofG. sucrofermentansB-11267inHSmedium(HS),thinstillage (TS)andwhey. 0 1 2 3 4 5 6 7 0 1 2 3 BC (g / L )

Culture time (days)

pH 3,95 pH 5 pH 6

Fig.4–BCproductionbyG.sucrofermentansB-11267at differentinitialpHoftheculturemediumwiththinstillage.

theHSmediumintogluconicacid,whichalsoresultedinpH decrease.36

TheeffectofinitialpHoftheculturemediumwiththin stil-lageoncelluloseproductionwasinvestigated.Whenculturing thebacteriumG.sucrofermentansB-11267onTS,thehighestBC productionof6.19g/LwasobservedatinitialpH3.95(Fig.4).

Structureofbacterialcellulose

It is known that the composition of the nutrient medium impacts not only performance but also the structure of cellulose.37

Thispapershowsthatintheprocess ofcultivating bac-teriumindynamicconditions,agglomeratesofcellulosewith differentshapesandsizesareformed(Fig.5).Itisfoundthat theTSandwhey,comparedtostandardHSmedium,yieldﬁner andhomogeneousstructures.

Tostudy the microscopicdetailsofthe polymer,atomic force microscopy (AFM)was used.AFMimagingshows the biopolymermicro-morphology,topographyandmicroscopic detailsofitssurface.Theouterpartoftheagglomerateswas analyzed.ThesamplesobtainedinTS,wheyandtheclassic environmentHSwerecompared(Fig.6).

The morphology of BCsamples from G. sucrofermentans B-11267displayednano-scale networkstructure(Fig.6).BC generatedinfermentationusingwastewaterhadamore com-pact structure. There are also differences in the width of the microfibrils (Table 1). Narrow microfibrils of 40–70nm are formed on the TS, while more widely microfibrils of 100–180nmformonwhey.ThewidthofBCmicrofibrilsformed onstandardHSmediumaveraged60–90nm.

Themorphological changes inBCcan inﬂuencevarious propertiesandmicrostructuressuchascrystallinity.Inorder

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Fig.5–BCproducedbyG.sucrofermentansB-11267inagitatedcultureconditionsusingHSmedium(A),whey(B)andthin stillage(C). 1.00 um 500.00 um 1.00 um 0.00 0.00 0.00 0.00 0.50 0.50 1.00 1.00 1.50 1.50 0.00 0.00 0.00 0.00 0.50 1.00 1.50 0.50 1.00 1.50 408.05 [nm] 448.35 [nm] 3.12 x 3.12 um 3.75 x 3.75 um 1.87 x 1.87 um 0.00 305.80 [nm] 0.50 1.00 1.50 0.00 0.50 0.00 1.87 x 1.87 [um] Z 0.00 - 305.80 [nm] 1.87 x 1.87 [um] Z 0.00 - 448.45 [nm] 1.87 x 1.87 [um] Z 0.00 - 304.24 [nm] 1.00 1.50 305.80 [nm]

A

B

C

448.45 [nm] 304.24 [nm] 448.45 [nm] 304.24 [nm]

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Table1–WidthofcellulosemicroﬁbrilsbyAFMand crystallinitybyX-raydiffraction.

Medium Width(nm) Crystallinity(%)

HS 60–90 79.7

TS 40–70 82.3

Whey 100–180 50.2

toevaluatethecrystallinestructureandthechangein

crys-tallinityofBCproducedfromdifferentculturemedia,X-ray

diffractionwasusedandtheX-raypatternsofBCare

exhib-itedinFig.7.TheobtainedX-raypatternsshowcellulosewith thesamechemicalstructurebutwithdifferentcrystallinity degrees(Table1).

ThediffractiondiagramsofBCproducedbyallBCsamples showedthreemainpeaksat214.4◦,16.8◦ and22.5◦ (Fig.7), correspondingtothecrystallographicplanesof(100),(010)and (110),respectively.38_The_intensity_of_the₁₀₀_reflection_is_larger thanthatofthe010onewhenthefilmisparalleltotheX-ray beamandtheeffectisreversedintheperpendicular orienta-tion.Thisrevealsastronguniplanaritythatisduetothefact thatthecelluloseribbonsarepreferentiallyorientedparallel tothefilmsurfaceduringdrying.38

The crystallinity index values of BC from TS medium (82.3%)wereslightlyhigherthanthatofBCfromHSmedium (79.7%), which is similar to that reported in a previous studyforBCobtainedthroughthesameprocess.30_The crys-tallinity index of BC was reduced (50.2%) when grown in whey.

TostudythechemicalstructureofBC,weanalyzedFTIR spectra atwavelengths ranging from 400 to 4000cm−1. As showninFig.8,thefunctionalgroupsofBCsamplesobtained fromfermentationusingbothwastewatersandHSmedium werealmostthesame.Thecharacteristicbandsofcellulose (type I) appearedat3352and 3246cm−1 forthe stretching vibration of hydroxyl groups (–OH), at 2897cm−1 for the asymmetricstretchingvibrationofmethylenebridge(–CH2–), at2855cm−1forthesymmetricstretchingvibrationofmethyl (–CH3),andat1061cm−1fortheC–O–CandC–O–Hstretching vibrations of the sugar ring.34 The signals near 3240 and 750cm−1 were assigned to the triclinic I␣ allomorph, and

the signals near 3270 and 710cm−1 were assigned to the monoclinic I␤ form. Therefore, the synthesized cellulose containedbothallomorphs.

Discussion

In thisstudy,thehighestBCyieldof6.19g/Lwasobserved using TS, which is almost 3 times higher than on stan-dard HS medium(2.14g/L). The use ofwheyas a nutrient mediummakesitpossibletoobtain5.45g/LBCundersimilar conditionsofcultivation.FermentationontheTSandwhey inﬂuencedthemicro-morphologyandcrystallinityofBCbut exhibitednoeffectonitschemicalstructure.

Aspreviouslyreported,BCismadeofsubfibrilsof1.5nm assembled into nanofibrils of2–4nm(but up 25nm) width composed of 10–250 single polymeric chains and 1–9nm lengthequivalentfrom 2000up 18,000–20,000glucoseunits organizedintonanoribbonsof40–60nmwidth.8_In_this_study, thesynthesizedmicrofibrilsareclearlyribbon-shapedwitha widthrangingbetween40and90nmforHSmediumandTS. Suchobservationsagreewiththetypicalmorphologyreported forBC.8_More_widely_{microfibrils}_are_formed_on_whey.

Furthermore,thecrystallinityindexoftheBCfromTSis higherthanthatfromothermedia.ThisrevealsthatTScan affect the aggregationand crystallizationofBCmicroﬁbrils andincreasethecrystallinityindex.

Stillage contains organic and inorganic compounds, someofwhichmay bevaluablefermentationcoproducts.31 Ratanapariyanuch et al. used HPLC to analyze the com-ponents of TS.31 _They _found _that _the _major compo-nents in wheat thin stillage were dextrin (8.47–11.65g/L), glycerol (2.39–7.87g/L), lactic acid (5.07–7.41g/L), acetic acid (0.56–2.72g/L), succinic acid (0.63–0.93g/L), ethanol (0.23–1.31g/L), maltotriose(0.14–1.10g/L), maltose monohy-drate(0.03–1.05g/L),glycerophosphorylcholine(0.91–1.11g/L) andbetaine(0.8–1.03g/L).31

Wheycontainslactose(approximately70%ofalldry sub-stances).Inadditiontolactose,othercarbohydrates,suchas glucose, galactose, lactulose, and arabinose, are present in wheyinsmallamounts.Thehighbiologicalvalueofwheyis duetoproteins(mainlyalbuminsandglobulins),aminoacids, vitamins,andorganicacids(lactic,citric).35

100000 80000 60000 Intensity (counts) 40000 20000 10 15 20 25 30 35 Diffraction amgel 2θ (°) 40 45 50 TS HS Whey

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(1) (2) (3) 3352 3246 2920₂₈₉₇2855 1061 600 900 1200 1500 1800 2100 2700 3300 3900

Fig.8–FTIRspectraofBCproducedfromdifferentculturemedia:1–HSmedium;2–thinstillage;3–whey.

It is known that the choice of carbon source and its quantity is one of the main factors ininﬂuencing the BC formation.5,17,18

According to research literature ﬁndings, organic acids haveapositiveeffectonBCbiosynthesis.36_Thus,_the_efﬁciency ofitsformationisenhancedwhenthemediumcontainsacetic acid,whichreducestheformationofgluconicacid.37

Aceticacidisoneofmetabolizedproductsinthemetabolic networkofG.xylinus.37_It_can_be_transformed_into_acetyl-CoA andmetabolizedintricarboxylicacid(TCA)cycletogenerate ATP.Inaddition, aceticacidcanbeusedinthepathwayof gluconeogenesis,increasingglucoseavailability.39

Thereareﬁndingsthatstatethepositiveinﬂuenceof lac-ticacidonthesynthesisoftheBC.40 _Lactate_stimulates_the growthofcellsatanearlystageofculturedevelopment.40_It_is assumedthatitactsasanacceleratoroftheKrebscycle, result-ingintheincreasedformationofcelluloseandcellgrowth.40

Thesigniﬁcant effectofsuccinic acid on BCproduction enhancementhasbeenreportedpreviously.29,30_In_addition_to succinicacid,otherorganicacidsinTSsuchasgluconicacid, citricacidandmalicacidarealsobeneﬁcialforBCproduction enhancement.29,30_These_organic_acids_are_all_located_in_the pathwayofTCAcycle.

According to literature, the pH of the culture medium isacritical factor forBC productivity.41–43 _Previous_studies demonstratedthattherangeofpHvalueforBCproduction was approximately 4–7 and the optimum pH for BC pro-ductionvaries withthe bacterium strains, but had usually beenattributedtobewithinaneutraltoslightlyacidic pH range.5,41,43,44

Areasonable question is therefore if similar conditions favorBCproductioninthepresentedsystem.TSusedinthis researchwasacidic(pH3.95)duetothepresenceoforganic acids.Thisresultwas consistentwiththe work of Ratana-pariyanuchetal.,whoreportedthattherangeofpHofW-TS was3.76–3.97.31

WhenusingTS,themaximumBCproductionofG. sucro-fermentansB-11267reached6.19g/LwheninitialpHwas3.95. Theavailabilityoforganicacidsinthewastethatcontribute toitshighacidityalsohasapositiveimpactontheformation ofbiopolymer.TheinitialpHformaximumcellulose produc-tionwasdistinctivelylowcomparedtothepHtypicallyused inincubationwithbacteriaofthisgenus.Overall,thestrain G.sucrofermentansB-11267isacid-resistantandcanbeused

sustainableinfermentationprocessesforBCproductionusing acidicfoodindustryby-products.

Severalattemptshavebeenmadetoharvestorengineer strainsthatareresilienttolowpHmediaduringBC biosynthe-sis.Castroetal.reportedthatabacterialstrainisolatedfrom thefermentationofColombianhomemadevinegar, Gluconace-tobactermedellensis,ishighlytoleranttolowpH:anoptimum yield of4.5g/Lwas achieved at pH3.5, which is generally toolow forother bacterialspeciestofunction.38 _Kim_et _al. reportedthatcelluloseproducingbacterialstrain Gluconaceto-bactersp.gelSEA623-2isolatedfromcitrusfruitjuicefungus isalsohighlytoleranttolowpH.Inordertoinvestigatethe effectofinitialpHonBCproduction,SEA623-2wascultivated inthebiocelluloseproductionmediumwithdifferentpHlevels (2.5–5)understaticculture.TheoptimumpHforBCproduction was3.5.45

Recently,ithasbeenreportedthatBCcanbesynthesized underalkalineenvironment.44_A_bacterial_strain_isolated_from fermentedfruitjuice,KomagataeibacterintermediusFST213-1, canproduceBCwithinpH4–9andexhibitmaximumBC pro-duction(1.2g/L)atpH8inshort-term(4-day)cultivation.44

Changes in pH can indicate the biochemical reactions occurring in the culture.43 _BC _synthesis _is _an energy-dependentprocess,andtherefore,inHSmediumwithonly glucose as energy source, the oxidation of glucose to glu-conate prevails overthe synthesisofBC. Theformationof gluconicacidsubtractsthecarbonskeleton(glucose)fromthe BCproduction.46_Furthermore,_the_development_of_gluconate signiﬁcantlydecreasesthepHofthemediumandthisisan additionallimitationtotheBCproduction.

CulturingthebacteriumG.sucrofermentansB-11267onTS andwhey,apHincreasewasobserved,whichismostlikely duetotheconsumptionoforganicacidsofstillageandwhey. The concentration ofmonocarboxylic acids inTS medium after3daysofcultivationwasnegligible,suggestingthatthey weremetabolizedviaTCAcycleorthattheformed oxaloac-etate wasdecarboxylatedinto pyruvatethatisusedbythe gluconeogenesispathwaytoproduceglucose,aspreviously statedforK.xylinus.39

ThisresultwasconsistentwiththeworkofHwangetal., whostatedthatduringthecellgrowth/celluloseproduction phase,celluloseproductionwasaccompaniedbycellgrowth andconsumptionofthegluconicacid.47_At₃₅_h,_the_cell_mass andcelluloseconcentrationweremaximalandthepHofthe

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culturebrothincreasedfrom thelowestvalueof4.3to6.5. Inthestationaryphase,however,gluconicacidcontinuedto beconsumedwithoutfurtherproductionofcellulose,andthe pHoftheculturebrothincreasedcontinuouslyfrom6.5to7.0. Itwasconsideredfrom thisresultthattheculturemedium mustcontainacertainlevelofgluconicacidforcellulose pro-duction.However,furtherstudywillbeneededtoclarifythis point.

Conclusion

Onthebasisoftheobtainedresults,itcanbeconcludedthat acidicby-productsofthealcoholanddairyindustries,such asthin wheatstillage andwhey,are apromisingsubstrate forobtainingBC.Theselow-costby-productscansigniﬁcantly contribute to the reduction of production costs. Further-more,BCcanbeproducedbyemployingthinwheatstillage toachievehigh yieldsand qualityofthe product. Remark-ably,the strainG.sucrofermentansB-11267investigatedhere yieldsalargeamountofBCinlowpHmedia,indicatingits tolerancetoacidicenvironmentswhileoptimallyproducing cellulose.Suchsituationishighlydesirableinindustrially rele-vantfermentationprocessesforBCproductionwiththeadded advantagethatitlimitsmicrobialcontamination.Thus,the resultsofthisstudypresentanexcellentviewpointtoproduce ananomaterialwithdifferentpossibleapplicationsbyusing acidicfoodindustryby-products.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgments

TheauthorswouldliketothankDrK.N.NishchevandDrV.M. KyashkinforhelpinAFMandXRDstudies.

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