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  Biological   treatment   of    wastewater   contaminated   with    p-cresol   using Pseudomonas    putida immobilized   in   polyvinyl   alcohol   (PVA)   gel RihamSurkatti,MuftahH.El-Naas* Chemical   and   Petroleum   Engineering    Department,   UAE    University,   P.O.   Box   15551,    Al-Ain,   United    Arab   Emirates 1.   Introduction Phenolic   compounds   such   as   cresols   are   major   toxic   pollutantsthat   are   commonly   observed   in   the   effluent   of    various   industrialwastewaters.    p-Cresol   is   widely   used   in   the   production   of antioxidants,   light-resistance,   dyes,   pigments   and   antiseptics[1,2].   It   is   highly   toxic,   potentially   carcinogenic,   and   can   cause,even   at   low   concentrations,   adverse   effect   on   the   nervous,cardiovascular   and   respiration   systems   [2–4].    p-Cresol   has   beenclassified   as   pollutant   of    Group   C   (possible   human   carcinogens)and   listed   as   a   priority   pollutant   by   the   US   EnvironmentalProtection   Agency   (EPA)   [5–7].The   World   Health   Organization(WHO)   recommends   0.001   mg/l   as   the   acceptable    p-cresol concentration   in   potable   water.   Therefore,   there   is   an   urgent   needto   reduce   the   concentrations   of     p-cresol   to   the   acceptable   limitbefore   discharging   contaminated   wastewater   to   any   water   body.Traditionally,   complex   and   expensive   treatment   techniqueshave   been   employed   for   wastewater   treatments   including:   carbonadsorption   [8],ion   exchange   [9],activated   solvent   extraction   [10],and   chemical   oxidation   [11].   These   alternatives   are   usuallyassociated   with   numerous   drawbacks   such   as   high   cost   and   theformation   of    hazardous   by-products   [12,13].   Biological   treatmenthas   been   gaining   attention   as   an   efficient   technology   that   isversatile,   environmental   friendly,   inexpensive   and   can   offercomplete   mineralization   of    organic   pollutants   [13,14].   Manymicroorganisms   have   shown   high   potential   in   utilizing   cresol   assole   source   of    carbon   and   energy,   under   aerobic   and   anaerobicoperating   conditions,   even   at   relatively   high   concentrations   [15–19].   Aerobic   microorganisms   are   more   favorable   in   the   degradationof    toxic   compounds   since   they   grow   faster   and   can   achievecomplete   conversion   of    organic   pollutant   to   inorganic   compounds(CO 2 ,   H 2 O)   [20]. Pseudomonas    putida   is   well   known   Gram-negative,aerobic   bacterium   that   grows   optimally   at   room   temperature   [21].It   is   a   member   of    the   fluorescent    pseudomonad   group   that   has   greatpotential   in   the   areas   of    bioremediation   and   biocatalysts   [22].Theuse   of    free   bacteria   in   wastewater   treatment   usually   createsnumerous   problems;   consequently   cell   immobilization   in   biologi-cal   wastewater   treatment   is   gaining   attention   by   many   investi-gators   [23–25].   The   immobilization   of    biomass   has   severaladvantages   including   increasing   the   biodegradation   rate;   enhanc-ing   the   control   of    the   bioprocess;   improving   biocatalyst   stabilityand   advancing   the   tolerance   against   harsh   environmental   condi-tions   [26,27].   Among   immobilization   carrier,   PVA   is   a   type   of polymer   that   has   been   widely   used   in   the   area   of    biocatalysts   [28].It   is   inexpensive   and   non-toxic   synthetic   polymer,   which   can   bephysically   cross-linked   by   iterative   freezing–thawing   process,  Journal   of    Water   Process   Engineering   1   (2014)   84–90 A   R    T   I   C   L    E   I   N   F   O  Articlehistory: Received   22   December   2013 Receivedinrevisedform23March2014 Accepted   24   March   2014 Availableonline24April2014 Keywords: Biodegradation P-Cresol Spouted   Bed   Bioreactor   (SBBR) P.    putida ImmobilizationContinuous   flow A   B   S   T   R    A   C   T Inthisstudy,thebiodegradationofsimulatedwastewatercontaining  p-cresol hasbeencarriedoutusing Pseudomonasputida immobilizedinPVAgel,inbatchandcontinuousreactors.Theeffectsofinitialconcentration,temperature,pHandPVAvolumefractiononthebiodegradationof   p-cresol wereevaluatedinabatchSBBR.   Continuousexperimentswerealsocarriedouttostudytheeffect   ofotheroperatingparameterssuchasairflowrateandresidencetimeonthebiodegradationefficiency.Thebatchexperimentalresultsindicatedthatthebiodegradationcapabilitiesof  P.putida arehighlyaffectedbytemperature,pHandPVAvol.%andoptimumperformancewereobtainedat35 8 C,8and40%,respectively.Thebiomassdidnotseemtobeinhabitedbyhighconcentrationof   p-cresol andthebiodegradationdatafittedwelltheMonodnon-inhibitoryModel.ThekineticsdataobtainedfromtheMonodmodelwereutilizedinmodelingthecontinuousbiodegradationprocessandgaveverygoodagreementwithexperimentalresults.    2014ElsevierLtd.Allrightsreserved. * Corresponding   author.   Tel.:   +971   3   713   5188. E-mail   address:   (M.H.   El-Naas). Contents   lists   available   at   ScienceDirect  JournalofWaterProcessEngineering jo   urn   alhom   ep   age:   www.elsevier   .com/locat   e/jw   p   e    2014   Elsevier   Ltd.   All   rights   reserved.  producing   a   gel   that   has   a   fibril   network   with   highly   porousstructure,   high   mechanical   strength,   and   elastic   nature   [24,29,30].The   porous   structure   increases   the   diffusion   of    the   substrate   andoxygen   to   enhance   the   biodegradation   process.   It   is   well   knownthat   this   method   also   decreases   the   toxicity   and   increasesmechanical   strength   for   the   prepared   matrix   [31,32].The   aim   of    this   study   is   to   evaluate   the   biodegradation   of     p-cresol   by   P.    putida   immobilized   in   poly   vinyl   alcohol   (PVA)   gel   andto   investigate   the   effect   of    several   operating   conditions   on   thebiodegradation   process.   Batch   and   continuous   experiments   werecarried   out   in   a   specially   designed   Spouted   Bed   Bioreactor   (SBBR)to   evaluate   the   effect   of    initial    p-cresol   concentration,   operatingtemperature,   solution   pH,   PVA   volume   fraction,   air   flow   rate   andliquid   flow   rate   on   the   biodegradation   rate. 2.   Materials   and   methods  2.1.   Preparation   and   acclimatization   of    immobilized   bacteria   in   PVA gel A   special   strain   of    the   bacterium   P.    putida   was   obtained   in   anAMNITE   cereal   form   (P300)   from   Cleveland   Biotech   Ltd.   The   cerealcontained   a   consortium   of    microorganisms,   with   P.    putida   as   thedominate   strain.   Bacterial   cells   were   extracted   from   the   cereal,immobilized   in   PVA   gel   by   freezing–thawing   process,   andacclimatized   to    phenol   solution   with   concentrations   up   to200   mg/l.   More   details   about   the   bacterial   extraction   andimmobilization   were   reported   in   previous   studies   by   El-Naaset   al.   [28,33].The   immobilized   bacteria   were   then   graduallyacclimatized   to    p-cresol   concentration   up   to   200   mg/l   over   a   periodof    7   days   by   placing   the   PVA   particles   in    p-cresol   synthetic   solutionwith   mineral   nutrients   in   Spouted   Bed   Bioreactor   (SBBR).  2.2.   Spouted   Bed   Bioreactor    (SBBR) The   Spouted   Bed   Bioreactor   was   made   of    Plexiglas   with   a   totalvolume   of    300   ml   and   fitted   with   a   surrounding    jacket   fortemperature   control.   Air   was   continuously   introduced   at   aspecified   flow   rate   into   the   reactor   to   enhance   mixing   and   atthe   same   time   provide   excess   oxygen   to   maintain   aerobicconditions   [14].   The   temperature   of    the   reactor   content   wascontrolled   to   the   desired   value   by   the   surrounding    jacket.   The   SBBR is   characterized   by   a   systematic   intense   mixing   due   to   the   cyclicmotion   of    particles   within   the   bed,   that   is   generated   by   a   single   air jet   injected   through   an   orifice   in   the   bottom   of    the   reactor   [34].   Aschematic   diagram   of    the   Spouted   Bed   Bioreactor   (SBBR)   is   shownin   Fig.   1.  2.3.   Reagents Analytical   grade    p-cresol   was   purchased   from   Sigma–Aldrich   ina   powder   form.   All   other   chemicals   and   PVA   powder   were   of analytical   grade   and   were   obtained   from   BDH,   UK.  2.4.   Synthetic    wastewater     preparation Synthetic   wastewater   of     p-cresol   was   prepared   for   the   desiredconcentrations   in   mineral   nutrient   solution   before   each   experi-mental   run.   The   solutions   were   always   kept   in   a   brown   flask   toavoid   light   oxidation   of    the    p-cresol .  2.5.    Analytical   methods Analysis   for    p-cresol   was   carried   out   using   Chrompack   GasChromatograph,   Model   CP9001   with   flame   ionization   detection(GC/FID).   The   GC   was   equipped   with   capillary   column   (Stabilwax,30   m,   0.25   mm   ID)   and   a   flame   ionization   detector   which   was   set   at250   8 C.   A   sample   of    1   m l   was   filtered   through   syringe   filter   withpore   size   of    0.45   m m   and   injected   into   the   GC.   The   temperatureprogram   started   at   100   8 C   and   increased   at   a   rate   of    20   8 C   min  1 to180   8 C.   The   analyses   were   conducted   in   duplicates,   and   a   standardsolution   was   used   to   recheck   the   accuracy   of    the   GC. 3.   Results   and   discussions  3.1.   Batch   biodegradation   of    p-cresolBatch   experiments   were   carried   out   in   order   to   study   the   effectof    initial   concentration,   temperature,   pH   and   PVA   vol.%   on   thebiodegradation   process.   All   experimental   results   reported   in   thenext   sections   were   based   on   averaging   results   of    repeatedexperimental   runs   (duplicates).  3.1.1.   Effect    of    p-cresol   initial   concentration Batch   experiments   were   carried   out   at   various   initial    p-cresol concentrations   (25,   50,   100,   150   and   200   mg/l)   to   evaluate   theeffect   of    initial   concentration   on   the   biodegradation   rate.   From   thestart,   a   yellow   color   was   observed   for   a   short   period   of    time   during Fig.   1.   A   schematic   diagram   of    the   spouted   bed   bioreactor   (SBBR). R.   Surkatti,   M.H.   El-Naas    /     Journal   of    Water    Process   Engineering    1   (2014)   84–90   85  all   experimental   runs.   This   is   believed   to   be   due   to   the   formation   of   2-hydroxymuconicsemialdehyde ,   which   has   been   produced   from   themetabolizing   of     3-methyl   catechol   by   meta   pathway   [35,36].   Theconcentration   of     p-cresol   at   different   time   intervals   was   measuredquantitatively   by   GC   (FID)   as   mentioned   in   Section   2.5.   Fig.   2   showsthe   experimental   data   of    the   reduction   in    p-cresol   concentration.Clearly   the   reduction   seems   to   be   linear   with   time,   which   indicatesconstant   biodegradation   rates.   The   biomass   did   not   seem   to   beinhibited   by   the    p-cresol   as   indicated   by   the   exponential   increase   of the   biodegradation   rate   with   initial   concentration   (Fig.   3).   The   lackof    inhibitory   effect   even   at   high   concentrations   of    200   mg/l   couldbe   attributed   to   the   biomass   immobilization   in   PVA   gel,   which   actsas   a   protective   shelter   against   substrate   toxicity   [23].   However,   theinhibitory   effect   of     p-cresol   has   been   reported   at   400   mg/l   byBasheer   and   Farooqi   [4]   in   the   biodegradation   of     p-cresol   usingactivated   sludge.  3.1.2.   Effect    of    temperature Experiments   were   carried   out   at   temperatures   ranging   from   25to   45   8 C   to   study   the   effect   of    temperature   on   the   degradation   of     p-cresol .   Other   conditions   such   as   initial   concentration   and   pH   werekept   constant   at   200   mg/l   and   7,   respectively.   Fig.   4   presents   theexperimental   results   of    the   effect   of    temperature   on   thebiodegradation   rate.   Increasing   the   operating   temperature   from25   to   30   8 C   seems   to   enhance   the   biodegradation   capability   of    P. putida ,   reaching   an   optimum   between   30   and   40   8 C.   Temperatureshigher   than   40   8 C   or   lower   than   25   8 C   tend   to   have   negative   effecton   the   biodegradation   rate.   Low   temperatures   usually   result   inslowing   down   the   activity   of    the   bacteria,   while   raising   thetemperature   to   high   values   (higher   than   40   8 C)   leads   to   deactiva-tion   of    the   main   biodegradation   enzymes   [37,38].  3.1.3.   Effect    of     pH  Being   proteins,   enzymes   are   stabilized   by   weak   hydrogen   bondsand   are   generally   affected   by   the   variation   of    pH   [39].In   order   tostudy   the   effect   of    pH   on   the   biodegradation,   experiments   wereconducted   at   pH   ranging   from   5.0   to   8.0.   Initial    p-cresol concentration   and   temperature   were   fixed   at   200   mg/l   and30   8 C.   It   is   believed   that   most   organisms   cannot   tolerate   pH   levelsbelow   4.0   or   above   9.5,   and   the   optimum   pH   for   mostmicroorganisms   lies   in   this   range.   As   shown   in   Fig.   5,thebiodegradation   rate   of     p-cresol   is   diminished   at   pH   5   and   increasessharply   at   higher   values.   The   experimental   results   indicate   that   thebiodegradation   of     p-cresol   reaches   optimum   at   a   pH   value   of    8.However,   any   pH   variation   between   6   and   8   does   not   seem   to   haveany   negative   effect   on   the   biodegradation   rate.   Similar   results   werereported   by   Singh   et   al.   [40]   for   the   biodegradation   of     p-cresol   using Gliomastix   indius . Time (min) 403020100   p  -  c  r  e  s  o   l   C  o  n  c  e  n   t  r  a   t   i  o  n   (  m  g   /   l   ) 050100150200250 25 mg/l50 mg/l100 mg/l150 mg/l200 mg/l Fig.   2.   Variation   of     p-cresol   concentration   with   time;   PVA   vol.%   =   30%   and   T    =   30   8 C.  p-cresol Concentration (mg/l) 220200180160140120100806040200    B   i  o   d  e  g  r  a   d  a   t   i  o  n   R  a   t  e   (  m  g   /   l .   h   ) 180200220240260280300320340360 Fig.   3.   Biodegradation   rate   at   different    p-cresol   initial   concentrations;   PVAvol.%   =   30%   and   T    =   30   8 C. Temperature (ºC ) 50454035302520    B   i  o   d  e  g  r  a   d  a   t   i  o  n   R  a   t  e   (  m  g   /   l .   h   ) 200220240260280300320 Fig.   4.   Variation   of    biodegradation   rate   with   temperature.   Initial   pH   7;   PVAvolume   =   30%   of    total   volume;   initial    p-cresol   concentration   =   200   mg/l. pH 987654    B   i  o   d  e  g  r  a   d  a   t   i  o  n   R  a   t  e   (  m  g   /   l .   h   ) 220230240250260270280290300310320330340 Fig.   5.   Effect   of    the   initial   solution   pH   on   the   biodegradation   rate;   PVA   Vol.%   =   30;initial    p-cresol   concentration   =   200   mg/l;   T    =   30   8 C. R.Surkatti,   M.H.   El-Naas    /Journal   of    Water    Process   Engineering    1   (2014)   84–90 86   3.1.4.   Effect    of    PVA%    in   the   total   operating    volume The   amount   of    PVA   pellets   can   be   considered   as   an   indirectmeasure   of    the   amount   of    active   biomass   available   for   thebiodegradation   of     p-cresol ,   and   consequently   it   is   one   of    the   mostimportant   factors   that   can   affect   the   biodegradation   process.Experiments   were   conducted   to   study   the   effect   of    PVA   volume,   asa   fraction   of    the   total   operating   reactor   volume,   on   thebiodegradation   rate.   The   initial    p-cresol   concentration,   solutionpH   and   temperature   were   kept   at   200   mg/l,   7,   and   30   8 C,respectively.   The   total   operating   volume   of    the   bioreactor   wasmaintained   at   300   ml.   Fig.   6   shows   the   biodegradation   rate   forthree   PVA   volume   fractions   (20,   30   and   40%).   It   is   noticeable   thatthe   biodegradation   rate   of     p-cresol   tends   to   increase   linearly   withincreasing   the   PVA   volume   fraction   in   the   reactor.   This   is   expectedas   the   amount   of    PVA   is   directly   related   to   the   amount   of    bacteria   inthe   bioreactor,   and   hence   the   presence   of    more   PVA   particlesmeans   the   presence   of    more   bacterial   cells.   In   addition,   the   volumefraction   of    the    p-cresol   solution   is   reduced.   Although   larger   PVAvolume   fractions   were   not   tested,   it   is   expected   that   more   PVAparticles   in   the   reactor   will   hinder   the   movement   and   mixing   of    thepellets,   and   consequently,   reduce   the   biodegradation   rate.  3.2.   Continuous   biodegradation    for    p-cresolAlthough   the   batch   experimental   work   provided   essential   dataregarding   the   effect   of    certain   operating   parameters   on   thebiodegradation   rate,   continuous   operation   is   vital   in   assessingthe   potential   industrial   application   of    the   biodegradation   process.Experiments   were   carried   out   to   study   the   continuous   biodegra-dation   process   by   feeding   a   synthetic   wastewater   containing    p-cresol   with   different   initial   concentrations   to   the   bioreactor   using   aperistaltic   pump   (Watson   Marlow,   Model   323)   for   a   period   of    4   h,or   until   the   biodegradation   rate   reaches   steady   state.   The   reactortemperature   and   pH   were   kept   constant   at   8   and   35   8 C,which   arethe   optimum   conditions   obtained   in   the   batch   study.   In   allexperiments   the   volume   fraction   of    the   PVA   pellets   was   kept   at   30%of    the   total   operating   volume.   Again,   all   experiments   were   carriedout   in   duplicates   and   average   values   are   reported   in   the   followingsections.   The   standard   deviation   ranged   between   2   and   5%.  3.2.1.   Effect    of    initial   concentration The   effect   of     p-cresol   concentration   on   the   biodegradation   ratewas   studied   at   four   initial   concentrations:   50,   100,   150   and200   mg/l.   Both   liquid   and   air   flow   rates   were   fixed   at   5   ml/min   and2   l/min,   respectively.   Since   the   SBBR    was   operated   under   well-mixed   conditions,   the   substrate   concentration   in   the   reactor   wasthe   same   as   that   of    the   outlet.   Samples   were   collected   from   theeffluent   stream   and   analyzed   at   different   time   intervals.   Thereduction   of     p-cresol   concentration   as   a   function   of    time   at   differentinitial   concentrations   is   shown   in   Fig.   7,and   indicates   that   thebiodegradation   rate   is   highly   dependent   on   the   initial   concentra-tion.   The   residence   time   inside   the   reactor   (60   min)   was   sufficientto   completely   consume   the   substrate   at   low   concentration   (50   mg/l).   However,   at   higher   concentrations,   the   substrate   was   notcompletely   degraded,   but   steady   state   was   achieved   within   oneresidence   time.   It   must   be   mentioned   here   that   during   the   startupof    the   biodegradation   process,   sharp   reduction   in    p-cresol concentration   was   observed,   followed   by   a   period   of    slowreduction   before   stabilizing.   A   faster   sorption   step   due   to   thephysiochemical   interactions   between   the   organic   chemicals   andmicrobial   cell   walls   is   believed   to   have   resulted   in   the   first   stage   of the   biodegradation   process   [41,42].During   this   phase,   the   removalby   the   biodegradation   is   less   significant.   However,   in   the   secondphase,   the   entire   exposed   surface   of    the   PVA   pellets   gets   saturatedwith   the   substrate   or   reach   closed   to   saturation,   then   the   removalby   adsorption   becomes   less   significant.   In   this   phase,   thecontribution   of    biodegradation   becomes   significant.   The   overallpercent   removal   is   plotted   as   a   function   of    initial   concentration   inFig.   8.In   all   cases,   the   percent   removal   reached   more   than   85%,which   indicates   that   the   immobilized   bacteria   have   very   highpotential   for   the   biodegradation   of     p-cresol . PVA% 45403530252015    B   i  o   d  e  g  r  a   d  a   t   i  o  n   R  a   t  e   (  m  g   /   l .   h   ) 150200250300350400450500 Fig.   6.   Variation   of    biodegradation   rate   with   PVA%   volume;   T    =   35   8 C;   initial    p-cresol concentration   =   200   mg/l;   pH   =   7. Time (min) 250200150100500   p  -  c  r  e  s  o   l   C  o  n  c  e  n   t  r  a   t   i  o  n   (  m  g   /   l   ) 050100150200 50 mg/l100 mg/l150 mg/l200 mg/l Fig.   7.    p-Cresol   concentration   as   a   function   of    time   for   different   initialconcentrations.   Reactor   temperature   =   35   8 C;pH   =   8;   air   flow   rate   2   l/min;   liquidflow   rate   =   5   ml/min.  p-cresol Initial Concentration (mg/l) 200150100500    %    R  e  m  o  v  a   l 708090100 Fig.   8.    p-Cresol   removal   %   as   a   function   of    initial   concentration.   Reactortemperature   =   35   8 C;   air   flow   rate   2   l/min;   liquid   flow   rate   =   5   ml/min. R.   Surkatti,   M.H.   El-Naas    /     Journal   of    Water    Process   Engineering    1   (2014)   84–90   87
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