research article removal of olive mill wastewater...

Hindawi Publishing CorporationJournal of Waste ManagementVolume 2013 Article ID 630209 7 pageshttpdxdoiorg1011552013630209

Research ArticleRemoval of Olive Mill Wastewater Phenolics with the Use ofa Polyphenol Oxidase Homogenate from Potato Peel Waste

Florin Daniel Demian1 and Dimitris P Makris2

1 Laboratory of Chemistry of Natural Products Mediterranean Agronomic Institute of Chania (MAICh)PO Box 85 73100 Chania Greece

2 School of Environment University of the Aegean Mitr Ioakim Street Lemnos 81400 Myrina Greece

Correspondence should be addressed to Dimitris P Makris dmakrisaegeangr

Received 17 September 2013 Accepted 1 November 2013

Academic Editor Milva Pepi

Copyright copy 2013 F D Demian and D P Makris This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Olive mill wastewater (OMW) originating from a two-phase olive oil producing plant was treated with a crude polyphenoloxidase (PPO) homogenate prepared from potato waste peels The treatments carried out were based on a 23-full-factorialcentral composite design (CCD) in order to identify optimal operational conditions with regard to polyethylene glycol (PEG)concentration pH and treatment duration The treatment performance was assessed by estimating the reduction in totalpolyphenol (TP) concentration The model obtained produced a satisfactory fitting of the data (1198772 = 096 119875 = 00017) Theutilisation of the predictivemodel enabled the theoretical calculation of the optimal set of conditions whichwere pH = 4 119905 = 357 hand [PEG] = 900mg Lminus1 Under these conditions the optimal theoretical removal calculated was 54 plusmn 9 Examination of thetreated samples with high-performance liquid chromatography (HPLC) showed that the potato homogenate afforded changes inthe polyphenolic profile Based on the experimental evidence oxidation pathways were proposed

1 Introduction

Olive mill wastewater (OMW) is a highly polluting effluentof olive oil production and its disposal is a serious envi-ronmental peril The main negative effect associated withits dumping is the high toxicity to plants microorganismsand aquatic organisms [1] Thus OMW is not appropriatefor watering and fertilizing purposes and it is recalcitrant tobiodegradation by bacteria and fungi The toxicity of OMWis largely attributed to its exceptionally high polyphenolicburden which may reach up to 80 g Lminus1 This lends OMWa CODBOD

5ratio of 25ndash5 which makes it 5ndash80 times

stronger pollutant than domestic sewage [1]The remediation of OMW has been a subject of several

studies dealing with catalysis-based oxygenation with vari-ous inorganic catalysts [2] and biological treatment mainlywith laccase- or peroxidase-producing fungi [3ndash7] The useof enzymes in bioremediation processes has gained a wideacceptance because of the recognition that enzymes fromvarious plant and microbial sources have several advantages

over conventional physical and chemical treatments Theseadvantages include selective removal of particular pollutantsapplication to xenobiotic recalcitrant compounds high reac-tion rates operation over a wide range of pH and salinityreduction in sludge volume and simplicity of controlling theprocess [8 9]

Among the enzymatic processes for waste treatmentperoxidase- and polyphenol oxidase-catalysed treatmentsof phenols are probably the most comprehensively studied[10] Plant food wastes and byproducts including trimmingsand peels might contain a range of enzymes capable oftransforming bioorganic molecules and thus they may havepotential uses in bioremediation processes [11] PPO frompotato in particular has been shown to effectively polymeriselignin fragments in artificial wastewater in combinationwith peroxidase [12] detoxify bisphenol A [13] decolourisedying effluents [14 15] degrade pentachlorophenol [16] andremove phenol [17] and other halogenated phenols [18] fromaqueous effluents

2 Journal of Waste Management

To the best of our knowledge potato PPO has neverbeen investigated for its potential to removeOMWphenolicsOn such a conceptual basis this study was undertaken toexamine the prospect of using crude potato peel PPO toremove OMW phenolics by the application of a 23-full-factorial design and response surfacemethodology includingpH treatment time and polyethylene glycol (PEG) as criticalfactors

2 Materials and Methods

21 Reagents and Chemicals Bradford reagent trans-chloro-genic acid (CGA) trichloroacetic acid (TCA) and 4-amino-antipyrine (4-AAP)were fromSigmaChemical Co (St LouisMO USA) NN-Dimethylformamide (DMF) and potassiumferricyanide were fromMerck (Darmstadt Germany)

22 Olive Mill Wastewater (OMW) TheOMWwas obtainedfrom a two-phase processing plant located within theprefecture of Chania (Crete Greece) The plant processesorganically cultivated olives for the production of extravirginolive oil The OMWwas collected immediately after disposalto avoid any changes in the polyphenolic composition Uponreception the waste was stored at 4∘C in the dark until usedfor no longer than a week

23 Preparation of the Potato Peel Homogenate Brown-skinpotatoes (Solanum tuberosum L) were purchased from agrocery (Chania Crete) The tubers were transferred tothe laboratory and manually peeled and the peels wereimmediately frozen with liquid nitrogen The frozen tissuewas ground with a pestle and a mortar and mixed withcharcoal to remove phenolics Typically 1 g of charcoal wasused for approximately 25 g of ground tissue The charcoal-treated tissuewas thenmixedwith 150mLof phosphate buffer(50mM pH 66) and homogenised in a domestic blender toform slurry The homogenate was centrifuged at 4000 g for15min and filtered through paper filter The clear filtrate wasused as crude enzyme source

24 Polyphenol Oxidase Activity The assay mixture con-tained 025mL 4-AAP (10mM in water) 01mL substrate(100mMCGA in DMF) 06mL of phosphate buffer (50mMpH 66) and 01mL enzyme extract The formation ofchromogen (quinoneimine dye) adduct of 4-AAPCGA wasmonitored by recording the absorbance at 510 nm (119860

510)

for over 3min against suitable blank One enzyme unit(U) was defined as 120583moles of quinoneimine dye formedper min For all determinations 120576 = 12 000 was usedfor the quinoneimine dye [19] Control reactions by usingheat-inactivated homogenate were also carried out For alldeterminations a computer-controlled HP 8452A diode-array spectrophotometer was used Protein content wasdetermined according to Bradford 1976 [20] using bovineserum albumin as the standard

25 Enzymatic Treatment The waste was homogenised byvigorous shaking then filtered through filter paper and

Table 1 Experimental values and coded levels of the independentvariables used for the 23-full-factorial design

Independent variables Code units Coded variable levelminus1 0 1

[PEG] (mg Lminus1) 1198831

100 500 900pH 119883

24 6 8

Time (h) 1198833

1 3 5

diluted 1 20 with tap water The OMW was adjusted at vari-ous pH levels (Table 1) using either 01 NHCl or 01 NNaOHThe medium (final volume 20mL) was composed of 19mLOMW 05mL crude PPO extract (final total protein concen-tration 155120583gmLminus1 142U) and 05mL PEG solution Thefinal mixture was placed in a 30mL screw-cap glass reactorbearing a teflon-coated magnetic stirrer and sparged withair for 5min prior to treatments to ascertain the presenceof sufficient amount of oxygen Treatments were carried outunder stirring with a magnetic stirrer operating at 400 rpmControl treatments with heat-inactivated homogenate werealso carried out

26 Total Polyphenol Determination A sample (1mL) waswithdrawn from the reaction medium and placed in a 15mLEppendorf tube The sample was mixed with 01mL TCAvortexed and then centrifuged in a table centrifugator at10000 rpm An aliquot of 05mL of appropriately dilutedOMWwasmixed with 03mL deionised water 01mL 4-AAP(20mM in 025M NaHCO

3) and 01mL potassium ferri-

cyanide (85mM in 025M NaHCO3) Absorbance readings

were carried out at 510 nm against suitable blank [21] Resultswere expressed as caffeic acid equivalents (CAE) using acalibration curve of 119860

510against caffeic acid concentration

(125ndash200mg Lminus1)

27 Experimental Design and Statistical Analyses A 23-full-factorial experimental design was used to identify therelationship existing between the response function andprocess variables as well as to determine those conditionsthat optimised the PPO-catalysed oxidation process Theresponse function considered was the removal of TP fromthe reaction medium The three independent variables orfactors considered were PEG (119883

1 varying between 100 and

900mg Lminus1) pH (1198832 varying between 4 and 8) and time (119883

3

varying between 1 and 5 h) Each variable to be optimised wascoded at three levels minus1 0 and 1 (Table 1) The value rangesused for PEGwere based on preliminary experiments and theliterature data [21 22]

The three independent variables were coded according tothe following equation

119909119894=119883119894minus 1198830

998779119883119894

119909119894= 1 2 3 (1)

where 119909119894and 119883

119894are the dimensionless and the actual value

of the independent variable 119894 1198830is the actual value of

the independent variable 119894 at the central point and Δ119883119894

Journal of Waste Management 3

Table 2 Measured and predicted TP removal values determinedfor individual design points

Design point Independent variables Response ( TP removal)119883111988321198833

Observed Predicted1 minus1 minus1 minus1 426 4222 minus1 minus1 1 421 4153 minus1 1 minus1 357 3434 minus1 1 1 340 3295 1 minus1 minus1 400 3976 1 minus1 1 255 2557 1 1 minus1 485 4778 1 1 1 340 3299 minus1 0 0 0 3510 1 0 0 0 2211 0 minus1 0 230 24312 0 1 0 196 24013 0 0 minus1 404 43314 0 0 1 328 35615 0 0 0 200 14716 0 0 0 209 147

is the step change of 119883119894corresponding to a unit variation

of the dimensionless value Response at each design pointwas recorded (Table 2) Data from the central compositeexperimental design were subjected to regression analysisusing least square regression methodology to obtain theparameters of the mathematical models

Studentrsquos 119905-test permitted the checking of the statisticalsignificance of the regression coefficients deriving from themodel Analysis of variance (ANOVA) was applied to evalu-ate the statistical significance of the model Response surfaceplots were obtained using the fitted model by keeping theindependent variables simultaneous All determinationswerecarried out at least in triplicate and values were averaged andgiven along the standard deviation (plusmn SD) For all statisticsMicrosoft Excel 2000 SigmaPlot 11 and JMP 8 were used

28 HPLC Analysis The equipment utilized was an HP 1090Series II Liquid Chromatograph coupled with an HP 1090diode-array detector and controlled by Agilent ChemStationsoftware The column was a Phenomenex Synergi HydroRP18 4120583m 250 times 46mm protected by a guard volumepacked with the same material Both columns were main-tained at 40∘C Eluent (A) and eluent (B) were 005 aqueoustrifluoroacetic acid (TFA) and acetonitrile (MeCN) contain-ing 005 TFA respectively The flow rate was 1mLminminus1and the elution programme used was as follows 5min 5B 65min 50 B Monitoring of the eluate was performed at275 nm

3 Results and Discussion

31 Factorial Design Optimisation Values of the independentprocess variables (119883

1 1198832 and 119883

3) are considered and

measured and predicted values for the response ( TP

0

10

20

30

40

50

000510

00

05

10

TP re

mov

al (

)

minus05

minus05

minus10

minus10

X1

X2

Figure 1 Response-surface plot illustrating the effect onpolyphe-nol removal upon simultaneous variation of PEG concentration(1198831) and pH (119883

2)

removal) are analytically given in Table 2 The experimentalvalues of the response were analysed by multiple regressionto fit the following second-order polynomial equation

TP Removal = 1471 minus 0641198831minus 014119883

2minus 388119883

3

+ 40011988311198832minus 335119883

11198833minus 015119883

21198833

minus 11841198832

1+ 946119883

2

2+ 2476119883

2

3

(2)

The quality of fit was ascertained using the regressioncoefficients (1198772) The experimental data obtained showed agood fit with the equations (1198772 = 096 119875 = 00017)This fact indicated a satisfactory agreement between observedand predicted responses and that the equation found canadequately predict the experimental results

After removal of the nonsignificant factors as revealedby the ANOVA analysis the theoretical model could besimplified as follows


11198833

minus 11841198832

1+ 946119883

2

2+ 2476119883

2

3

(3)

The utilisation of the predictivemodel enabled the theoreticalcalculation of the optimal set of conditions which werepH = 4 119905 = 357 h and [PEG] = 900mg Lminus1 Under theseconditions the optimal theoretical removal calculated was538 plusmn 94 The trends revealed in each case were recorded inthe form of three-dimensional plots (Figures 1 2 and 3)

ThepHoptima reported for potato PPOwithCGAwhichis a physiological substrate were 43 [23] 5 [24] 65 [25]and 66 [26] These discrepancies might be rather attributedto various isoforms of the enzyme that exist in potato [27]The theoretically optimal pH 4 calculated lies at the lowerextreme of these values However enzyme activity is not the


0

10

20

30

40

50

0005 00

05

10

TP re

mov

al (

)

minus05

minus10 minus05

minus10

minus10

X1

X3

Figure 2 Response-surface plot illustrating the effect onpolyphe-nol removal upon simultaneous variation of PEG concentration(1198831) and time (119883

3)

0005 00

05

10

TP re

mov

al (

)

minus05

minus05

minus10

minus10

X2

X3

45

40

35

30

25

20

15

10

5

Figure 3 Response-surface plot illustrating the effect onpolyphe-nol removal upon simultaneous variation of pH (119883

2) and time (119883

3)

only crucial parameter that could affect polyphenol removalAn important factor implicated could be how easily oxidisedphenolics polymerise and precipitate

In fact previous studies demonstrated that a decrease inpH resulted in decreased solubility of phenol oxidation prod-ucts generated through horseradish peroxidase- (POD-)mediated oxidation [28] Further a decrease in pHwas shownto enhance aggregation of chlorophenol oligomers thus facil-itating their subsequent precipitation upon treatment withhorseradish POD [29] Therefore it could be hypothesisedthat disappearance of polyphenol oxidation products pre-sumably through precipitation might be promoted at pH 4

0 10 20 30 40 50 60

0

5

10

15

20

(mAU

)

Untreated

(a)

0 10 20 30 40 50 60

0

5

10

15

20

(mAU

)

PPO-treatedPPO-treated

minus5

(b)

Figure 4 HPLC traces of original (untreated) and PPO-treatedOMW Monitoring of the eluent was performed at 275 nm

hence the higher removal observed Further to that the removal observed should be considered as the integration of removal of the various phenolics that may occur in OMWsince not all substances are equally removed Immobilizedlaccase-catalyzed treatment of OMW indicated that removalof individual phenolics may vary from 88 to 99 [30]

32 Putative Pathways and Products The HPLC analysis ofthe sample exhibiting the highest polyphenol removal(no 7 Table 2) revealed that the PPO treatment broughtabout significant alterations in the trace of the treated waste(Figure 4) In particular it was observed that the major sub-stances detected at 275 nm practically disappeared but it wasalso noticed that there was nomajor qualitative change in theprofile an indication that removal of phenolics was probablyachieved through the formation of insoluble polymers andthat there was no formation of soluble oxidation products atleast at a detectable level

The principal phenolics detected in various OMW havebeen reported to be hydroxytyrosol tyrosol and caffeicacid [31ndash33] hydroxytyrosol tyrosol and protocatechuicacid [34] verbascoside and its derivatives [35] and severalother substances most of them are possessing an o-diphenolfeature [36 37] PPO-catalysed oxidation of o-diphenolsproceeds through a four-electron transfer mechanism andsubsequent quinone formation [38] Quinones in turn canspontaneously polymerise to yield insoluble polymers [39]Although the activity of potato PPO on monophenols is


rather trivial [24 40] monophenolase activity could bestimulated in the presence of o-diphenols [41] In additionmonophenols could either react with the o-quinones formed[42] or oxidised through coupling reactions [43] In any casethe outcomewould be probably the generation of large insol-uble oligomers andor polymers which would be removedthrough precipitation as demonstrated in several similarcases [44 45] Hydroxytyrosol dimers were isolated followingperoxidaseH

2O2-mediated hydroxytyrosol oxidation and

their formation was attributed to quinone dimerisation [46]It is certain that the exceptionally high concentration

of OMW in phenolics would not permit a crude enzymepreparation to function to a satisfactory extent The useof enzymes such as potato PPO cannot be regarded as aprincipal process that could efficiently remediate OMW butrather as an assisting complementary means of removingtoxic substances thus contributing to an integrated treatmentof OMWW and similar effluents In this view the exploita-tion of potato solid byproducts for producing PPO-activehomogenates might merit a higher attention as a materialwith promising prospect in bioremediation

4 Conclusions

Themost important findings of this study can be summarisedas follows

(i) The implementation of a 23-full-factorial design forthe optimisation of TP removal from OMW usinga crude PPO preparation from potato peels showedthat a predicted value of 538 can be achievedthe optimal conditions being pH 4 357 h and PEGconcentration of 900mg Lminus1

(ii) The optimum pH value of the process which differsfrom the pH optimum potato PPO might indicatethat the catalytic activity of the enzyme even lowis necessary to form precursors (quinones) whichthen can spontaneously polymerise and be removedby precipitation at pH lower than that of the enzymeoptimal

(iii) The chromatographic profile of the PPO-treated sam-ple as well as optical examination revealed impor-tant decrease in the OMW polyphenols which ispresumably attributed to polymer formation andprecipitation

(iv) Further work is needed to examine factors pertainingto improve the process including enzyme-to-wasteratio and temperature In addition analytical deter-minations should be undertaken to clarify the exactnature of the oxidation products

Abbreviations

4-AAP 4-aminoantipyrineCCD Central composite designDMF DimethylformamideHPLC High-performance liquid chromatographyPEG Polyethylene glycol

PPO Polyphenol oxidasePOD PeroxidaseOMW Olive mill wastewaterTP Total polyphenols

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] M Niaounakis and C P Halvadakis Processing Waste Manage-mentmdashLiterature Review and Patent Survey Elsevier 2006

[2] D Mantzavinos and N Kalogerakis ldquoTreatment of olive milleffluents part I Organic matter degradation by chemical andbiological processesmdashan overviewrdquo Environment Internationalvol 31 no 2 pp 289ndash295 2005

[3] M Asgher H N Bhatti M Ashraf and R L Legge ldquoRecentdevelopments in biodegradation of industrial pollutants bywhite rot fungi and their enzyme systemrdquo Biodegradation vol19 no 6 pp 771ndash783 2008

[4] A Ben Sassi N Ouazzani G M Walker S Ibnsouda M ElMzibri and A Boussaid ldquoDetoxification of olive mill wastew-aters by Moroccan yeast isolatesrdquo Biodegradation vol 19 no 3pp 337ndash346 2008

[5] A Tsioulpas D Dimou D Iconomou and G Aggelis ldquoPheno-lic removal in olive oil mill wastewater by strains of Pleurotusspp in respect to their phenol oxidase (laccase) activityrdquoBioresource Technology vol 84 no 3 pp 251ndash257 2002

[6] I Sampedro A DrsquoAnnibale J A Ocampo S R Stazi andI Garcıa-Romera ldquoBioconversion of olive-mill dry residue byFusarium lateritium and subsequent impact on its phytotoxic-ityrdquo Chemosphere vol 60 no 10 pp 1393ndash1400 2005

[7] M Saavedra E Benitez C Cifuentes and R Nogales ldquoEnzymeactivities and chemical changes in wet olive cake after treatmentwith Pleurotus ostreatus or Eisenia fetidardquo Biodegradation vol17 no 1 pp 93ndash102 2006

[8] J Karam and J A Nicell ldquoPotential applications of enzymes inwaste treatmentrdquo Journal of Chemical Technology and Biotech-nology vol 69 pp 141ndash153 1997

[9] K Ikehata and J A Nicell ldquoCharacterization of tyrosinase forthe treatment of aqueous phenolsrdquo Bioresource Technology vol74 no 3 pp 191ndash199 2000

[10] N Duran and E Esposito ldquoPotential applications of oxidativeenzymes and phenoloxidase-like compounds inwastewater andsoil treatment a reviewrdquo Applied Catalysis B vol 28 no 2 pp83ndash99 2000

[11] D Lopez-Molina A N P Hiner J Tudela F Garcıa-Canovasand J N Rodrıguez-Lopez ldquoEnzymatic removal of phenolsfrom aqueous solution by artichoke (Cynara scolymus L)extractsrdquo Enzyme and Microbial Technology vol 33 no 5 pp738ndash742 2003

[12] A Guerra A Ferraz A R Cotrim and F T Da Silva ldquoPoly-merization of lignin fragments contained in a model effluentby polyphenoloxidases and horseradish peroxidasehydrogenperoxide systemrdquo Enzyme andMicrobial Technology vol 26 no5-6 pp 315ndash323 2000

[13] Y J Xuan Y Endo and K Fujimoto ldquoOxidative degradation ofbisphenol A by crude enzyme prepared from potatordquo Journal of


Agricultural and Food Chemistry vol 50 no 22 pp 6575ndash65782002

[14] A A Khan and Q Husain ldquoDecolorization and removal oftextile and non-textile dyes from polluted wastewater anddyeing effluent by using potato (Solanum tuberosum) solubleand immobilized polyphenol oxidaserdquo Bioresource Technologyvol 98 no 5 pp 1012ndash1019 2007

[15] N Loncar N Bozic I Andelkovic et al ldquoRemoval of aqueousphenol and phenol derivatives by immobilized potato polyphe-nol oxidaserdquo Journal of the Serbian Chemical Society vol 76 no4 pp 513ndash522 2011

[16] M-F Hou X-Y Tang W-D Zhang L Liao and H-F WanldquoDegradation of pentachlorophenol by potato polyphenol oxi-daserdquo Journal of Agricultural and Food Chemistry vol 59 no 21pp 11456ndash11460 2011

[17] J Shao L-L Huang and Y-M Yang ldquoImmobilization ofpolyphenol oxidase on alginate-SiO

2hybrid gel stability and

preliminary applications in the removal of aqueous phenolrdquoJournal of Chemical Technology and Biotechnology vol 84 no4 pp 633ndash635 2009

[18] N Loncar B Janovic M Vujcic and Z Vujcic ldquoDecolorizationof textile dyes and effluents using potato (Solanum tuberosum)phenoloxidaserdquo International Biodeterioration and Biodegrada-tion vol 72 pp 42ndash45 2012

[19] H Saruta Y Ashihara and M Sugiyama ldquoColorimetric deter-mination of carboxypeptidase A activity in serumrdquo ClinicalChemistry vol 32 no 5 pp 748ndash751 1986

[20] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[21] YWu K E Taylor N Biswas and J K Bewtra ldquoComparison ofadditives in the removal of phenolic compounds by peroxidase-catalyzed polymerizationrdquo Water Research vol 31 no 11 pp2699ndash2704 1997

[22] J A Nicell K W Saadi and I D Buchanan ldquoPhenol polymer-ization and precipitation by horseradish peroxidase enzyme andan additiverdquoBioresource Technology vol 54 no 1 pp 5ndash16 1995

[23] D A Abukharma and H U Woolhouse ldquoThe preparation andproperties of o-diphenol oxygen oxidoreductase from potatotubersrdquo New Phytologist vol 65 pp 477ndash478 1966

[24] S S Patil and M Zucker ldquoPotato phenolases Purification andpropertiesrdquo Journal of Biological Chemistry vol 240 no 10 pp3938ndash3943 1965

[25] A Sanchez-Ferrer F Laveda and F Garcıa-Carmona ldquoPartialpurification of soluble potato polyphenol oxidase by partition-ing in an aqueous two-phase systemrdquo Journal of Agricultural andFood Chemistry vol 41 no 8 pp 1219ndash1224 1993

[26] J Batistuti and E J Lourenco ldquoIsolation and purificationof polyphenol oxidase from a new variety of potatordquo FoodChemistry vol 18 no 4 pp 251ndash263 1985

[27] P W Thygesen I B Dry and S P Robinson ldquoPolyphenoloxidase in potato A multigene family that exhibits differentialexpression patternsrdquo Plant Physiology vol 109 no 2 pp 525ndash531 1995

[28] QHuang J Tang andW JWeber Jr ldquoPrecipitation of enzyme-catalyzed phenol oxidative coupling products background ionand pH effectsrdquo Water Research vol 39 no 13 pp 3021ndash30272005

[29] K Yamada T Shibuya M Noda et al ldquoInfluence of positionof substituent groups on removal of chlorophenols and cresols

by horseradish peroxidase and determination of optimumconditionsrdquo Bioscience Biotechnology and Biochemistry vol 71no 10 pp 2503ndash2510 2007

[30] A DrsquoAnnibale S R Stazi V Vinciguerra and G GiovannozziSermanni ldquoOxirane-immobilized Lentinula edodes laccase sta-bility and phenolics removal efficiency in olivemill wastewaterrdquoJournal of Biotechnology vol 77 no 2-3 pp 265ndash273 2000

[31] H Azaizeh F Halahlih N Najami D Brunner M Faulstichand A Tafesh ldquoAntioxidant activity of phenolic fractions inolive mill wastewaterrdquo Food Chemistry vol 134 no 4 pp 2226ndash2234 2012

[32] A El-Abbassi H Kiai and A Hafidi ldquoPhenolic profile andantioxidant activities of olive mill wastewaterrdquo Food Chemistryvol 132 no 1 pp 406ndash412 2012

[33] A A DeebA M K Fayyad and M A Alawi ldquoSeparation ofpolyphenols from Jordanian olive oil mill wastewaterrdquo Chro-matography Research International vol 1 pp 1ndash8 2012

[34] A Scoma C Pintucci L Bertin P Carlozzi and F FavaldquoIncreasing the large scale feasibility of a solid phase extractionprocedure for the recovery of natural antioxidants from olivemill wastewatersrdquo Chemical Engineering Journal vol 198-199pp 103ndash109 2012

[35] A Cardinali S Pati F Minervini I DrsquoAntuono V Linsalataand V Lattanzio ldquoVerbascoside isoverbascoside and theirderivatives recovered from olive mill wastewater as possiblefood antioxidantsrdquo Journal of Agricultural and Food Chemistryvol 60 no 7 pp 1822ndash1829 2012

[36] H K Obied M S Allen D R Bedgood P D Prenzler KRobards and R Stockmann ldquoBioactivity and analysis of bio-phenols recovered from olivemill wasterdquo Journal of Agriculturaland Food Chemistry vol 53 no 4 pp 823ndash837 2005

[37] T Jerman Klen and B Mozetic Vodopivec ldquoUltrasonic extrac-tion of phenols from olive mill wastewater comparison withconventional methodsrdquo Journal of Agricultural and Food Chem-istry vol 59 no 24 pp 12725ndash12731 2011

[38] R Yoruk and M R Marshall ldquoPhysicochemical properties andfunction of plant polyphenol oxidase a reviewrdquo Journal of FoodBiochemistry vol 27 no 5 pp 361ndash422 2003

[39] G Toscano M L Colarieti and G Greco Jr ldquoOxidativepolymerisation of phenols by a phenol oxidase from greenolivesrdquo Enzyme and Microbial Technology vol 33 no 1 pp 47ndash54 2003

[40] D Ni Eidhin P Degn and D Orsquobeirne ldquoCharacterization ofpolyphenol oxidase from rooster potato (Solanum tuberosum cvRooster)rdquo Journal of Food Biochemistry vol 34 no 1 pp 13ndash302010

[41] S P Kowalski N T Eannetta A T Hirzel and J C SteffensldquoPurification and characterization of polyphenol oxidase fromglandular trichomes of Solanum berthaultiirdquo Plant Physiologyvol 100 no 2 pp 677ndash684 1992

[42] P Sarni-Manchado V Cheynier andMMoutounet ldquoReactionsof polyphenoloxidase generated caftaric acid o-quinone withmalvidin 3-O-glucosiderdquo Phytochemistry vol 45 no 7 pp1365ndash1369 1997

[43] K Robards P D Prenzler G Tucker P Swatsitang andW Glover ldquoPhenolic compounds and their role in oxidativeprocesses in fruitsrdquo Food Chemistry vol 66 no 4 pp 401ndash4361999

[44] S Kobayashi and H Higashimura ldquoOxidative polymerizationof phenols revisitedrdquo Progress in Polymer Science (Oxford) vol28 no 6 pp 1015ndash1048 2003


[45] S Mukherjee B Basak B Bhunia A Dey and B MondalldquoPotential use of polyphenol oxidases (PPO) in the bioremedi-ation of phenolic contaminants containing industrial wastew-aterrdquo Reviews in Environmental Science and Biotechnology vol12 pp 61ndash73 2013

[46] M De Lucia L Panzella A Pezzella A Napolitano andM DrsquoIschia ldquoOxidative chemistry of the natural antioxidanthydroxytyrosol hydrogen peroxide-dependent hydroxylationand hydroxyquinoneo-quinone coupling pathwaysrdquo Tetrahe-dron vol 62 no 6 pp 1273ndash1278 2006

Submit your manuscripts athttpwwwhindawicom

Forestry ResearchInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Environmental and Public Health

Journal of



EcosystemsJournal of


MeteorologyAdvances in

EcologyInternational Journal of


Marine BiologyJournal of


Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

Advances in


Environmental Chemistry

Atmospheric SciencesInternational Journal of



Waste ManagementJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal of

Geophysics


Geological ResearchJournal of

EarthquakesJournal of


BiodiversityInternational Journal of


ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

OceanographyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of Computational Environmental SciencesHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


ClimatologyJournal of


To the best of our knowledge potato PPO has neverbeen investigated for its potential to removeOMWphenolicsOn such a conceptual basis this study was undertaken toexamine the prospect of using crude potato peel PPO toremove OMW phenolics by the application of a 23-full-factorial design and response surfacemethodology includingpH treatment time and polyethylene glycol (PEG) as criticalfactors

2 Materials and Methods

21 Reagents and Chemicals Bradford reagent trans-chloro-genic acid (CGA) trichloroacetic acid (TCA) and 4-amino-antipyrine (4-AAP)were fromSigmaChemical Co (St LouisMO USA) NN-Dimethylformamide (DMF) and potassiumferricyanide were fromMerck (Darmstadt Germany)

22 Olive Mill Wastewater (OMW) TheOMWwas obtainedfrom a two-phase processing plant located within theprefecture of Chania (Crete Greece) The plant processesorganically cultivated olives for the production of extravirginolive oil The OMWwas collected immediately after disposalto avoid any changes in the polyphenolic composition Uponreception the waste was stored at 4∘C in the dark until usedfor no longer than a week

23 Preparation of the Potato Peel Homogenate Brown-skinpotatoes (Solanum tuberosum L) were purchased from agrocery (Chania Crete) The tubers were transferred tothe laboratory and manually peeled and the peels wereimmediately frozen with liquid nitrogen The frozen tissuewas ground with a pestle and a mortar and mixed withcharcoal to remove phenolics Typically 1 g of charcoal wasused for approximately 25 g of ground tissue The charcoal-treated tissuewas thenmixedwith 150mLof phosphate buffer(50mM pH 66) and homogenised in a domestic blender toform slurry The homogenate was centrifuged at 4000 g for15min and filtered through paper filter The clear filtrate wasused as crude enzyme source

24 Polyphenol Oxidase Activity The assay mixture con-tained 025mL 4-AAP (10mM in water) 01mL substrate(100mMCGA in DMF) 06mL of phosphate buffer (50mMpH 66) and 01mL enzyme extract The formation ofchromogen (quinoneimine dye) adduct of 4-AAPCGA wasmonitored by recording the absorbance at 510 nm (119860

510)

for over 3min against suitable blank One enzyme unit(U) was defined as 120583moles of quinoneimine dye formedper min For all determinations 120576 = 12 000 was usedfor the quinoneimine dye [19] Control reactions by usingheat-inactivated homogenate were also carried out For alldeterminations a computer-controlled HP 8452A diode-array spectrophotometer was used Protein content wasdetermined according to Bradford 1976 [20] using bovineserum albumin as the standard

25 Enzymatic Treatment The waste was homogenised byvigorous shaking then filtered through filter paper and

Table 1 Experimental values and coded levels of the independentvariables used for the 23-full-factorial design

Independent variables Code units Coded variable levelminus1 0 1

[PEG] (mg Lminus1) 1198831

100 500 900pH 119883

24 6 8

Time (h) 1198833

1 3 5

diluted 1 20 with tap water The OMW was adjusted at vari-ous pH levels (Table 1) using either 01 NHCl or 01 NNaOHThe medium (final volume 20mL) was composed of 19mLOMW 05mL crude PPO extract (final total protein concen-tration 155120583gmLminus1 142U) and 05mL PEG solution Thefinal mixture was placed in a 30mL screw-cap glass reactorbearing a teflon-coated magnetic stirrer and sparged withair for 5min prior to treatments to ascertain the presenceof sufficient amount of oxygen Treatments were carried outunder stirring with a magnetic stirrer operating at 400 rpmControl treatments with heat-inactivated homogenate werealso carried out

26 Total Polyphenol Determination A sample (1mL) waswithdrawn from the reaction medium and placed in a 15mLEppendorf tube The sample was mixed with 01mL TCAvortexed and then centrifuged in a table centrifugator at10000 rpm An aliquot of 05mL of appropriately dilutedOMWwasmixed with 03mL deionised water 01mL 4-AAP(20mM in 025M NaHCO

3) and 01mL potassium ferri-

cyanide (85mM in 025M NaHCO3) Absorbance readings

were carried out at 510 nm against suitable blank [21] Resultswere expressed as caffeic acid equivalents (CAE) using acalibration curve of 119860

510against caffeic acid concentration

(125ndash200mg Lminus1)

27 Experimental Design and Statistical Analyses A 23-full-factorial experimental design was used to identify therelationship existing between the response function andprocess variables as well as to determine those conditionsthat optimised the PPO-catalysed oxidation process Theresponse function considered was the removal of TP fromthe reaction medium The three independent variables orfactors considered were PEG (119883

1 varying between 100 and

900mg Lminus1) pH (1198832 varying between 4 and 8) and time (119883

3

varying between 1 and 5 h) Each variable to be optimised wascoded at three levels minus1 0 and 1 (Table 1) The value rangesused for PEGwere based on preliminary experiments and theliterature data [21 22]

The three independent variables were coded according tothe following equation

119909119894=119883119894minus 1198830

998779119883119894

119909119894= 1 2 3 (1)

where 119909119894and 119883

119894are the dimensionless and the actual value

of the independent variable 119894 1198830is the actual value of

the independent variable 119894 at the central point and Δ119883119894











1 1198832 and 119883



0

10

20

30

40

50

000510

00

05

10

TP re

mov

al (

)

minus05

minus05

minus10

minus10

X1

X2


2)



2minus 388119883

3

+ 40011988311198832minus 335119883

11198833minus 015119883

21198833

minus 11841198832

1+ 946119883

2

2+ 2476119883

2

3

(2)




11198833

minus 11841198832

1+ 946119883

2

2+ 2476119883

2

3

(3)




0

10

20

30

40

50

0005 00

05

10

TP re

mov

al (

)

minus05

minus10 minus05

minus10

minus10

X1

X3


3)

0005 00

05

10

TP re

mov

al (

)

minus05

minus05

minus10

minus10

X2

X3

45

40

35

30

25

20

15

10

5


2) and time (119883

3)



0 10 20 30 40 50 60

0

5

10

15

20

(mAU

)

Untreated

(a)

0 10 20 30 40 50 60

0

5

10

15

20

(mAU

)


minus5

(b)










4 Conclusions






Abbreviations





References

























































Journal of












Volume 2014

Advances in









Geophysics
























1 1198832 and 119883



0

10

20

30

40

50

000510

00

05

10

TP re

mov

al (

)

minus05

minus05

minus10

minus10

X1

X2


2)



2minus 388119883

3

+ 40011988311198832minus 335119883

11198833minus 015119883

21198833

minus 11841198832

1+ 946119883

2

2+ 2476119883

2

3

(2)




11198833

minus 11841198832

1+ 946119883

2

2+ 2476119883

2

3

(3)




0

10

20

30

40

50

0005 00

05

10

TP re

mov

al (

)

minus05

minus10 minus05

minus10

minus10

X1

X3


3)

0005 00

05

10

TP re

mov

al (

)

minus05

minus05

minus10

minus10

X2

X3

45

40

35

30

25

20

15

10

5


2) and time (119883

3)



0 10 20 30 40 50 60

0

5

10

15

20

(mAU

)

Untreated

(a)

0 10 20 30 40 50 60

0

5

10

15

20

(mAU

)


minus5

(b)










4 Conclusions






Abbreviations





References

























































Journal of












Volume 2014

Advances in









Geophysics















0

10

20

30

40

50

0005 00

05

10

TP re

mov

al (

)

minus05

minus10 minus05

minus10

minus10

X1

X3


3)

0005 00

05

10

TP re

mov

al (

)

minus05

minus05

minus10

minus10

X2

X3

45

40

35

30

25

20

15

10

5


2) and time (119883

3)



0 10 20 30 40 50 60

0

5

10

15

20

(mAU

)

Untreated

(a)

0 10 20 30 40 50 60

0

5

10

15

20

(mAU

)


minus5

(b)










4 Conclusions






Abbreviations





References

























































Journal of












Volume 2014

Advances in









Geophysics



















4 Conclusions






Abbreviations





References

























































Journal of












Volume 2014

Advances in









Geophysics

























































Journal of












Volume 2014

Advances in









Geophysics














research article removal of olive mill wastewater...

Documents