optimisation by response surface methodology of fungal lipase production on olive mill wastewater

Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 81:1586–1593 (2006)

Optimisation by response surfacemethodology of fungal lipase productionon olive mill wastewaterAlessandro D’Annibale,∗ Viviana Brozzoli, Silvia Crognale, Anna Maria Gallo,Federico Federici and Maurizio PetruccioliDipartimento di Agrobiologia e Agrochimica, University of Tuscia, I-01100 Viterbo, Italy

Abstract: The suitability of olive mill wastewater (OMW) as a growth medium for lipase production was assessedusing Penicillium citrinum NRRL 1841, a versatile strain capable of producing lipase on several OMW typologiesdiffering widely in their chemical oxygen demand, polyphenol and total sugar contents. Lipase production byP. citrinum in OMW-based media was significantly stimulated by nitrogen addition, with ammonium chlorideproving to be the most effective source. In contrast, the addition of vegetable oils did not significantly affect lipaseproduction. On the basis of these results, lipase production was subsequently optimised in shaken flasks by acentral composite design. To this aim, the impact of three crucial variables, namely initial pH and concentrationsof ammonium chloride and yeast extract, was investigated. Maximum lipase activity (ca 1230 U dm−3) was reachedafter 188 h of fermentation. The process was subsequently assessed in stirred tank and bubble column reactors ofidentical working capacity (3 dm3), leading to lower lipase production (735 and 430 U dm−3 respectively) than inshaken flasks. 2006 Society of Chemical Industry

Keywords: olive mill wastewater valorisation; Penicillium citrinum; lipase production; medium optimisation

INTRODUCTIONThe olive oil manufacturing process produces largeamounts (ca 3.0 × 107 m3 annually) of a dark-coloured effluent, usually referred to as olive millwastewater (OMW), characterised by a rather variablechemical composition depending on harvesting period,cultivar and extraction process.1 Owing to the highchemical oxygen demand (up to ca 150 g dm−3)of the wastewater and the presence of recalcitrantorganic compounds (i.e. polyphenols), OMW disposalrepresents a large-scale environmental problem,particularly in the Mediterranean region.1,2

However, OMW might also be regarded as apromising resource owing to the presence of simpleand complex sugars, which make it a potential basisfor fermentation processes.1,3–7 In particular, OMWcould be a putative candidate as a potentially suitableliquid growth medium for the production of microbiallipases by virtue of its content of residual lipids, theamount of which depends mainly on the extractionprocess efficiency.5

For these reasons, the objective of the presentstudy was to assess the suitability of OMW as agrowth medium for the production of lipases (glycerolester hydrolases, EC 3.1.1.3), a group of enzymescharacterised by a wide range of applications.8 Tothis aim, Penicillium citrinum NRRL 1841 was usedowing to its previously reported capability to grow

on undiluted OMW and produce lipase.9 This studyreports the optimisation of lipase production byP. citrinum NRRL 1841 in shaken cultures andthe subsequent assessment of the process in bothmechanically and pneumatically agitated bioreactors.

MATERIALS AND METHODSOlive mill wastewaters and micro-organismOMW samples, the composition of which is shownin Table 1, were withdrawn from different oliveoil extraction plants that used either conven-tional or modified three-phase extraction technology.Unless indicated otherwise, OMW was centrifuged(3800 × g, 20 min) and supplemented with yeastextract (0.5 g dm−3) and (NH4)2SO4 (1.0 g dm−3).Before sterilisation (121 ◦C, 20 min) the pH of OMW-based media was adjusted to 6.1 with a few drops of1.0 mol dm−3 NaOH.

Penicillium citrinum NRRL 1841 was obtained fromthe North Regional Research Laboratory (NRRL,Peoria, IL, USA) culture collection. The strain wasmaintained at 4 ◦C and periodically subcultured onpotato dextrose agar (PDA).

Shaken flask experimentsFive-day-old PDA slant cultures were suspended in5 cm3 of sterile deionised water and used as the

∗ Correspondence to: Alessandro D’Annibale, Dipartimento di Agrobiologia e Agrochimica, University of Tuscia, Via San Camillo De Lellis, I-01100 Viterbo, ItalyE-mail: [email protected]/grant sponsor: INCA (Consorzio Interuniversitario per la Chimica dell’Ambiente)(Received 1 October 2005; revised version received 10 November 2005; accepted 10 November 2005)Published online 12 July 2006; DOI: 10.1002/jctb.1554

2006 Society of Chemical Industry. J Chem Technol Biotechnol 0268–2575/2006/$30.00

Fungal lipase from olive mill wastewaters

Table 1. Characteristics of olive mill wastewater samples used in the present studya

OMWbCOD

(g dm−3)

Total sugars(g dm−3)

COD/totalsugars

Total phenols(g dm−3)

Lipids(g dm−3) pH

OMW-1 26.7 ± 1.2a 9.2 ± 0.5a 2.90 ± 0.07a 2.24 ± 0.10a 0.24 ± 0.02a 4.88OMW-2 51.5 ± 1.9b 25.1 ± 2.0b 2.05 ± 0.09b 3.45 ± 0.3b 1.08 ± 0.04b 4.82OMW-3 36.4 ± 1.5c 8.2 ± 0.3a 4.43 ± 0.22c 3.24 ± 0.2b 0.38 ± 0.02c 4.90OMW-4 49.1 ± 2.3b 18.8 ± 1.3c 2.61 ± 0.14a 2.52 ± 0.4a 0.40 ± 0.03c 5.13

a Data are shown as mean ± standard deviation of three determinations. Values within a column followed by the same letter are not significantlydifferent (P ≤ 0.05, Tukey test).b OMW-1 and OMW-4 came from a traditional three-phase extraction system, while OMW-2 and OMW-3 came from a modified three-phase systemwith low water consumption. OMW-1 and OMW-3 were withdrawn from storage tanks, while OMW-2 and OMW-4 were taken from the decanterduring the manufacturing process.

inoculum for pre-cultures to obtain an initial sporeconcentration of ca 1 × 106 cm−3. Incubations werecarried out at 28 ◦C for 72 h under orbital shaking(180 rpm) in Erlenmeyer flasks (500 cm3) containing95 cm3 of twofold diluted OMW-1. Pre-cultures(5 cm3) were then used to inoculate Erlenmeyer baffledflasks (500 cm3) containing 95 cm3 of OMW-basedmedia, which were incubated for 212 h as above.Culture samples were withdrawn on a daily basisstarting from the 48th hour of fermentation. Allexperiments were performed in triplicate.

The effect of nitrogen was studied in shaken cultureby adding to OMW-1 one of the following compounds:NH4Cl, (NH4)2SO4, NaNO3 or urea. To facilitatecomparisons, these compounds were added to givethe same final nitrogen content in the OMW-basedmedium (0.63 g nitrogen dm−3). The influence of oiladdition was evaluated by adding to OMW-1 one ofthe following oils (3.0 g dm−3): olive oil, corn oil orsoybean oil.

Experimental designThe composition of the OMW-based medium was fur-ther optimised by response surface methodology usinga central composite design.10,11 Three independentquantitative variables, namely NH4Cl concentration(X1), yeast extract concentration (X2) and initial pH(X3), were investigated in this study. The experimentaldesign included a set of 14 variable combinations andthree centre points. The actual values of the variableswere coded as dimensionless terms using the equation

Xi = (Ai − A∗0)/�A (1)

where Xi is the coded value and Ai the actual value ofthe variable, A∗

0 is the actual value of the same variableat the centre point and �A is the step change in thevariable. Data were subjected to analysis of variance(ANOVA) and fitted according to the second-orderpolynomial model

Y = β0 +∑

βiXi +∑

βiiX2i +

∑βijXiXj (2)

where Y is the predicted response variable, β0 is theintercept, βi and βii are linear and quadratic coeffi-cients respectively, βij is the interaction coefficient andXi and Xj are the coded forms of the input variables.

The factorial design was replicated twice. Statisticalexamination of the results and a response surface studywere carried out using the statistical software packageModde 5.0 (Umetrics AB, Umea, Sweden).

Reactor experimentsLipase production was subsequently assessed in biore-actors using the OMW-based medium previously opti-mised in shaken culture. Specifically, fermentationswere carried out in a 3 dm3 jacketed benchtop stirredtank reactor (STR) (Applikon Dependable Instru-ments, Schiedam, The Netherlands) and a 3 dm3

bubble column reactor (BCR Steroglass, Perugia,Italy), each filled with 2 dm3 of medium. The STRwas equipped with a top stirrer bearing two six-bladeRushton-type turbines. The following probes wereinstalled on the top plate: a dissolved oxygen sensor(Ingold, Urdorf, Switzerland), a double reference pHsensor (Phoenix, AZ, USA) and a PT 100 temperaturesensor (Applikon). Standard bioprocess conditions inthe STR were as follows: inoculum density 5% (v/v);impeller speed 500 rpm (tip speed 118 cm s−1), aera-tion rate 1.0 vol vol−1min−1 (vvm); temperature 28 ◦C;initial dissolved oxygen concentration 100% of satura-tion. Experiments in the BCR (diameter 12 cm, height27 cm) were performed under the following condi-tions: inoculum density 5% (v/v); aeration rate 1.0vvm; silicon antifoam 1 ml dm−3; temperature 28 ◦C.Air was injected through a fritted glass sparger (diame-ter 11 cm). Fermentation parameters were monitoredin both bioreactors by an adaptative/PID digital con-troller (ADI 1030, Applikon). Each condition wastested in triplicate. The volumetric mass transfer coef-ficient (KLa) in both reactors was determined by thestatic method of gassing out.12

Lipase assayLipase activity was determined spectrophotometricallyat 35 ◦C using β-naphthylmyristate as substrate, asdescribed elsewhere.13 One unit (1 U) of lipase activitywas defined as the amount of enzyme producing1 µmol product min−1 under the assay conditions.

Analytical methodsMicrobial biomass concentration was measured bydry weight estimation. Broth samples were filtered

J Chem Technol Biotechnol 81:1586–1593 (2006) 1587DOI: 10.1002/jctb

A D’Annibale et al.

on preweighed Whatman GF/C filter discs (diameter47 mm), the harvested biomass was washed twice withdistilled water and the filter was dried at 105 ◦C for24 h, cooled in a desiccator and weighed. Chemicaloxygen demand (COD), total phenols and totalsugars were determined by standard methods.14 Lipidswere determined gravimetrically after petroleum etherextraction.15

RESULTSEffect of nitrogen and oil supplementation onlipase and biomass productionFigure 1A shows that nitrogen addition to OMW ledto a significant increase in lipase activity regardlessof the nitrogen source. However, ammonium chlorideproved to be the most effective in supporting lipaseproduction. The activity peaks obtained on mediacontaining the other nitrogen sources did not differsignificantly from one another (Fig. 1A). Nitrogen

addition did not lead to a statistically significantincrease in biomass with respect to non-supplementedOMW (Fig. 1A).

On the other hand, although the addition of oils tonitrogen-supplemented OMW-1 resulted in a markedincrease in biomass, it did not significantly affect lipaseproduction (Fig. 1B). The same effect due to theaddition of oils was also observed in the wastewaterwhich had not been previously supplemented withnitrogen (data not shown).

Effect of different OMWPenicillium citrinum NRRL 1841 was grown on fourOMW typologies to test its versatility to grow andproduce lipase on wastewaters differing widely in theirchemical composition. Table 2 shows that the mostprominent lipase productions were obtained on theeffluents characterised by the highest organic loads,i.e. OMW-2 and OMW-4. In particular, lipase activityobtained in the former and the latter wastewater

Figure 1. Effect of (A) nitrogen source and (B) oil addition on biomass and lipase production by Penicillium citrinum NRRL 1841 on OMW-1. Valuesare means of three independent experiments; error bars indicate standard deviations. The same letter above bars of the same colour indicates theabsence of significant differences (P < 0.01, Tukey test).

1588 J Chem Technol Biotechnol 81:1586–1593 (2006)DOI: 10.1002/jctb


Table 2. Effect of different OMW typologies (see also Table 1) on

lipase and biomass production by Penicillium citrinum NRRL 1841

and on COD and total phenol removala

OMW

Lipaseactivity

(U cm−3)

Mycelialgrowth

(g dm−3)

CODremoval

(%)

Totalphenolremoval

(%)

OMW-1 610 ± 11a 5.96 ± 0.22a 63.2 ± 1.5a 71.5 ± 2.4aOMW-2 1030 ± 65b 8.32 ± 0.51b 63.6 ± 2.8a 75.7 ± 3.1aOMW-3 616 ± 35a 4.55 ± 0.43a 48.4 ± 2.7b 62.4 ± 1.1bOMW-4 955 ± 45b 7.81 ± 0.55b 68.1 ± 3.1a 49.8 ± 3.2c

a Data are shown as mean ± standard deviation of three independentexperiments. Values within a column followed by the same letter arenot significantly different (P ≤ 0.05, Tukey test).

amounted to 1030 and 955 U dm−3 respectively.Similarly, the highest biomass productions wereobtained on OMW-2 and OMW-4, reaching 8.32and 7.81 g dm−3 respectively. Furthermore, andirrespective of initial organic load and total phenols,the fungus brought about a marked COD reduction(Table 2).

Optimisation of lipase productionIn order to optimise lipase production by P. citrinumNRRL 1841 in OMW-based media, the combinedeffect of three variables (i.e. concentrations of NH4Cland yeast extract and initial pH) was assessed usinga central composite design, a variant of fractionalfactorial design. Table 3 shows the structure of theexperimental design and the responses obtained foreach combination of the quantitative variables understudy. It can be noted that lipase activity valuesvaried over a wide range (from 208 to 1272 U dm−3)depending on the selected variable combination. Datawere best fitted by a polynomial quadratic equation,as can be inferred from the good agreement ofexperimental data with those estimated by the model(Table 3). The correlation coefficient (R2) adjustedfor degrees of freedom was 0.93, indicating that thestatistical model can explain 93% of the variabilityin the response (Table 4). The model F value of36.7 (P < 0.001) indicates that model terms werehighly significant. In addition, the FME value of 7.11(P = 0.129), calculated as the ratio between meansquares of model error and replicate error, indicatesthat the probability for lack of fit of the modelwas not statistically significant (Table 4). Table 4also shows that the intercept, first-order and second-order coefficients of independent variables were highlysignificant, with the exception of the second-ordercoefficient for yeast extract. Figure 2 shows thecontour diagrams for lipase production as a functionof combinations of the variables taken two at a time.The highest values of activity were obtained withcombinations of the variables under study which werevery close to that of the central points.

As a matter of fact, the combination optimisedby the model was as follows: 2.7 g dm−3 NH4Cl,

Table 3. Central composite design showing coded and actual values

of the independent variables ammonium chloride (X1) and yeast

extract (X2) concentrations and initial medium pH (X3) and observed

and predicted values of lipase activity by Penicillium citrinum NRRL

1841 grown on undiluted OMW-4

Independentvariablesa

Lipase activity(U dm−3)

Experiment X1 X2 X3 Observedb Predicted

1 −1 (2) −1 (0.5) −1 (5.1) 680 684.062 +1 (4) −1 (0.5) −1 (5.1) 208 190.413 −1 (2) +1 (1.5) −1 (5.1) 869 806.264 +1 (4) +1 (1.5) −1 (5.1) 300 312.615 −1 (2) −1 (0.5) +1 (7.1) 537 592.616 +1 (4) −1 (0.5) +1 (7.1) 580 561.467 −1 (2) +1 (1.5) +1 (7.1) 759 714.818 +1 (4) +1 (1.5) +1 (7.1) 600 683.669 −1 (2) 0 (1.0) 0 (6.1) 1100 1147.24

10 +1 (4) 0 (1.0) 0 (6.1) 945 884.8411 0 (3) −1 (0.5) 0 (6.1) 1091 1067.4412 0 (3) +1 (1.5) 0 (6.1) 1179 1189.6413 0 (3) 0 (1.0) −1 (5.1) 790 853.6414 0 (3) 0 (1.0) +1 (7.1) 1070 993.4415 0 (3) 0 (1.0) 0 (6.1) 1202 1249.9416 0 (3) 0 (1.0) 0 (6.1) 1272 1249.9417 0 (3) 0 (1.0) 0 (6.1) 1250 1249.94

a Actual values (in parentheses) of variables X1 and X2 are expressedin g dm−3.b Values represent means of two independent experiments; standarddeviations were lower than 10%.

Table 4. Least squares estimates of coefficients of input variables

NH4Cl (X1) and yeast extract (X2) concentrations and initial pH (X3).

Statistical parameters measuring the correlation and significance of

the model are shown in the last two columns

Coefficient Valuea Parameterb Value

Intercept β0 1227 ± 35.8∗∗∗ R2 0.96β1 −131.2 ± 27.4∗∗∗ R2

adj 0.93β2 61.1 ± 17.4∗ F 36.7∗∗∗β3 69.9 ± 27.4∗ FME 7.11NSβ2

1 −275.1 ± 49.9∗∗∗ Confidence level 0.95β2

3 −367.6 ± 49.9∗∗∗β1β3 115.6 ± 30.7∗∗

a Estimated coefficient ± standard error and significance level:∗ P ≤ 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; NS, not significant.b R2, correlation coefficient; R2

adj, correlation coefficient adjusted fordegrees of freedom; F, ratio between mean squares of regressionand residuals; FME, ratio between mean squares of model error andreplicate error.

1.1 g dm−3 yeast extract, pH 6.15. To corroboratethese findings and to assess whether they might alsobe extended to OMW with similar organic loads,shaken flask experiments were performed with bothOMW-4 and OMW-2 using the above-mentionedoptimal combination of variables. Interestingly, in theformer and the latter wastewater, maximum lipaseactivity values obtained after 192 h were very close tothat predicted by the model (1232 and 1208 U dm−3

respectively vs 1242 U dm−3; data not shown).



Figure 2. Contour diagrams for lipase production by Penicillium citrinum NRRL 1841 on OMW-4 as a function of (A) NH4Cl concentration and initialpH, (B) yeast extract concentration (YE) and initial pH and (C) NH4Cl concentration and YE. Plots A–C are referred to coded levels (−1, 0 and +1) ofthe remaining variable.

Lipase production in bioreactorsTo gain information on the possible upscaling of theprocess, further experiments were performed in 3 dm3

laboratory-scale reactors. Specifically, pneumaticallyagitated (BCR) and mechanically agitated (STR)systems were employed for this purpose usingthe optimised OMW-based medium. Agitation andaeration conditions in the two reactors were setin order to obtain similar KLa values (0.125and 0.131 s−1 in the BCR and STR respectively).Figure 3A and B show typical fermentations in theBCR and STR respectively. In both cases, extracellularlipase peaked 188 h after inoculation. However, theonset of enzyme activity was anticipated in the STR,reaching 133 U dm−3 after 20 h, and maximum activitywas significantly higher than in the BCR (735 vs430 U dm−3 respectively). Regardless of the bioreactortype, the time courses of lipase production were notfound to be correlated with biomass production. In the

STR, maximal biomass production was anticipatedwith respect to the BCR (Fig. 3).

DISCUSSIONPrevious studies showed that OMW might bea valuable growth medium for lipase productionby yeasts such as Yarrowia lipolytica and Candidacylindracea NRRL Y-17506.5,9 However, the widevariability in the chemical composition of thiswastewater is a technical constraint to its biologicalupgrading. For this reason, it is of paramountimportance to use micro-organisms showing goodversatility in growth and production in a wastewater,the composition of which may easily vary from onebatch to another.

In the present study, P. citrinum NRRL 1841was found to grow optimally and to producelipase in OMW with an organic load higher than



Figure 3. Time course of growth and lipase production by Penicillium citrinum NRRL 1841 cultured on OMW-4 at pH 6.15, supplemented withNH4Cl (2.7 g dm−3) and yeast extract (1.1 g dm−3), in (A) a 3 dm3 bubble column reactor and (B) a 3 dm3 stirred tank reactor. The depletion profilesof total sugars and phenols are also shown. Values are means of two independent experiments.

45 000 mg dm−3 and a total phenol content as high as3400 mg dm−3. This is not an obvious result, since it isknown that OMW may exert concentration-dependentinhibitory effects on fungi, mainly due to phenols.16,17

This had implied the need to dilute OMW to allowfungal growth,18,19 while P. citrinum NRRL 1841 wasable to grow and produce lipase on four differentOMW typologies without the need for any dilution(present study). It is worth noting that a studyconducted on microbiota in OMW disposal lagoonsshowed that fungi belonging to the genus Penicilliumwere widely distributed and had the ability to growin undiluted effluent in the absence of nutritionalsupplements.20 However, the present study shows thatlipase production by P. citrinum NRRL 1841 tookadvantage from the addition of nitrogen to the OMW-based medium, even though such addition did notsignificantly increase fungal biomass production. Thebest nitrogen sources for P. citrinum NRRL 1841 lipaseproduction were found to be NH4Cl and, to a lesserextent, (NH4)2SO4. These results are in agreementwith those obtained by Duran and co-workers,21

who studied lipase production by P. citrinum inan oil refinery waste and reported that ammoniumsalts, NH4Cl in particular, were the optimal nitrogensources, while urea depressed lipase production. The

present study also shows that the addition of vegetableoils to OMW, albeit leading to a marked increase infungal biomass, did not result in a significant increasein lipase production. In this respect, several studiesindicate that the stimulatory effect of triacylglycerols(TAGs) on P. citrinum extracellular lipase productionis preferentially obtained at low concentrations.22,23 Inthis respect it has been hypothesised that the additionof high TAG concentrations to the media mightfavour the accumulation of fatty acids throughout thefermentation, thus leading to the inhibition of enzymeproduction.22

With regard to the subsequent optimisation phase oflipase production by P. citrinum NRRL 1841, it has tobe pointed out that this is the first report dealing withthe response surface method to specifically optimiselipase production in an OMW-based medium. Theselection of the quantitative variables investigated inthis study was done on the basis of several studiesemphasising the importance of pH and nitrogensources for lipase production by P. citrinum.22–26 As amatter of fact, all the variables under study significantlyaffected lipase production. The mathematical modelprediction of P. citrinum NRRL 1841 lipase activitytitres was in good agreement with experimentalobservations, as can be inferred from the high



statistical significance of the model. In addition, thesubsequent validation trials carried out on two OMWtypologies using the best combination of the variablesconfirmed the good predictive power of the model.With regard to the effect of initial pH on lipaseproduction, the optimal value (6.15) was found tobe lower that reported by Sztajer and Maliszewska.24

The addition of yeast extract to ammonium-containingmedia increased lipase production, in agreement withprevious studies conducted with another P. citrinumstrain.23,26

Most studies available on the production of fungallipases in bioreactors have been carried out inSTRs with wide variations in agitation and aerationconditions.27–29 However, a recent study showed thatthe lipase productivity by Geotrichum candidum inan airlift reactor was about 60% higher than in anSTR, probably owing to the lower-shear-stressingenvironment.30 In fact, the same authors suggestedthe use of agitation rates of ca 300–350 rpm in anSTR, in agreement with another study conductedwith Aspergillus terreus.31 For this reason, the lipaseproduction process by P. citrinum NRRL 1841 onthe optimised OMW-based medium was preliminarilyassessed in both an STR and a pneumaticallyagitated system, i.e. a BCR. For ease of comparison,bioreactors with identical working capacity andconditions of aeration/agitation leading to the samevalues of KLa were employed. Lipase productions andproductivities obtained with both reactors were foundto be significantly lower than those in shaken flasks;moreover, lipase activity obtained in the STR washigher than in the BCR.

CONCLUSIONSOMW valorisation by its use as a growth mediumfor lipase production by P. citrinum NRRL 1841appears to be possible and promising. This strainproved to be able to cope with OMW typologiescharacterised by markedly different compositions, thusshowing its versatility. The lipolytic activity of thestrain was favoured by the addition of low amountsof NH4Cl and yeast extract to OMW with a COD ofca 50 000 mg dm−3. Oil addition did not result in asignificant increase in lipase activity, thus confirmingthat the stimulatory effect due to oils in P. citrinumcultures is exerted at low concentrations of lipids whichare compatible with their typical contents in OMW.Preliminary results obtained in both mechanicallyand pneumatically agitated bioreactors suggest theneed for further investigation to optimise oxygentransfer, which appears to be a critical factor for lipaseproduction by fungi.30,31

ACKNOWLEDGEMENTThe research was supported by a grant fromINCA (Consorzio Interuniversitario per la Chimicadell’Ambiente), project ‘Agro-Food National Plan’.

REFERENCES1 Ramos-Cormenzana A, Monteoliva-Sanchez M and Lopez MJ,

Bioremediation of alpechın. Int Biodet Biodeg 35:249–268(1995).

2 Casa R, D’Annibale A, Pieruccetti F, Stazi SR, GiovannozziSermanni G and Locascio B, Reduction of the phenoliccomponents in olive-mill wastewater and its impact on durumwheat (Triticum durum Desf.) germinability. Chemosphere50:959–966 (2003).

3 Federici F, Montedoro GF, Servili M and Petruccioli M, Pecticenzyme production by Cryptococcus albidus var. albidus on oliveoil vegetation waters enriched with sunflower calathide meal.Biol Wastes 25:291–301 (1988).

4 Montedoro GF, Begliomini AL, Servili M, Petruccioli M andFederici F, Pectinase production from olive vegetation watersand its use in the mechanical olive oil extraction processto increase oil yield and improve quality. Ital J Food Sci4:355–362 (1993).

5 Scioli C and Vollaro L, The use of Yarrowia lipolytica to reducepollution in olive mill wastewaters. Water Res 31:2520–2524(1997).

6 Crognale S, Federici F and Petruccioli M, β-Glucan productionby Botryosphaeria rhodina on undiluted olive-mill wastewaters.Biotechnol Lett 25:2013–2015 (2003).

7 Fenice M, Giovannozzi Sermanni G, Federici F andD’Annibale A, Submerged and solid-state bioprocesses forlaccase and manganese-peroxidase production by Panustigrinus on olive-mill wastewater-based media. J Biotechnol100:77–85 (2003).

8 Jaeger KE and Eggert T, Lipases for biotechnology. Curr OpinBiotechnol 13:390–397 (2002).

9 D’Annibale A, Giovannozzi Sermanni G, Federici F andPetruccioli M, Olive mill wastewaters: a promising substratefor microbial lipase production. Bioresouce Technol in press.

10 Montgomery DC, Design and Analysis of Experiments (4th edn).Wiley, New York, NY (1997).

11 Burkert JFM, Maugeri F and Rodrigues MI, Optimization ofextracellular lipase production by Geotrichum sp. usingfactorial design. Bioresource Technol 91:77–84 (2004).

12 Wise WS, The measurement of the aeration of culture media.J Gen Microbiol 5:167–177 (1951).

13 Versaw WK, Cuppett SL, Winters DD and Williams LE, Animproved colorimetric assay for bacterial lipase in non-fat drymilk. J Food Sci 54:1557–1558 (1989).

14 APHA/AWWA/WEF, Standard Methods for Examination of Waterand Wastewater (19th edn). American Public Health Associa-tion/American Water Works Association/Water EnvironmentControl Federation, Washington, DC (1995).

15 IRSA, Metodi Analitici per i Fanghi. III. Parametri Chimico-fisici(Quaderni Istituto Ricerca sulle Acque 64). CNR, Rome (1985).

16 Sayadi S and Ellouz R, Screening of white rot fungi for thetreatment of olive mill waste-water. J Chem Technol Biotechnol57:141–146 (1993).

17 Sayadi S, Allouche N, Jaoua M and Aloui F, Detrimental effectsof high molecular-mass polyphenols on olive mill wastewatersbiotreatments. Process Biochem 35:725–735 (2000).

18 Vinciguerra V, D’Annibale A, Delle Monache G and Giovan-nozzi Sermanni G, Correlated effects during the bioconver-sion of waste olive waters by Lentinus edodes. BioresourceTechnol 51:221–226 (1995).

19 Martirani L, Giardina P, Marzullo L and Sannia G, Reductionof the phenol content and toxicity in olive mill wastewaterwith the ligninolytic fungus Pleurotus ostreatus. Water Res30:1914–1918 (1996).

20 Millan B, Lucas R, Robles A, Garcia T, de Cienfuegos GAand Galvez A, A study on the microbiota from olive-millwastewater (OMW) disposal lagoons, with emphasis onfilamentous fungi and their biodegradative potential. MicrobiolRes 155:143–147 (2000).

21 Miranda OA, Salgueiro AA, Pimentel MCB, Lima Filho JL,Melo EHM and Duran N, Lipase production by a Brazilian



strain of Penicillium citrinum using an industrial residue.Bioresource Technol 69:145–147 (1999).

22 Maliszewska I and Mastalerz P, Production and some propertiesof lipase from Penicillium citrinum. Enzyme Microb Technol14:190–193 (1992).

23 Pimentel MCB, Krieger N, Coelho LCCB, Fontana JO,Melo EHM, Ledingham WM, et al, Lipase from a Brazilianstrain of Penicillium citrinum. Appl Biochem Biotechnol49:59–74 (1994).

24 Sztajer H and Maliszewska I, The effect of culture conditionson lipolytic productivity of Penicillium citrinum. Biotechnol Lett11:895–898 (1989).

25 Pimentel MCB, Melo EHM, Lima Filho JL and Duran N,Production of lipase free of citrinin by Penicillium citrinum.Mycopathologia 133:119–121 (1996).

26 Pimentel MCB, Melo EHM, Lima Filho JL, Ledingham WMand Duran N, Lipase from a Brazilian strain of Penicilliumcitrinum cultured in a simple and inexpensive medium:heat-denaturation, kinetics, and pH stability. Appl BiochemBiotechnol 66:185–195 (1997).

27 Jacobsen T, Jensen B, Olsen J and Allermann K, Extracellularand cell-bound lipase in relation to growth of Geotrichumcandidum. Appl Microbiol Biotechnol 32:256–261 (1989).

28 Freire DM, Teles EMF, Bon EPS and Lippel San’t Anna G,Lipase producton by Penicillium restrictum in a bench-scalefermentor: effect of carbon and nitrogen nutrition, agitationand aeration. Appl Biochem Biotechnol 63–65:409–421 (1997).

29 Ibrahim OC, Nishio N and Nagai S, Production of athermostable lipase by Humicola lanuginosa grown onsorbitol–corn steep liquor medium. Agric Biol Chem51:2145–2151 (1987).

30 Burkert JFD, Maldonado RR, Maugeri F and Rodrigues MI.Comparison of lipase production by Geotrichum candidumin stirring and airlift fermenters. J Chem Technol Biotechnol80:61–67 (2005).

31 Gulati R, Saxena RK and Gupta R, Fermentation and down-stream processing of lipase from Aspergillus terreus. ProcessBiochem 36:149–155 (2000).


optimisation by response surface methodology of fungal lipase production on olive mill wastewater

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