effect of agitation and aeration on the reduction of pollutant load of olive mill wastewater by the...
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Biochemical Engineering Journal 29 (2006) 243–249
Effect of agitation and aeration on the reduction of pollutant load ofolive mill wastewater by the white-rot fungus Panus tigrinus
Alessandro D’Annibale, Daniele Quaratino,Federico Federici, Massimiliano Fenice ∗
Dipartimento di Agrobiologia & Agrochimica, Universita degli Studi della Tuscia,Via San Camillo de Lellis snc, 01100 Viterbo, Italy
Received 21 March 2005; accepted 4 January 2006
Abstract
The white-rot fungus Panus tigrinus CBS 577.79 was cultivated both in mechanical (stirred tank, STR) and pneumatically (bubble column,BCB) agitated bioreactors and investigated for its ability to reduce the polluting load of olive-mill wastewater (OMW). Both aeration and agitationstrongly influenced treatment efficiency. Best pollutants biodegradation performances were achieved in the bubble column bioreactor. Using thisbg©
K
1
a(aolmsioN
lfHt
ec
1d
ioreactor, COD reduction, dephenolization and decoloration were 60.9, 97.2 and 75%, respectively. In contrast, lower depollution efficiency wasenerally observed in STR due to the possible occurrence of shear stress.
2006 Elsevier B.V. All rights reserved.
eywords: Olive mill wastewater; Biodegradation; Lignin modifying enzymes; Bubble column bioreactor; Aeration; Agitation
. Introduction
During the process of olive oil extraction large amounts ofn effluent, which is often referred to as olive mill wastewaterOMW), are generated. Around 3.0 × 107 m3 of OMW are annu-lly produced in the Mediterranean basin representing 98–99%f the entire world olive oil production [1,2]. OMW uncontrolledand spreading on agricultural soils may cause serious environ-
ental pollution with unpredictable effects on the soil–plantystem [3]. Problems in OMW disposal were formerly confinedn the historical oil-producing countries but are now diffusing tother countries such as USA (California in particular), Australia,ew Zealand and Chile.In the last 20 years, many processes to reduce the pollutant
oad of OMW have been described [3,4]. OMW land spreadingor ferti-irrigation purposes is now allowed in several countries.owever, this practice could have big limitations due to some
oxic characteristics of the waste.The main risks associated with the release of OMW in the
nvironment are due to its high organic load and to the signifi-ant presence of phenolic components [5,6], the concentration of
which may easily reach 5–10 g l−1, depending on cultivar, har-vesting season and type of extraction process. Phytotoxic effectsas well as antibacterial activity have been associated with thephenolic fraction [7,8]. For this reason, the fate of phenols is afactor of paramount importance in assessing the efficacy of anOMW disposal treatment. Generally, a microbial pre-treatmentof OMW can positively affects its composition [3,9] often solv-ing toxicity problems. Therefore, the selection of appropriatebiological agents to perform OMW treatment has to take intoaccount their intrinsic capability to degrade aromatics [10].White-rot fungi (basidiomycetes) show a non-specific radical-based degradation mechanism of aromatic substances occurringin the extracellular environment [11]. The non-specific ligni-nolytic system produced by these microorganisms can degrademany persistent pollutants [12]. In addition, several white-rotspecies and/or their oxidases proved to be capable of degrad-ing OMW phenols and to efficiently decolourise the effluent[13–17]. The degradation mechanisms involve some extracellu-lar oxidases, such as laccase (E.C. 1.10.3.2 benzenediol: oxygenoxidoreductase) and manganese-dependent peroxidase (E.C.1.11.1.13 Mn(II): hydrogen peroxide oxidoreductase).
∗ Corresponding author. Tel.: +39 0761 357318; fax: +39 0761 357242.E-mail address: [email protected] (M. Fenice).
In comparison with other white-rot fungi such as thosebelonging to some species of Pleurotus, Trametes, Phane-rochaete, etc., Panus tigrinus, has been less investigated. How-ever, starting from the early works of Leontievsky et al. [18], the
369-703X/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.bej.2006.01.002
244 A. D’Annibale et al. / Biochemical Engineering Journal 29 (2006) 243–249
various information concerning this fungus show that this organ-ism is a good producer of lignin-modifying enzymes (LME) (i.e.laccase and manganese peroxidase) [19–21] even if grown onOMW [22,23]. However, almost nothing is known about theeffect of submerged fermentation parameters, such as agita-tion and aeration, on growth and/or performance (i.e. lignin-modifying enzymes production) of this microorganism. On thecontrary, such information has been reported to be very impor-tant for other white-rot fungi [24–26].
In the present study, in order to make a good choice of fer-mentor for possible applications, the effect of agitation and/oraeration on the efficiency of pollutant removal from OMW byP. tigrinus CBS 577.79 was investigated comparing liquid sub-merged fermentations in mechanically (stirred-tank reactor) andpneumatically (bubble column bioreactor) agitated systems.
2. Materials and methods
2.1. Materials
The OMW employed in this study was collected from local(three phases) olive mills. Its main chemical and physical char-acteristics were as follows: pH 5.4, COD 43,000 mg l−1, totalsuspended solids 36 g l−1, total sugars 7.5 g l−1, total phenols2.9 g l−1, total nitrogen 1.1 g l−1, soluble phosphorus 0.18 g l−1,sodium 34 mg l−1, calcium 24 g l−1, potassium 530 mg l−1, cop-p2
2
trw
2
sSmRbitppPwitcptwl
was injected through a fritted glass sparger (diameter 10 cm).The fermentation parameters (temperature, pH and dissolvedoxygen) were monitored in both bioreactors by an adapta-tive/PID digital controller, ADI 1030 (Applikon DependableInstruments, Schiedam, The Netherlands). Each condition wastested in triplicate using two identical reactors for a total of sixfermentations.
2.4. KLa measurements
The volumetric mass-transfer coefficient (KLa) of the biore-actors was determined by the static method of gassing out ofWise [27] using both distilled water and OMW at different com-binations of aeration rates (0.5, 1.0 and 1.5 vvm) and stirrerspeeds (250, 500, 750 rpm) for the STR. KLa measurementsfor the BCB were performed at 0.1, 0.3, 0.5, 0.8, 1.0 vvm. Testswere carried out at 28 ◦C.
2.5. Enzymatic assays and analytical methods
Laccase and MnP activity were assayed as previouslyreported [28]. Colour and chemical oxygen demand were deter-mined according to standard methods. Total phenols were deter-mined as previously described [5]. Total sugars were deter-mined by the phenol/sulphuric acid method [29]. Ethyl acetate-epp
2
ttAf
TVi
As
S101101
B00001
Ci
er 1.1 mg l−1, magnesium 2.3 mg l−1, colorimetric units (CU)500.
.2. Organism and inoculum production
P. tigrinus (strain 577.79) was from the CBS culture collec-ion (Baarn, The Netherlands). The strain was maintained andoutinely sub-cultured on potato dextrose agar slants. Inoculaere produced as previously described [22].
.3. Liquid submerged fermentation (LSF) in bioreactors
Fermentations were carried out in a 3-l jacketed bench-toptirred tank reactor (STR) (Applikon Dependable Instruments,chiedam, The Netherlands) filled with 2 l of medium. The fer-entor was equipped with a top stirrer bearing two six-bladeushton-type turbines (diameter 4.5 cm, blade width 1.4 cm,lade length 1.4 cm) and three baffles (width 1.4 cm). Air wasnjected through a perforated pipe sparger located under the bot-om turbine. The following probes were installed on the toplate: dissolved oxygen sensor (Ingold, CH), double referenceH sensor (Phoenix Electrode Company, Houston, TX, USA),T 100 temperature sensor. Standard bioprocess conditionsere as follows: inoculum size 0.9 g l−1 mycelium dry weight;
mpeller speed 500 rpm (impeller tip speed = 118 cm s−1), aera-ion rate 1.0 vvm; temperature 28 ◦C; initial dissolved oxygenoncentration 100% of saturation. Further experiments wereerformed in a jacketed bubble column bioreactor (BCB) (3 lotal volume, maximum diameter 12 cm, height 30 cm) filledith 2 l of the medium under the following conditions: inocu-
um size 0.9 g l−1; aeration rate 0.3 vvm; temperature 28 ◦C. Air
xtractable phenols were obtained and analysed by reversedhase high performance liquid chromatography (RP-HPLC) asreviously reported [23].
.6. Statistical analysis of data
One-way analysis of variance (ANOVA) and pair-wise mul-iple comparison procedure (Tukey test) were carried out usinghe software SigmaStat (Jandel Scientific, CA, USA). One-wayNOVA was applied to percent removal data, previously trans-
ormed into arcsin of the square root.
able 1olumetric mass-exchange (KLa) for the STR and the BCB bioreactors measured
n distilled water and in OMW
eration rate/stirringpeed (vvm/rpm)
KLa in water (s−1) KLa in OMW (s−1)
TR.0/250 0.0645 ± 0.001d 0.0647 ± 0.004d
.5/500 0.0814 ± 0.011e 0.0802 ± 0.008e
.0/500 0.1317 ± 0.002bc 0.1314 ± 0.003bc
.5/500 0.1430 ± 0.001c 0.1431 ± 0.003c
.5/750 0.1471 ± 0.0004bc 0.1456 ± 0.008bc
.0/700 0.1476 ± 0.0005bc 0.1461 ± 0.005bc
CB.1/0 0.1020 ± 0.0010ab 0.1011 ± 0.0010ab
.3/0 0.1248 ± 0.0007bc 0.1235 ± 0.0010bc
.5/0 0.1251 ± 0.011bc 0.1241 ± 0.010bc
.8/0 0.1253 ± 0.030bc 0.1248 ± 0.022bc
.0/0 0.1253 ± 0.001bc 0.1250 ± 0.002bc
olumn and line means followed by the same superscript letter were not signif-cantly different (P < 0.05) as determined by the Tukey test.
A. D’Annibale et al. / Biochemical Engineering Journal 29 (2006) 243–249 245
3. Results
3.1. Oxygen transfer in the bioreactors
Preliminary KLa measurements were done to compare theoxygen transfer capabilities of the two bioreactors. Table 1shows that no differences were recorded between water andOMW.
In the STR maximum volumetric mass-transfer (0.1317 s−1)was achieved at 500 rpm and 1 vvm, while in the BCB it wasreached at 0.3 vvm (0.125 s−1). For both bioreactor systems anyfurther increase of stirring and/or aeration did not result in sig-nificant (P = 0.005) increase of volumetric mass-transfer.
3.2. Submerged fermentations in STR and BCB
With regard to STR, the effects of agitation and aeration onthe OMW depollution efficiency were investigated at 250, 500and 750 rpm (impeller tip speed 59, 118, 177 cm s−1, respec-
tively) and 0.5, 1.0 and 1.5 vvm. Experiments in BCB werecarried out only at the aeration rate necessary to obtain maxi-mal volumetric mass-transfer (0.3 vvm). Table 2 summarizes theresults obtained in reactor experiments including some fermen-tation parameters such as enzyme productions, productivities,and specific activities.
Best results in STR were obtained at 500 rpm and 1.0 vvm,with this combination COD, phenol and colour reductions were60.4, 84 and 76%, respectively. Under this condition, the high-est maximal dephenolation rate (MDR), and maximal colorremoval rate (MCR) (11.27 mg l−1 h−1 and 8.79 CU l−1 h−1,respectively) were achieved. Stirring speed higher than 500 rpmand aeration rate higher than 1.0 vvm led to lower efficiency ofpollutant reduction. In particular, at 500 rpm and 1.5 vvm COD,phenol and colour reduction were 51.4, 78.5 and 54%, respec-tively. In addition, stirrer speed of 750 rpm in combination withany aeration rate (0.5 and 1.0 vvm) led to further decrease ofdepollution efficiency (Table 2). Maximum organic load removalyield (ORY) was obtained in STR at 250 rpm and 1 vvm (9.83 mg
Table 2Maximal COD and total phenols removals, decolorization and parameters of process performances in a 3-l stirred tank (STR) or in a 3-l bubble column (BCB) reactor
Stirring/aeration(rpm/vvm)
CODreduction (%)
Dephenolization (%) Decolorization (%) DS MDR(mg l−1 h−1)
ORR(mg l−1 h−1)
ORY MCR(CU l−1 h−1)
STR
B
D d at tO d remr dardr ame sT
TM ivity a
S(
S
S
B
Dpaa
250/1.0 57.8a [12] 87.5a [14] 64a [12]500/0.5 58.5a [9] 86.9a [10] 73b [9]500/1.0 60.4a [8] 84a [9] 76b [9]500/1.5 51.4c [12] 78.5b [9] 54c [13]750/0.5 48.8b [13] 77.8b [14] 52.5c [12]750/1.0 49.2b [13] 84a [14] 53c [12]
CB0/0.3 60.9a [8] 97.2c [6] 75b [6]
S: dephenolization selectivity (maximal phenols removal per COD consumeRR: organic load removal rate (mg COD removed l−1 h−1); ORY: organic loa
ate (colorimetric unit l−1 h−1). Data are the mean of six fermentations and stanemoval is indicated between square brackets. Column means followed by the sukey test.
able 3aximal biomass production and laccase and MnP volumetric activity, product
tirring/aerationrpm/vvm)
Biomass (g l−1) Laccase
Maximal activity−1
Productivity−1 −1
(IU l ) (IU l h ) (ITR250/1.0 2.8a [7] 4600a ± 98 [13] 14.74 1500/0.5 3.5c [9] 2889d ± 57 [9] 13.37500/1.0 3.8b [9] 3900c ± 249 [9] 18.05 1500/1.5 4.1b [8] 1953b ± 83 [7] 11.62750/0.5 3.4c [7] 2050b ± 123 [14] 6.10750/1.0 3.3c [7] 2160b ± 153 [14] 6.43
CB0/0.3 3.8b [7] 4300ac ± 23 [7] 25.59 1
ata between square brackets indicate time (days) required to attain either maximalroduction; specific activity: maximal enzyme activity/biomass production (recordednd standard deviations, when not specified, were less than 10%. Column means fols determined by the Tukey test.
0.103a 7.55a 85.36a 9.83a 5.55a
0.100a 10.50c 116.45d 7.19b 8.45b
0.093a 11.27b 135.27b 6.83b 8.79b
0.103a 10.50c 82.47a 5.66c 4.33a
0.107b 6.71a 67.26c 6.17b 4.56a
0.115b 7.25a 67.80c 6.61b 4.61a
0.126b 19.57d 155.9b 7.07b 13.0c
hat time); MDR: maximal dephenolation rate (mg phenols removed l−1 h−1);oval yield (mg COD removed mg−1 biomass); MCR: maximal color removaldeviations were less than 10%. The time (days) required to attain the maximaluperscript letter were not significantly different (P < 0.05) as determined by the
nd specific activity in a 3-l stirred tank (STR) or in a 3-l airlift (ALR) reactor
Mn-peroxidase
pecific activity−1
Maximal activity−1
Productivity−1 −1
Specific activity−1
U g ) (IU l ) (IU l h ) (IU g )642.8 65a ± 9 [10] 0.27 23.2825.4 216d ± 16 [9] 1.0 61.7026.3 360b ± 20 [9] 1.67 94.7476.3 123c ± 2 [9] 0.57 30602.9 136c ± 12 [8] 0.71 40654.5 158c ± 9 [8] 0.82 47.9
131.6 410b ± 22 [7] 2.44 107.9
biomass production or maximal enzyme activity. Biomass: maximal biomassat the time of maximal enzyme activity). Data are the mean of six fermentationslowed by the same superscript letter were not significantly different (P < 0.05)
246 A. D’Annibale et al. / Biochemical Engineering Journal 29 (2006) 243–249
Fig. 1. Depletion profiles of COD (�), phenols (©) and color (�) by P. tigrinus CBS 577.79 grown on OMW in a bubble column reactor.
of COD removed per milligram of biomass) due to the lowestfungal growth (2.8 g l−1).
In BCB, OMW depollution was generally higher than thatobtained in STR. Particularly important were the improvedvalues of all parameters related to phenol removal, such asdephenolization (97.2%), dephenolization selectivity (0.126)and maximal dephenolization rate (19.57 mg l−1 h−1). In addi-tion, under this condition, the highest colour removal rate wasattained (13.0 CU l−1 h−1). As a whole, OMW depollution inBCB was definitely faster than in STR. In particular, the timerequired to reach maximal colour and phenols removal in BCBwas markedly reduced compared with the best STR condition(6 days versus 9 days, respectively) (Table 2).
The superiority of the pneumatic system, shown for the OMWdepollution, was generally recorded also for some fermenta-tion parameters. Particularly high, due to a very fast enzymeonset and early achievement of the activity peak (Day 7),were the productivities of laccase (25.59 IU l−1 h−1), and MnP(2.44 IU l−1 h−1) (Table 3). The enzymes specific activities inBCB were always higher than those in STR; the sole exceptionwas that of laccase at 250 rpm/1 vvm (1916.1 IU g−1) that wasthe highest (Table 3).
Fig. 1 shows the time courses of colour, phenols and CODobserved in a typical fermentation run on BCB, which provedto be the best system for OMW depollution.
After a slow start, the effluent’s organic decreased rapidlyflth6do
m
ethyl acetate OMW extracts from both 6-day-old cultures inBCB and related incubation controls. Fifteen phenolic com-pounds were identified in OMW incubation control (Fig. 2),the most abundant components of which were catechol 4(263 mg l−1), gallic acid 1 (210 mg l−1), tyrosol 7 (190 mg l−1)and hydroxytyrosol 2 (76 mg l−1) (data not shown). Fig. 2 alsoshows that most of them were totally depleted with the exceptionof gallic acid, catechol, vanillin, para-coumaric acid and ferulicacid, the concentrations of which were reduced by 76, 85, 95, 5and 60%, respectively.
4. Discussion
In the present study, the reduction of OMW pollutantsby P. tigrinus CBS 57.79 was investigated in two reactorsystems. The efficiency of the fungus in removing COD,colour and phenols from the effluent was found to be nega-tively affected in STR both by stirring and aeration regimeshigher than 500 rpm and 1.0 vvm, respectively. Best resultswere obtained in BCB at an aeration rate (0.3 vvm) pro-viding the highest volumetric mass transfer in that system(Table 1). The superiority of the pneumatically-agitated sys-tem with respect to STR was shown by the reduction intime required to attain maximal COD, phenols and colourremovals. Percent reduction and removal rate of OMW phe-nols in BCB were significantly higher than those obtained intvlCsgmat
rom the first day of fermentation being depleted at an almostinear rate up to Day 4. Subsequently, COD reduction was slowero stop thereafter (61%). Dephenolation started within the firstours of fermentation and continued almost linearly up to Daywhen more than 97% where removed. In contrast, strong
ecolourization started on Day 2 reaching a maximum (75%)n Day 6.
To evaluate the impact of the fungal treatment on OMWonocyclic phenols, RP-HPLC analyses were performed on
he best STR condition (97.2% versus 86.9%, 19.57 mg l−1 h−1
ersus 10.5 mg l−1 h−1, respectively). In addition, depheno-ization selectivity (i.e. the amount of phenols removed perOD consumed), was improved in the pneumatically-agitated
ystem suggesting its better suitability in addressing the fun-al degradation machinery towards phenols breakdown. As aatter of fact, MnP and laccase activity peaks in BCB were
chieved earlier than in STR (Table 2). The anticipated produc-ion of both lignin-modifying enzymes, potentially involved in
A. D’Annibale et al. / Biochemical Engineering Journal 29 (2006) 243–249 247
Fig. 2. Reversed-phase HPLC chromatograms at 254 nm of OMW incubation control (A) and treated for 6 days with P. tigrinus liquid cultures in a 3-l bubble-column reactor (B). The following compounds have been numbered according to their increasing retention times: (1) gallic acid; (2) hydroxytyrosol; (3) 3,4-dihydroxyphenylacetic acid; (4) catecol; (5) 2,5-dihydroxybenzoic acid; (6) 3-hydroxyphenylacetic; (7) tyrosol; (8) 3-hydroxybenzoic acid; (9) 4-methylcatecol;(10) syringic acid; (11) vanillin; (12) 3-methoxyphenol; (13) para-coumaric acid; (14) ferulic acid; (15) synapic acid; is internal standard.
phenols breakdown [18,20], might explain why MDR and DSin BCB were generally higher than in STR. In addition, nolag phase was practically observed in BCB as shown by thetime courses of biomass production and COD consumption(Fig. 1).
In STR, the combination of agitation and aeration regimes(250 rpm and 1 vvm), providing the lowest volumetric masstransfer coefficient (KLa 0.0647 s−1), led to reduced fun-gal growth (2.8 g l−1), MnP production and productivity(65 ± 9 IU ml−1 and 0.27 IU l−1 h−1, respectively). In contrast,
248 A. D’Annibale et al. / Biochemical Engineering Journal 29 (2006) 243–249
under this conditions, depollution performances, laccase pro-duction and productivity were not negatively affected in com-parison with other STR experiments.
At the highest stirrer speed (750 rpm), time requirements toobtain maximal removal of pollutant parameters were markedlyhigher than those needed at 500 rpm. Consequently, all thekinetic parameters related to OMW depollution (e.g. MDR, ORRand MCR) were significantly lowered.
The lower adequacy of STR than BCB in supporting OMWbiodegradation by P. tigrinus, especially at high stirring and aer-ation regimes, might be due to the occurrence of shear stress phe-nomena. Several studies report the negative effect of shear stresson the degradative metabolism of white-rot fungi [25,30,31].Mycelial damage and morphological changes caused by shearstress were observed when the white-rot fungus Trametes multi-color was grown in a stirred-tank reactor, leading to low laccaseproduction [32]. For P. tigrinus, in particular, it was reportedthat the ligninolytic capacity of strain 8/18 was suppressed bythe agitation [19].
Enzyme denaturation caused by the mechanical forces gen-erated at high speed regimes [30] and/or vigorous aeration [26]might be an additional explanation for the lowest LMEs produc-tion observed in this study.
Differences in KLa between the two reactors tested wererecorded only under conditions of low aeration (1 vvm/250 rpmand 0.5 vvm/500 rpm for the STR and 0.1 vvm for the BCB).SdSruosmrtamh[sa
r≥aoa[p
bsaamb
The results of this study show that the up-scaling of OMWdepollution treatment by P. tigrinus CBS 577.79 should be car-ried out in reactors providing good oxygen transfer with minimalshear effects such as the BCB. Moreover, due to the low aera-tion necessary for good mixing and mass-transfer the use of asimple Bubble Column Bioreactor would have a positive impacton process costs.
Acknowledgements
This work was financially supported partially by theConsorzio Interuniversitario della Chimica per l’Ambiente(INCA) within the frame of the project “Piano AgroalimentareNazionale” and partially by the European Union, project“Medusa Water” (contract no. ICA3-CT-1999-00010).
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[
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