Anaerobic digestion challenge of raw olive mill wastewater

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    The adopted solution in many countries is the evaporation inopen ponds which requires large areas and generates several prob-lems such as bad odour, methane emissions, inltration into thesoil and insect proliferation (Roig et al., 2006; Jarboui et al.,2010). This means that common cost-effective practices appliedto OMW management are not an operative solution to solve thisproblem.

    tions 204400 times higher than the ordinary urban wastewater(Azbar et al., 2009; Xing et al., 2000) and, consequently, it repre-sents a signicant energy potential (Gelegenis et al., 2007). Apartfrom the renewable energy generation in the form of biogas, anaer-obic digestion presents some other appealing advantages since itallows small amounts of sludge generation, low nutrient require-ments, reduction of greenhouse gases emissions and productionof a liquid fertilizer. However, several OMW characteristics suchas the acid pH, low alkalinity, low nitrogen content and the pres-ence of a lipidic fraction and phenolic compounds derived fromthe olive stones and pulp, make this wastewater a potential toxic

    Corresponding author. Tel.: +351 210924600; fax: +351 217127195.E-mail addresses: (M.A. Sampaio), marta.goncal

    Bioresource Technology 102 (2011) 1081010818

    Contents lists available at

    T (M.R. Gonalves), (I.P. Marques).production did not contribute to nd a solution in order to properlymanage the resulting efuent (McNamara et al., 2008). OMWcannotbe treated in a domestic wastewater treatment plant due totechnical limitations (Rozzi and Malpei, 1996). On the other hand,the application of untreated OMW on soils and crops causesphytotoxic and biotoxic effects whichmake it unsuitable for furtheruse as fertilizer or as irrigation water (Niaounakis and Halvadakis,2006).

    this situation. Biological processes are considered environmentallyfriendly and, in many cases, a cost-effective procedure (McNamaraet al., 2008).

    Anaerobic digestion has been reported as one of the most prom-ising technologies for the disposal of OMW (Paraskeva andDiamadopoulos, 2006; Marques, 2000). Comprising a high organiccontent (45220 g COD L1), this efuent is classied among thestrongest industrial liquid wastes that corresponds to concentra-1. Introduction

    Olive oil production is expandinghealth-promoting effects. Most counphase centrifugation system, fromwhreddish-brown liquid called olive m(Morillo et al., 2009; McNamara etbecome a serious environmental proil increasing production and induprocess that generates larger amoun2006). Moreover, the scattered and0960-8524/$ - see front matter 2011 Elsevier Ltd. Adoi:10.1016/j.biortech.2011.09.001wide as a result of itsake use of the three-

    ge quantities of a strongstewater are obtained008). This efuent hasas a result of the oliveation of the extractionMW (Kapellakis et al.,nal nature of olive oil

    OMW has been the subject of many waste treatment studiesinvolving chemical and physical treatment (coagulation/occula-tion and chemical oxidation), biochemical treatment (fermentation,aerobic process, composting) and combined processes/techniques(Roig et al., 2006; El-Gohary et al., 2009; Sarika et al., 2005). How-ever, no satisfactory solution has yet been found for the safe OMWdisposal mainly due to technical and economical limitations(Morillo et al., 2009). As a result, signicant OMW volumes in Med-iterranean area are discharged directly into watercourses (Azbaret al., 2009; El-Gohary et al., 2009) and it is urgent to adopt technol-ogies that allow maximizing the benet/price ratio and overcomeAnaerobic digestion challenge of raw oliv

    M.A. Sampaio, M.R. Gonalves, I.P. Marques Bioenergy Unit, National Laboratory of Energy and Geology I.P. (LNEG), Estrada Pao do

    a r t i c l e i n f o

    Article history:Received 30 May 2011Received in revised form 26 August 2011Accepted 1 September 2011Available online 10 September 2011

    Keywords:Raw olive mill efuentBiogasAnaerobic hybrid digesterPhenolic compoundsOrganic shocks operation

    a b s t r a c t

    Olive mill wastewater (OMHigh concentrations (545OMW adverse characteristCH4) and 8182% COD remperformance was also obseing piggery efuent and ODeveloped biomass (350

    efuents complementarityobtained. Unlike what is reexpenses to correct it or d


    journal homepage: www.ll rights reserved.mill wastewater

    iar 22, 1649-038 Lisboa, Portugal

    was digested in its original composition (100% v/v) in an anaerobic hybrid.g COD m3), acid pH (5.0) and lack of alkalinity and nitrogen are someLoads of 8 kg COD m3 d1 provided 3.73.8 m3 biogas m3 d1 (6364%l. An efuent with basic pH (8.1) and high alkalinity was obtained. A goodd with weekly load shocks (2.74.1, 8.410.4 kg CODm3 d1) by introduc-alternately. Biogas of 3.03.4 m3 m3 d1 (6369% CH4) was reached.s) was neither affected by raw OMW nor by organic shocks. Through thencept, a stable process able of degrading the original OMW alone wasred, OMW is an energy resource through anaerobiosis without additionalase its concentration/toxicity.

    2011 Elsevier Ltd. All rights reserved.

    SciVerse ScienceDirect


    evier .com/locate /bior tech

  • 2. Methods

    2.1. Substrates: agro-livestock and industrial efuents

    The OMW tested in this study resulted from the olive oil cam-paign of 2010. It was collected in an olive oil production plantequipped with a three-phase olive oil extraction process, locatedin Rio Maior (Portugal). The unit is characterized by an averageolive oil production capacity of 42 m3 year1. Piggery efuentwas obtained from a pig farming facility located in the vicinity ofthe olive oil mill, also in Rio Maior. Both substrates were character-ized (Table 1) and stored at 4 C. They were digested in their origi-nal form; which means that they were not subjected to anyalteration.

    2.2. Analytical and chromatograph methods

    Characterization of the efuents used in the hybrid feed.

    echnology 102 (2011) 1081010818 10811substrate and not suitable for anaerobiosis. To overcome theseproblems several synthetic nutrient, chemical additions and pre-treatments (chemical and biochemical) have been reported toenable OMW anaerobic digestion (Dareioti et al., 2009; El-Goharyet al., 2009; Martinez-Garcia et al., 2009; Azbar et al., 2009,Gelegenis et al., 2007). But again, these pre-treatments involveinputs which raise the cost-benet ratio and also lead to organicload reductions and, consequently, to a decrease of the availablemethanogenic potential for energy production.

    This work is part of a broader plan that aims to make the ener-getic valorisation of the raw OMW, by anaerobiosis, simpler, moreexible and cheaper. So, the concept of OMW complementaryefuent was applied in order to reduce the treatment processingsteps by elimination of the operational phases related with OMWcorrections and/or pre-treatments. This feeding approach wastested by combining progressive increases of OMW volumes witha complementary efuent during the experimental period. The rstresults have showed to be possible to treat anaerobically the rawOMW using another efuent and digesting them simultaneously(Marques et al., 1997, 1998).It was also proved that the piggeryefuent can work as a good complementary efuent of OMW.Afterwards, the strategy of combining OMW with other wasteswas used by different authors. A study about the effect of differentsubstrates (manure, household waste and sewage sludge) revealedthat OMWandmanure were the best co-digestion option (Angelidakiand Ahring, 1997). More recently, a combination of OMW anddiluted poultry manure was degraded in a cylindrical down owanaerobic reactor with 18 days of hydraulic retention time (HTR)(Gelegenis et al., 2007). However, under a critical OMW percentageof 28% (v/v) the methane production rate dropped rapidly and1 m3 m3 d1 was registered as a maximum. In a two-stage CSTRanaerobic reactor, Dareioti et al. (2009) used a mixture of 55%OMW and 40% cheese whey and 5% (v/v) liquid cow manure. Efu-ent was successfully degraded and a methane production rate ofabout 1.35 m3 m3 d1 was obtained using a HTR of 19 days. Othermixture (75% OMW plus 25% pig slurry) was pre-treated byCandida tropicalis and digested in a xed bed reactor (HTR of11 days) to give 1.61 m3 m3 d1 of biogas (Martinez-Garciaet al., 2009). The up-ow xed bed digester (anaerobic lter), pre-viously studied by Marques (2001), was also tested with severalfeed mixtures but without recourse to any operational action be-fore the anaerobic digestion phase (pre-treatments, chemical cor-rections or supplementations). Working with 83% OMW and 17%piggery efuent (v/v) and about 6 days of HTR, a production rateof 1.31 m3 CH4 m3 d1 was registered. Following the work per-formed and aiming to make the process even simpler and cheaper,it was decided to test other up-ow digester type. An anaerobichybrid digester was used instead of the anaerobic lter (Gonalveset al., 2009). Similar feed mixtures were provided and amounts of83% OMW (v/v) were treated without any inhibition (HRT = 6 days)providing a methane production rate of 1.96 m3 m3 d1 (Gonalveset al., submitted for publication).

    Based on the team results a biogas plant working all year withthe complementary substrate can advantageously receive increas-ing amounts of OMW without affecting the system stability. Bear-ing in mind the seasonality of the OMW production (three or fourmonths a year) and the large volumes of efuents that are gener-ated (730 million m3 every year: Niaounakis and Halvadakis,2006), the present work aims to test the ability of the anaerobicunit to digest a single substrate. In order to reduce the storage timeof OMW, a feeding consisting only of the original OMW was pro-vided to study its effect in the digester behaviour. Being possibleand advantageous to operate the reactor with two substrates

    M.A. Sampaio et al. / Bioresource T(OMW and the complementary efuent), the other goal of thisstudy is to evaluate the conditions of process stability when so dif-ferent efuents are alternately introduced.OMW PE

    pH 4.96(0.08) 6.99(0.01)Partial alkalinity (kg CaCO3 m3) 0 4.85(0.14)Total alkalinity (kg CaCO3 m3) 2.40(0.07) 8.63(0.39)COD (kg O2 m3) 55.28(2.3) 30.71(0.00)CODS (kg O2 m3) 50.81(0.57) 12.12(0.12)NH3 (kg N m3) 0 1.83(0.04)Total N (kg N m3) 0.21(0.02) 2.35(0.53)TSS (kg m3) 3.18(0.07) 19.60(1.65)TS (kg m3) 28.23(0.36) 23.31(0.05)VSS (kg m3) 0.53(0.00) 4.80(0.38)VS (kg m3) 15.85(3.22) 15.70(0.01)VFA (kg acetic acid m3) 2.64(0.38) 3.43(0.27)TPh (kg caffeic acid m3) 3.59(0.01) 0.38(0.00)Colour [390 nm] 22.42(0.68) 2.23(0.00)Aromatic compounds [254 nm] 128.12(1.93) 17.82(0.00)Total and soluble chemical oxygen demand (COD and CODS),total solids (TS), volatile solids (VS), total suspended solids (TSS),volatile suspended solids (VSS) and total ammonium nitrogen(TNH4 -N) concentrations were determined according to StandardMethods (APHA, 1998). The proportion of ammonium concentra-tions and free ammonia (NH4 versus NH3) were estimated accord-ing to Eq. (1) (El-Mashad et al., 2004), where T is the absolutetemperature (273373 K).

    NH3 N THN4 N 1 10pH=10 0:1075 2725=T


    pH measurements were performed in a WTW pH meter andprobe. Alkalinity was evaluated as partial alkalinity (PA) and totalalkalinity (TA) by titration to pH 5.75 and 4.50 with normalized0.1 N HCl, respectively. Total nitrogen (TN) was quantied viaMerck Nitrogen cell tests (10150 mg NL1). Colour and aromaticcompounds measurements were assayed by measurement of theabsorbance at 390 and 254 nm, respectively, using a HitachiU-2000 Spectrophotometer. Total phenolic compounds (TPh) con-centration values as caffeic acid were determined via a modiedFolinCiocalteu method (Singleton and Rossi, 1965). Volatile fattyacids (VFA: acetate, propionate, butyrate, iso-butyrate, iso-valerateand valerate) were analysed using a gas chromatograph (HewlettPackard 5890) equipped with a ame ionization detector and a2 m 2 mm Carbopack B-DA/4% Carbowax 20 M (80120 mesh)column. Nitrogen was used as carrier gas (30 mL mn1). Tempera-ture of the column, injector and detector was 170, 175 and 250 C,respectively. Total VFA concentrations were expressed as acetic

    Table 1OMW, olive mill wastewater; PE, piggery efuent; 0, below detection limit.Values are the averages of determinations. Values in brackets show standarddeviations.

  • acid. Soluble sampleswere obtained by centrifugation at 10000 rpmfor 10 min using a VWR Galaxy 7D Microcentrifuge and were usedfor CODS, colour, aromatic compounds, total phenolic compoundsand VFAs determination.

    The methane content of biogas collected in the digester head-space was measured by the injection of 0.5-mL bioreactor gassample into a gas chromatograph (Varian CP 38000) equipped witha thermal conductivity detector and a Porapack S column of1/8 3 m. Column, injector and detector temperatures were 50,60 and 100 C, respectively. Nitrogen was utilized as the carriergas (20 mL mn1).

    2.3. Reactor operation

    Experiments were carried out by using an up-ow anaerobichybrid digester that was previously used and described elsewhere(Gonalves et al., submitted for publication). It was initiallyremoved from the cold chamber at 4 C and then kept under mes-ophilic conditions of temperature (37 1 C) by using a water jack-et. It was fed in a semi continuous manner by means of a peristalticpump in order to obtain a HRT of about six days. The inuent con-sisted of a blend of the raw OMW and its complementary substrate(piggery efuent, PE) obtained by an increase of OMW contentalong the experimental time (Marques, 2001). Gas production

    (B) Digester feeding with the original OMW (from day 233 to287, Raw OMW feed). The hybrid reactor was fed with100% OMW and any kind of supplementation, correction ordilutions of the olive oil mill efuent were performed (Table2). On day 288 of the experiment, an interruption of thedigester operation took place. During a period of 11 days,the unit was preserved at mesophilic conditions of temper-ature and no feed was provided.

    (C) Feeding the digester by applying alternate pulses of each ofthe substrates (from day 300 to 350 days, Raw OMW or PE,alternated feed) (Table 2). The unchanged OMW wasdigested during the initial two weeks (C0) of this phase. Afterthat, ve weekly cycles were performed by using alternatelyeach substrate. During each cycle, 17% and 83% of the oper-ational time corresponded to PE and OMW supplies,respectively.

    3. Results and discussion

    The load applied to the hybrid unit was efciently convertedinto biogas. The hybrid stability and its capacity in convertingthe potential toxic matters of the inuent are documented by theremoval ability and methane production of the digester biomassover the 350 days of experiment.

    10812 M.A. Sampaio et al. / Bioresource Technology 102 (2011) 1081010818was evaluated by a wet gas meter and corrected to standard con-ditions for pressure and temperature (1 atm, 0 C). Volume ofdigested ow was registered every day in order to determine thehydraulic retention time (HRT) of the assay. Inuent and efuentsamples were taken one or two times a week along the trial time.The operational period can be described in three main experimen-tal phases:

    (A) The restarting of the hybrid digester and its operation usingOMW complemented with PE (up to day 232, Raw OMWand PE, mixture feed). During the rst 14 days of operation,the hybrid was fed with piggery efuent and a HRT of aboutsix days was set. Afterwards, the digester inuent mixturewas changed. OMW volumes of 53%, 69% and 83% (v/v) wereprovided. (Table 2)

    Table 2Operational conditions of hybrid digester.

    Phase Time (d) Substrates

    OMW (% v/v)

    A0 014 0A1 1582 53


    A2 137168 69A3 169232 83

    B 233287 100

    Stop 288299 C0 300313 100C1 314315 0

    315320 100C2 321322 0

    322327 100C3 328329 0

    329334 100C4 335336 0

    336341 100C5 342343 0

    343350 100Raw OMW and PE, mixture feed (Phase A), Raw OMW feed (Phase B) and Raw OMW orOMW, Olive Mill Wastewater; PE, piggery efuent; HRT, Hydraulic Retention Time; OLRValues are the averages of determinations. Values in brackets show standard deviations3.1. Phase A. Complementary substrates trial: raw OMW and PE,mixture feed

    Operating under organic loading rates (OLR) of 5.2 kg CODm3 d1 (Phase A2, Table 2) and 7.49.0 kg COD m3 d1 (PhaseA3), ranges of total COD removal of 75% and 8083% and biogas vol-umes of 2.7 and 2.83.6 m3 m3 d1, containing 67% and 6667%CH4, were obtained, respectively. The increase of the inuent con-centration till 57 kg COD m3 (Phase A3) did not cause instabilityneither decrease of the hybrid performance. The methane yieldof 0.429 m3 CH4 kg1 COD removal (Phase A2) evolved to0.3170.358 m3 CH4 kg1 COD removal (Phase A3, data not shown)indicating the unit capacity to degrade the organic matter accumu-lated and overcome the disequilibrium of the system.

    HRT (d) OLR (kg COD m3 d1)

    PE (% v/v)

    100 6.5(0.4) 47 6.2(0.5) 4.9(0.1)

    6.5(0.2) 18.5()6.3(0.5) 5.6(0.8)

    31 7.3(0.8) 5.2(0.6)17 6.6(0.2) 7.4(0.4)

    6.3(0.4) 9.0(0.3)0 6.4(0.7) 8.1(1.2)

    6.6(0.4) 8.0(0.3) 0 6.5(0.3) 10.4(0.7)

    100 6.0(0.8) 4.1(0.2)0 7.0(0.7) 8.8(1.3)

    100 5.2(0.2) 3.6(0.2)0 6.7(0.2) 8.4(0.9)

    100 5.5(0.2) 2.7(0.2)0 6.0(1.2) 8.5(1.1)

    100 6.1(0.9) 3.1(0.1)0 6.7(0.9) 8.9(1.1)

    100 5.7(0.2) 2.7(0.1)0 6.6(0.4) 8.6(0.6)PE, alternated feed (Phase C)., Organic Loading Rate..

  • The main results of the OMW digestion obtained in differentyears and provided from different mills (Gonalves et al., submit-ted for publication and current work) were presented against theload applied at 69% and 83% v/v OMW feeds (Fig. 1). From them

    it is possible to infer that the increase of OLR (5.29.0 kg CODm3 d1) promotes the production of the biogas volume(2.43.6 m3 m3 d1) and maintains its quality along the time ofoperation. Methane concentrations (6267%) were preserved in a

    y = 0,3519x + 0,5881R = 0,6414

    y = -0,4157x + 67,902R = 0,0647





















    4 5 6 7 8 9 10






    (m3 m


    1 )

    Organic loading rate (kg COD m-3d-1)

    Biogas 69% OMW Biogas 69% OMW, G.2011 Biogas 83% OMW Biogas 83% OMW, G.2011

    CH4 69% OMW CH4 69% OMW, G.2011 CH4, 83% OMW CH4 83%, G.2011

    Linear (Biogas) Linear (CH4)

    Fig. 1. Hybrid gas productivity: operation with 69% and 83% v/v OMW (Gonalves et al., submitted for publication).

    Table 3Anaerobic digestion of olive mill wastewater: operation methodologies using substrates mixtures.

    No. Reactor,temp. (C)

    Efuents(% v/v)


    HRT (d) OLR (kg COD m3 d1) Biogas methane (m3 m3 d1) CODremoval(%)


    1 AF, 35 OMW-83 None 67 7.79.6 3.64.0 7077 Marques et al. (1997)PE-17 2.32.6

    2 CSTR, 55 OMW-75 None 13 7.8 Angelidaki and Ahring(1997)Manure-25 1.55

    3 AF, 35 OMW-91 None 67 5.05.7 1.72.1 7375 Marques et al. (1998)PEdig.-9 1.11.4

    4 AF, 35 OMW-91 None 67 6.68.0 2.1 63.2 Marques (2001)PE-9 1.3OMW-83 8.310 3.42 73.6

    M.A. Sampaio et al. / Bioresource Technology 102 (2011) 1081010818 10813PEdig.-175 ,35 OMW-28 pH

    adjustments18 4.84

    diluted poultrymanure

    6 Fixed-bedreactor, 37

    OMW-75,cheese whey-25

    - sterilization- treatment:

    - 3.0C. tropicalis (a)- 26 g NaOH L1

    addition- NaHCO3addition

    7 Fixed-bedreactor, 37

    OMW-75, PE-25 11 5

    8 2 CSTR:acidogenic+ Methanogenic,35

    - OMW-55- cheese whey-40- cow manure-5

    - 0.26 g ureaL1feed)addition,acidogenicreactor

    - 14 g NaHCO3L1 addition,methanogenicreactor feed

    19 5.5

    9 OMW-20, liquidcow manure-80

    None 19 3.63

    10 AH, 37 OMW-83 None 6 7.1PE-17

    11 AH, 37 OMW-83 None 67 7.4PE-17

    12 AH, 37 OMW-100 None 67 8.01

    AF, anaerobic lter; CSTR, continuous stirred-tank reactor; AH, Anaerobic hybrid digesteranaerobically; HRT, hydraulic retention time; OLR, organic loading rate; COD, chemical

    a Grown in a complex culture medium and phenol.b 29 L d1/18 L = 1.61 L L reactor1 d1 (data from last four days of operation).2.21.53 Gelegenis et al. (2007)0.99

    1.25 83 Martinez-Garcia et al.(2007)1.61b 85 Martinez-Garcia et al.(2009)


    75.5 (% CODs) Dareioti et al. (2009)


    63.2 Dareioti et al. (2010)

    3.16 78.6 Gonalves et al.,submitted forpublication


    3.18 81.3 Present work2.123.742.36

    81.5 Present work

    ; OMW, olive mill wastewater; PE, Piggery efuent, PEdig., Piggery efuent digestedoxygen demand; CODs, soluble COD,

  • top

    ime (

    echPhase B: 100% OMW S








    230 240 250 260 270 280 290


    L (k

    g C



    d-1 );


    gas (



    d-1 )




    10814 M.A. Sampaio et al. / Bioresource Tnarrow range of the anaerobic process usual values. Regarding thedigester removal ability, the increase of phenols concentration ininuent (2.12.3 to 2.93.1 kg TPh m3) did not cause a relevantalteration on unit capacity (58.961.1% and 56.659.2% TPhremoval, respectively). Concerning the total COD, the more concen-trated inuent (4957 kg m3) corresponded to the highest CODremovals recorded (8083%).

    Several authors have presented studies on the use of differentefuents to digest OMW anaerobically. Table 3 summarizes themain data obtained from some operation methodologies that com-bine OMW with other efuents. This strategy has been used bydiverse authors but it was usually associated to several otherphases of operation mainly related to the inuent preparation tothe anaerobic step. Pre-treatment (C. tropicalis: Martinez-Garciaet al., 2007, 2009); pH adjustments (Gelegenis et al., 2007); ureaand alkali (14 g NaHCO3 L1) additions (Dareioti et al., 2009) aresome examples of the undertaken actions. Comparatively, thiswork group has been operated with the lower HRT and the highest





    230 240 250 260 270 280 290

    pH /


    (g L


    Time (

    pH in pH ef







    230 240 250 260 270 280 290


    in (



    Time (

    TPh in



    Fig. 2. Hybrid digester behaviour: Phase B and C (a) gas production, (b) pH andPhase C: organic pulses











    300 310 320 330 340 350 360





    Biogas CH4

    nology 102 (2011) 1081010818volume of the raw OMW in the inuent (83/91%) and, conse-quently, highest loading rates (510 kg COD m3 d1) were testedand higher volumes of gas (1.74.0 m3 biogas m3 d1; 1.12.6 m3

    CH4 m3 d1) were reached (Table 3: no. 1, 3, 4, 10 and 11).

    3.2. Phase B. Unbalance/toxic substrate trial: raw OMW feed

    When the digester was fed onlywith the original OMW (Phase B:233287 days), OLRs of about 8 kg COD m3 d1 provided a totalCOD removal of 8182% and a biogas volume of 3.73.8 m3 m3 d1

    (6364% CH4). The digester performance is presented in Fig. 2 andTables 4 and 5 (Phase B). The acid pH (4.95.1), the high concentra-tions of COD (5455 kg m3), VFA (2.84.0 kg m3) and phenoliccompounds (3.03.3 kg TPh m3) and, additionally, the null orreduced alkalinity and nitrogen contents (COD:N = 277:1 to 338:1,Tables 4 and 6), were some of the adverse characteristics of OMWthatwere registeredalong this assay. Evenunder theseunfavourableconditions, the hybrid feed was accepted and degraded by the


    300 310 320 330 340 350 360





    VFA ef VFA rem


    300 310 320 330 340 350 360





    TPh rem

    VFA, (c) TPh (phenol compounds), in, inuent; ef, efuent; rem, removal.

  • m. (






    echTable 4Hybrid data: COD removal and gas production.

    Phase Total COD Soluble COD

    Inf. (kg m3) Rem. (%) Inf. (kg m3) Re

    B 55.4(1.7) 82.0(1.0) 44.7(3.2) 8054.2(2.4) 81.0(1.7) 48.2(3.4) 80

    Stop C0 67.7(3.57) 81.0(0.6) 53.2(0.13) 76C1 22.0(1.3) 74.2(0.7) 14.5(0.3) 74

    57.8(0.0) 44.4(0.0)C2 17.9(0.9) 80.8(0.3) 13.0(0.3) 77

    M.A. Sampaio et al. / Bioresource Tdeveloped biomass as documented by the comfortable methaneyield reached (Y = 0.3610.377 m3 CH4 kg1 COD). The good perfor-mance of the unit under these operating conditions (100% v/v OMW,Table 3: no. 12) have never been referred before. On contrary, it wasshown that for anaerobic degradation of OMWalone, nitrogen addi-tionwas needed and a COD:N ratio of 61:1 to 42:1was necessary forthe optimal degradation process (Angelidaki et al., 2002). Effec-tively, in this case, the inuent was just composed by OMW thathas not undergone any alteration and presents characteristicspotentially adverse to a successful development of anaerobicprocess.

    Concerning the efuent, pH became basic (8.1) and alkalinityincreased to about 5.0 (PA) and 6.3 kg CaCO3 m3 (TA). Total

    56.5(0.6) 47.8(1.8)C3 15.5(0.9) 78.6(1.0) 10.0(0.3) 76.0(1.6

    53.4(2.4) 43.2(1.2)C4 16.8(0.4) 78.4(0.5) 9.7(0.6) 78.3(1.6

    55.9(0.6) 49.6(1.2)C5 15.0(0.7) 81.1(0.6 8.9(0.4) 80.3(0.7

    57.8(1.7) 48.4(1.2)

    () Single determination; Values are the averages of determinations taken at steady-sta

    Table 5Hybrid mean data: alkalinity and nitrogen contents.

    Phase Substrate Partial alkalinity (kg CaCO3 m3) Total alkalinity (kg CaCO

    in ef in ef

    B OMW 0 5.03(0.41) 2.42(0.06) 6.32(0.2OMW 0 5.05(0.11) 2.46(0.05) 6.20(1.1

    Stop C0 OMW 0 4.85(0.02) 2.37(0.11) 6.20(0.1C1 PE 3.13(0.18) 4.88(0.04) 6.48(0.11) 6.33(1)

    OMW 0 2.37(0.11)C2 PE 3.40(0.19) 5.19(0.55) 6.50(1) 6.71(0.4

    OMW 0 2.25(1)C3 PE 4.71(0.05) 5.69(0.1

    OMW 0 C4 PE 3.96(0.02) 4.70(0.35) 6.36(0.02) 5.61(0.0

    OMW 0 2.04(0.19)C5 PE 3.94(0.37) 4.75(0.07) 5.36(0.16) 5.55(0.1

    OMW 0

    In, inuent; ef, efuent; 0, below detection limit; (1) single determination; () standaa Calculated from Eq. (1).

    Table 6Conversion capacity of the hybrid digester: Phases A, B and C.

    Phase OLR (kg COD m3 d1) COD rem (%) Bioga

    Aa 8.20 81.3 3.18BC0 8.83 81.4 3.61C1C5b 7.72 79.0 3.25

    a 83% OMW + 17% PE (v/v).b 83% OMW + 17% PE (time feed).Biogas (m3 m3 d1) CH4 (%) Y (m3 CH4 kg1 COD)


    ) 3.78(0.26) 63.7(1.2) 0.361(0.054)) 3.70(0.16) 62.5(1.2) 0.377(0.067)

    ) 3.35(0.08) 63.1(1.0) 0.249(0.017)) 3.22(0.56) 63.2(-) 0.332(0.004)

    ) 3.39(0.65) 62.5(-) 0.350(0.006)

    nology 102 (2011) 1081010818 10815phenolic compounds removal of 46% was reached, being theremained fraction (1.41.5 kg m3) probably due to the presenceof polymerized phenolic matter since no colour clarication wasnoticed. Instead, a slight increase of the colour absorbance valueswas registered in digester efuent. Absorbance data of 2427 and3031 were recorded in the inuent and efuent, respectively.VFA were mostly consumed in the system and the VFA removalof 9698% resulted into an efuent of 0.080.1 kg m3 (Fig. 2b),being the acetic acid the main component (89% total VFA).Regard-ing the solids, inuent concentrations of 34.5 TS kg m3 (3.2) and1.04 VSS kg m3 (0.36) (269314 days), correspond to efuentamounts of 17.5 TS kg m3 (0.99) and 0.43 VSS kg m3 (0.24),respectively. These data indicate that the reactor was not subjected

    ) 3.43(0.88) 63.7(-) 0.371(0.023)

    ) 3.15(0.85) 68.7(-) 0336(0.006)

    ) 3.03(1.01) 62.9(-) 0.326(0.013)

    te period. Values in brackets shows standard deviations. Y, methane yield.

    3 m3) NH4 (kg N m3) NH3 (kg N m3)a Total N (kg N m3)

    in ef in ef in ef

    3) 0.01(0.01) 0.13(0.06) 0.20(0.01) 0.42(0.08)7) 0 0.02(0.02) 0.19(0.01) 0.31(0.04)

    8) 0 0 0.20(0.02) 0.24(0.02)

    1.74(1) 0.10(1) 0.081() 0.007() 1.99(0.51) 0.37(1)

    0 0.20(0.02)8) 1.69(0.02) 0.13(0.00) 0.070() 0.008() 1.83(1) 0.42(1)

    0 2) 1.60(0.04) 0.11(0.01) 0.139() 0.003() 2.25(1) 0.40(1)

    0 9) 1.55(0.05) 0.12(0.01) 0.130() 0.005() 0.43(0.01)

    0 1) 1.32(0.01) 0.11(0.01) 0.150() 0.004() 0.39(0.02)


    rd deviation that was not calculated.

    s (m3 m3 d1) CH4 (m3 m3 d1) Y (m3 CH4 kg1 COD)

    2.12 0.342.28 0.332.08 0.34

  • echto any washout process and that the solid biomass was maintainedin good conditions inside the unit.

    Table 6 provides a comparison of the average main dataobtained in different experimental phases. The results comparisonof the feeds digestion containing 83% and 100% of the raw OMW(Phases A and BC0) reveals that the OLR increase to 8.8 kg CODm3 d1 had a positive effect on gas production. Biogas and meth-ane productivity increased and the removal capacity was main-tained at previous levels. Table 6 gures reinforce the ndingalready made on the quality of the hybrid functioning regardingits great ability to degrade the raw OMW without the addition ofanother efuent.

    3.3. Phase C. Organic pulses trial: raw OMW or PE, alternated feed

    During the weekly cycles experiment (Phase C), PE and OMWwere alternatively supplied on its original composition accordingto Table 2. Successive changes of operating condition did not causethe disruption of the system. On the contrary, the performance ofthe hybrid was maintained as it is shown in Fig. 2 and Tables 4and 5 (Phase C). The introduction of PE followed by the OMW dur-ing the same experimental week corresponds to operate the unitunder constant shock of organic loads. The applied concentrationsof PE and OMW (1522 and 5358 kg COD m3, respectively) haveoriginated OLR alterations of three-four times (2.74.1 and8.48.9 kg COD m3 d1). The hybrid replied positively to theseconditions by providing gas volumes of 3.03.4 m3 m3 d1

    (6369% CH4) and methane yields of 0.3260.371 m3 CH4 kg1

    COD removal (Table 4). As it was already observed (Phase B), theoriginal OMW was also accepted by the present system that wassubject to intermittent working conditions.

    Comparing the values obtained during the operation of the mix-ture with 83% (v/v) OMW with those reached in this period (PhaseA versus Phases C1C5) it is noticed that the degree of inuenttreatment and the reached gas production in both trials are identi-cal (3.2 m3 biogas m3 d1 and 2.1 m3 CH4 m3 d1, Table 6). Effec-tively, the pulses experiment was predetermined based on theoperating time of each cycle and 17% and 83% of the functioningtime were used to introduced PE and OMW, respectively. So, sim-ilar amounts of each of the efuents have been supplied to the sys-tem in both operational situations. Data indicates that theoperation mode, feeding the digester with efuents mixture orefuents individually, is not a determinant factor for the properfunctioning of the anaerobic unit.

    3.4. OMW valorisation: resistance and adaptation capacities of theanaerobic system

    3.4.1. Feed suspension and storageThe hybrid digester used in this experiment was previously

    applied to digest several substrates (Gonalves et al., submittedfor publication) and it was preserved inside of a cold chamber dur-ing 18 months. Then, it was restarted to degrade substrates fromother processing units. The obtained results illustrate that theanaerobic digester biomass can be kept dormant for severalmonths as referred by Rozzi and Malpei (1996). The developedhybrid population did not lose the activity even after a period offeed suspension and storage at low temperature. Rather, it wasable to adapt and digest a new stock of substrates and convertthem into gas.

    Another period of feeding suspension took place after Phase B.Then, the hybrid was operated (Phase C0, day 300) with the originalOMWthatwas also used in the earlier period. It is interesting to note

    10816 M.A. Sampaio et al. / Bioresource Tthe response of the system. A rapid increase in gas production wasobserved in 6 days (Fig. 2a) and removals of 81% COD total(68 kg m3), 79% COD soluble (53 kg m3) and 99%VFA (5.2 kg m3)were registered (Fig. 2b and Table 4), indicating once again the greatcapacity of the system to degrade the unchanged OMW.

    3.4.2. Overloading and load shocks operationThe hybrid was accidentally subjected to an OLR increase from

    4.9 kg COD m3 d1 (inuent of 31 kg COD m3, 1582 days) to18.5 kgCODm3 d1 (inuent of 112.9 kgCODm3, 8394 days: Ta-ble 2). Additionally, in the nal phase of this period, the inuentwaschanged to a new stock of OMW and PE and an OLR of 5.6 kg CODm3 d1 (95136 days) was applied. As a result, the removal capac-ity of the unit (7783% COD, 83136 days) was not accompanied byits ability in converting the organicmatter into gas. Despite the goodquality of the biogas obtained (7072% CH4) indicating an activemethanogenic population, a low methane yield (0.1810.186 m3

    CH4 kg1 COD removal) was reached through these periods(83136 days) (data not shown). In order to assess the resilience ofthe changes in unit operation, it was decided to continue runningthe hybrid and increase the OMW amount into inuent (to 69%and 83% v/v) according to the work schedule (Table 2). The resultsreached subsequently and previously discussed in this paper allowinferring that the unit system had the ability to resist to adverseoccurrences. The hybrid resistance to an accidental overload andits capacity in preventing excessive loss of biomass was already no-ticed before (Gonalves et al., 2009, submitted for publication). Theadaptation capacity to different OMW stocks has also been veriedby other authors referring that the anaerobic biomass acclimatedto one substrate (particular phenol molecule) is simultaneouslyacclimated to other substrates with related structures (phenolmolecule) (Healy and Young, 1979).

    The anaerobic hybrid operated under successive organic pulsesby digesting PE andOMWalternately (Phases C1C5, 314350 days).The conversion efciency data and the working process stabilityobtained indicate that themicrobial communities developed a resis-tance capacity and were not disabled by the successive and pro-nounced alterations of organic loading rates (from 2.7 to 11 kgCOD m3 d1), pH and potential inhibiting/toxic compounds suchas phenolic matter, VFA and free ammonia contents.

    PE is characterized by high concentrations of total ammoniumand organic nitrogen (urea and proteins) and as the organic nitro-gen is degraded, the ammonium is released. The total ammoniumconcentration has a double effect in anaerobic digestion. It acts as apromoting agent of the buffer system by maintaining a high levelof bicarbonate but, on the other side, it can cause inhibition prob-lems that lead to an unstable process with a lowmethane yield anda high VFA level in the efuent (Murto et al., 2004; Zhang andJahng, 2010). The total ammonium inhibition has been suggestedto be directly related to the concentration of the undissociatedform (NH3) (Chen et al., 2008) being the effect more notable at highpH levels and temperatures (Eq. (1)). The aceticlastic methanogensare the most sensitive to ammonia toxicity and free ammoniaconcentrations of 25150 mg NH3-N L1 have been reported asinhibitory for mesophilic treatment. Levels up to 1.1 g NH3 L1

    can be tolerated if the culture has undergone a gradual adaptation(Guerrero et al., 1997; Murto et al., 2004).

    When the hybrid digester was fed only with OMW(233313 days) the biomass was probably accommodated to extre-mely low free ammonia concentrations; when the PE started to beused (314 day) an opposite situation takes place: the inlet freeammonia concentration of PE (72150 mg L1, Table 5) was in therange of inhibitors concentrations. The results (Fig. 2b and Table 4)indicate that biomass seems to have acquired the ability to withstand both adverse situations. So, it is possible to operate the anaer-obic hybrid under stable conditions using different efuents that are

    nology 102 (2011) 1081010818alternately applied.Another relevant aspect is the similarity of average data of the

    gas production and treatment efciency of the anaerobic process

  • individual substrates (Phase A versus Phases C1C5, Table 6). Thus,

    toxicity. The digested ow with a basic pH and high alkalinity and

    codigesting with diluted poultry-manure. Appl. Energy 84, 646663.

    concentrations. Bioresour. Technol. 61, 6978.

    echdevoid of VFA may be useful for agricultural applications. Theanaerobic hybrid is a sustainable and environmentally-attractivemeans of reducing OMW organic load, generating two products ofinterest (organic stream and a renewable energy source) and con-tributing for the greenhouse gas reduction.

    4. Conclusionsgiven that both feed models provide analogous results, the pulsesprocedure has the advantage of suppressing the efuents mixingstep. Taking into account a biogas plant, feeding the digester usingwastes separately, certainly contributes to make the process evensimpler and cheaper.

    3.4.3. Raw OMW as inuent of anaerobic hybrid digesterSeveral unfavourable characteristics of OMW that make it

    unsuitable for anaerobic process may be easily overcome by theapproach performed in this work: make use of an additionalefuent to complement OMW. A biomass adaptation process isprovided through the provision of increasing amounts of the con-centrate and potentially toxic substrate over time.

    The presences of inhibiting/toxic compounds have been men-tioned as a signicant problem for anaerobic digestion of OMW.Phenolic fraction had been described as inhibitory to methanogens.The water dilution of OMW has been used as method to reduce theconcentration of phenols and fatty acids. However, this fact resultsin spending on water consumption and larger volume of wastewa-ter to treat. With the prospect of making the process cheaper, thesubstrate concentration and its toxicity can be advantageouslyreduced by using another efuent produced in the vicinity. Swinemanure has been a problem in many regions of Portugal as in othercountries. Due to its high nitrogen content and high pH, freeammonia is usually the relevant inhibitor parameter of the anaer-obic system. Analysing both efuents and comparing them, it ispossible to observe that OMW presents opposite characteristicsof PE and it can run as OMW complement in terms of anaerobictreatment. Indeed, the use of another efuent is a way to providedilution but also an approach to compensate the system for gapsin the OMW composition, making the process much cheaper andappealing. Examples of compensation are the enhancement ofpH, alkalinity and nitrogen values.

    Intending to degrade the original OMW without any comple-mentation, all unfavourable characteristics inherent to the sub-strate will be present. It is well documented that OMW is anunbalance substrate and its concentration associated to its toxicitydo not permit the anaerobic process establishment (El-Goharyet al., 2009; Gelegenis et al., 2007). However, the results obtainedin this work suggest the opposite. The classic drawbacks of OMWwere somewhat mitigated and supported by the developed systeminside of the anaerobic hybrid along the time. The presence ofOMW toxic compounds did not prevent the biological conversionof most of the organic matter contained in the inuent. Theremaining colour is probably associated to the fraction of phenolicmatter that is not degraded under anaerobic conditions. For colourremoval purpose, an additional treatment can be used such as fer-mentative decolorization or electrochemical treatment (Aouidiet al., 2009; Papastefanakis et al., 2010).

    Accordingly, it is possible and advantageous to valorise energet-ically the raw OMW by anaerobiosis avoiding any previous alter-ation of the substrate in order to prevent its concentration and/orto carry out with a feed mixture of 83% OMW v/v or feed pulses of

    M.A. Sampaio et al. / Bioresource TThe high energy potential of OMW (5455 kg COD m3) isdirectly recoverable through anaerobic digestion, without requiringHealy, J.B., Young, L.Y., 1979. Anaerobic biodegradation of eleven aromaticcompounds to methane. Appl. Environ. Microbiol. 38 (1), 8489.

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    Guerrero, L., Omil, F., Mthdez, R., Lema, J.M., 1997. Treatment of saline wastewatersfrom sh meal factories in an anaerobic lter under extreme ammoniaanyprior correctionanddecreaseof its concentrationand/or toxicityand making the process simpler, more exible and cheaper.

    The hybrid worked under stable conditions when successiveshocks of loads (2.74.1, 8.310.7 kg COD m3 d1) were appliedby feeding alternately with different efuents.

    The strategy developed, on the basis of complementaryefuents concept, promoted the hybrid microbial communityadaptation to unfavourable characteristics of OMW. The unbal-anced composition and presence of toxic compounds did notprevent the proper hybrid functioning when 100% (v/v) OMWwas digested.


    The authors acknowledge the nancial support of the Fun-daopara a Cincia e a Tecnologia, FCT/MCTES, through the Pro-ject PTDC/ENR/69755/2006 and also through the grant given toMarta Gonalves SFRH/BD/40746/2007.


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    10818 M.A. Sampaio et al. / Bioresource Technology 102 (2011) 1081010818

    Anaerobic digestion challenge of raw olive mill wastewater1 Introduction2 Methods2.1 Substrates: agro-livestock and industrial effluents2.2 Analytical and chromatograph methods2.3 Reactor operation

    3 Results and discussion3.1 Phase A. Complementary substrates trial: raw OMW and PE, mixture feed3.2 Phase B. Unbalance/toxic substrate trial: raw OMW feed3.3 Phase C. Organic pulses trial: raw OMW or PE, alternated feed3.4 OMW valorisation: resistance and adaptation capacities of the anaerobic system3.4.1 Feed suspension and storage3.4.2 Overloading and load shocks operation3.4.3 Raw OMW as influent of anaerobic hybrid digester

    4 ConclusionsAcknowledgementsReferences


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