Bioresource Technology 101 (2010) 1628–1634

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Bioresource Technology

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Two-phase anaerobic co-digestion of olive mill wastes in semi-continuousdigesters at mesophilic temperature

Boubaker Fezzani *, Ridha Ben CheikhBiogas Laboratory, URSAM, Industrial Engineering Department, Ecole Nationale d’Ingénieurs de Tunis, Université Tunis El-Manar, BP. 37 le Bélvédère, 1002 Tunis, Tunisia

a r t i c l e i n f o a b s t r a c t

Article history:Received 3 June 2008Received in revised form 17 September2009Accepted 18 September 2009Available online 5 November 2009

Keywords:Olive mill wastewaterOlive mill solid wasteTwo-phase anaerobic co-digestionBiogasMesophilic temperature

0960-8524/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.biortech.2009.09.067

Abbreviations: AD, anaerobic digestion; CH4, meth(%); COD, chemical oxygen demand; HRT, hydraulicdihydrogeno sulphur (ppm); NH3, free ammonia nitammonium nitrogen (mg N/L) or (mg N/kg TS); OLR, oday); OMW, olive mill wastewater; OMSW, olive michemical oxygen demand (g COD/L); TCOD, total chemL); TKN, total Kjeldahl nitrogen (g N/L) or (g N/kgphosphorus (g P/L) or (g P/kg TS); TS, total solids (g/L(g/L); TVFA, total volatil fatty acids (g/L); VS, volatsuspended solids (g/L).

* Corresponding author. Tel.: +216 97 37 69 69.E-mail addresses: bfezzani2007@yahoo.fr, b.fezzan

This study investigates for the first time, on laboratory scale, the possible exploitation of the advantagesof two-phase anaerobic digestion for treating a mixture of olive mill wastewater (OMW) and olive millsolid waste (OMSW) using two sequencing semi-continuous digesters operated at mesophilic tempera-ture (37 ± 2 �C). The experiments were conducted at hydraulic retention times (HRTs) of 14 and 24 dayscorresponding to organic loading rates (OLRs) ranging from 5.54 to 14 g COD/L/day in the first stage (acid-ifier) and at HRTs of 18, 24 and 36 days corresponding to OLRs ranging from 2.28 to 9.17 g COD/L/day inthe second stage (methanizer). The results indicated that volatile fatty acids (VFA) concentrationsincreased with the increase of either HRT or feed concentration and their high values were obtained withthe most concentrated influent (196 ± 5 g COD/L) digested at the longest HRT (24 days) corresponded toan OLR of 8.17 g COD/L/d. Furthermore, two-phase anaerobic digestion system has given the best perfor-mances concerning methane productivity, soluble COD (SCOD) and phenol removal efficiencies and efflu-ent quality compared to those given by conventional one-phase anaerobic digestion (AD) reactors.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction content of both organic and phenolic compounds (up to 200 g

Olive mills wastewaters are produced in large quantities inMediterranean countries and cause considerable harmful effectsto the environment such as colouring of natural water, threat toaquatic life, causing surface and ground water pollution, changingsoil quality and plant growth and causing odours because of theirhigh concentration in organic and phenolic compounds (Lyberatoset al., 2002). One of the more effective ways to dispose of thesewastes is using the anaerobic digestion process (Boari et al.,1984, Borja et al., 1993). However, conventional anaerobicdigestion of olive mill wastewater (OMW) exhibits well knownproblems, related to OMW characteristics, that have limited itsapplication such as: intensive violet–dark red up to black colour,low alkalinity and pH, lack in ammonium nitrogen and high

ll rights reserved.

ane (%); CO2, carbon dioxideretention time (days); H2S,

rogen (ppm); NHþ4 —N, totalrganic loading rate (g COD/L/ll solid waste; SCOD, solubleical oxygen demand (g COD/

TS); TPO�4 —P, total mineral); TSS, total suspended solidsile solids (g/L); VSS, volatile

i@laposte.net (B. Fezzani).

COD/L and 15 gphenol/L, respectively: Fiestas et al., 1996; Angelidakiet al., 2002). To overcome these limitations several processes havebeen reported for upgrading OMW anaerobic digestion. The mostcost-effective process with energy recovery is employing the tech-nique of co-digestion of OMW with other substrates. Previousstudies showed that OMW could be treated successfully withouthigh dilution and without adding chemical substances if it wasco-digested with substrates containing high level of ammoniumnitrogen and alkalinity to compensate for their lack in OMW(Angelidaki and Ahing, 1997; Angelidaki et al., 2002; Fezzani andBen Cheikh, 2007). Furthermore, recent research has demonstratedthat two-phase anaerobic digestion (AD) offers an attractive alter-native to conventional one-phase anaerobic digestion because ofseveral potential advantages (Demirer and Chen, 2005). Firstly, itallows the selection and enrichment of different bacteria in eachphase. In the first phase, complex organic materials, carbohydrates,proteins, lipids, amino acids and long-chain fatty acids are de-graded by acidogenic bacteria to intermediary products such asvolatile fatty acids (VFA) and alcohols which are subsequentlyand easily metabolised and converted into CH4 and CO2 by metha-nogens or archaea in the second phase.

Secondly, it increases the stability of the process by controllingthe acidification-phase through optimisation of the hydraulicretention time (HRT) to prevent overloading and the build-upof toxic material. Also, the biomass concentration and otherconditions can be optimised independently for each stage. Thirdly,

B. Fezzani, R. Ben Cheikh / Bioresource Technology 101 (2010) 1628–1634 1629

the first stage may protect methanogens from large VFA produc-tion (especially acetic acid), decreasing of pH in the reactor and ra-pid acidification. Finally two-phase AD has given the bestperformance in methane productivity and COD removal efficiencycompared to one-phase conventional digesters (Bouallagui et al.,2004; Demirer and Chen, 2005). Many applications of two-phaseAD technique either at mesophilic or thermophilic conditions havebeen tested in the last few years and proven their success in treat-ing fruits and vegetables (Verrier et al., 1987; Bouallagui et al.,2004), urban wastewaters (Chanakya et al., 1992), activated sludge(Ghosh, 1991; Ghosh et al., 1995), industrial wastes (Ghosh et al.,1985), grass (Yu et al., 2002), coffee pulp juice (Calzada et al.,1984), food wastes (Koster, 1984), cane-molasses alcohol stillage(Yeoh, 1997), spent tea leaves (Goel et al., 2001), dairy wastewater(Lo and Liao, 1985, 1986a,b; Demirer and Chen, 2005) and abattoirwastes (Wang and Banks, 2003). However two-phase AD has neverbeen applied by any researcher to treat OMW mixed with olive millsolid waste (OMSW). Therefore, taking into account the potentialadvantages of two-phase AD and continuing the research ofupgrading the anaerobic co-digestion of OMW with OMSW theaim of the present work was to assess the performance of two-phase anaerobic co-digestion of OMW with OMSW in semi-contin-uous sequencing digesters operated at mesophilic temperature.This report discusses a laboratory scale investigation with empha-sis placed on the evaluation of optimal values of biogas production,methane percentage, chemical oxygen demand (COD), phenol andcolour removal efficiencies of effluents under different HRTs andOMW substrate concentrations.

2. Methods

2.1. Equipment

Five digesters of 18 L useful volume were used to investigatethe two-phase anaerobic co-digestion of OMW with OMSW. Twoof them were used for acidification phase and the three othersfor methanization phase. The heating of these digesters to

Fig. 1. Semi-continuou

37 ± 2 �C was performed with a water heater system equippedwith a thermostat and a pump. Agitation was done seven timesper hour by a motor agitator equipped with a time switch. Thedigesters were fed manually through the inlet ports once a dayand effluent were removed daily from the outlet ports at the samevolume as influent. The biogas was collected in a plastic bag. Thedigester used in this study is shown in Fig. 1 and has been de-scribed in detail in our previous work (Fezzani and Ben Cheikh,2007).

2.2. Substrates composition

2.2.1. Olive mill wastesThe OMSW and OMW used in this study were collected from

three-phase olive mill located in Kssar said at Tunis. OMSW wasstored in the fridge at temperature of 4 �C and OMW was storedat room temperature between 10 and 15 �C. Their main character-istics at the beginning of experiments, in average values, are sum-marised in Tables 1 and 2. The digesters were inoculated withactivated sludge collected from an aerobic wastewater treatmentplant located in Beja (northern Tunisia). Its composition, in averagevalues, is given in Table 3.

2.3. Experimental procedure

The two-phase anaerobic co-digestion experiments of OMWwith OMSW were carried out in ten runs using two sequencinglab-scale digesters operated at mesophilc temperature. The hydro-lysis–acidification step was carried out in the first digester whereasthe methanization step was carried out in the second digester.

At the beginning, all digesters were filled with aerobic activatedsludge as a source of biomass. The experiments were subsequentlyrun as following:

2.3.1. Hydrolysis and acidification stageThe operating conditions and the feed composition used to run

the experiments in the acidifiers are summarised in Table 4. Each

s feeding digester.

Table 1Average chemical composition of OMW used as main substrate.

Parameters Units Average values (*)

pH – 4.90 ± 0.1Colour (550 nm) – 7.95 ± 0.3TS g/L 85 ± 0.5VS g/L 55 ± 1.5TSS g/L 32 ± 0.5VSS g/L 23 ± 0.5TCOD g/L 130 ± 5SCOD g/L 82 ± 3TOC g/L 62 ± 1.5NHþ4 —N mg N/L 850 ± 50TKN g N/L 1.25 ± 0.05TPO�4 —P mg/L 950 ± 30Soluble phenols g C6H6O/L 7.5 ± 0.1methanol g/L 1.45 ± 0.25Acetic acid g/L 5.82 ± 0.5Propionic acid g/L 0.868 ± 0.2Butyric acid g/L 1.42 ± 0.2Valeric acid g/L 0.25 ± 0.2Caproic acid g /L 0.932 ± 0.2Alkalinity g CaCO3/L 3.24 ± 0.3

* Each value is an average of three replicates. ± shows standard deviations amongreplicates.

Table 2Average chemical composition of OMSW used as co-substrate.

Parameters Units Average values (*)

TS % 98 ± 1.5VS g/kg TS 970 ± 0.5TCOD g/kg TS 1180 ± 2TOC g/kg TS 560 ± 5NHþ4 —N g N/kg TS 1.1 ± 0.3TKN g N/kg TS 20 ± 1.5TPO�4 —P g/kg TS 1.57 ± 0.05

* Each value is an average of three replicates. ± shows standard deviations amongreplicates.

Table 3Average chemical composition of the activated sludge.

Parameters Units Average values (*)

pH – 7.4 ± 0.3TS g/L 40 ± 1.5VS g/L 27 ± 1.2TSS g/L 26 ± 1.5VSS g/L 21 ± 1.2TCOD g/L 35.5 ± 2.5SCOD mg/L 45 ± 5NHþ4 —N g N/L 1.5 ± 0.35TKN g N/L 2.3 ± 0.3TPO�4 —P g/L 1.25 ± 0.05Alkalinity g CaCO3/L 3.1 ± 0.72

* Each value is an average of three replicates. ± shows standard deviations amongreplicates.

Table 4Influent composition loaded into the acidifier at different OLRs.

Run HRT (day) Influent TCOD(g COD/L) (*)

OLR (g COD/L/d) (*) Operationaltime (day)

F

1 14 196 ± 5 14 ± 0.36 48 12 14 133 ± 3 9.5 ± 0.21 48 13 24 196 ± 5 8.17 ± 0.36 107 04 24 133 ± 3 5.54 ± 0.12 107 0

* Each value is an average of three replicates. ± shows standard deviations among repli

1630 B. Fezzani, R. Ben Cheikh / Bioresource Technology 101 (2010) 1628–1634

acidification reactor was loaded with an influent substrate concen-tration of 133 ± 3 or 196 ± 5 g COD/L/d at flow rates of 1.3 and0.75 L/day which corresponded to HRTs of 14 and 24 days, respec-tively. The quantity of the dry OMSW in all acidifier influents was56 g TS/L. The pH of the acidifier influent was adjusted to 6.0 ± 0.3then it was maintained at this level throughout each experimentby adding Ca(OH)2 as alkalinity (4–8 g/L) to maintain the optimumpH for acidogenic bacteria (Bouallagui et al., 2004). When not ad-justed, the pH inside the acidifier dropped rapidly to 5.0 ± 0.2,especially just at the end of the first feed period.

2.3.2. Methanization stageThe whole experiment in the methanizers was carried out over

six runs as shown in Table 5. At the beginning the methanizers(filled with aerobic activated sludge) were set in batch mode fer-mentation for 10–15 days until the start-up of biogas production.Then they were fed with effluent from the acidifiers (operated ata HRT of 24 days) at flow rates of 0.5, 0.75 or 1 L/day correspondingto HRTs of 36, 24 or 18 days, respectively. Alkalinity in the form ofCa(OH)2 was added to all effluents from the acidifiers (5–10 g/L ofOMW) to ensure a neutral medium (pH 7.0–7.4) for methanogenarchaea growth in the methanizer. Each run had a duration oftwo to three times the corresponding HRT. At the beginning of eachrun the amount of ammonium nitrogen inside the methanizer wasadjusted to 1.5 ± 0.35 g N/L by adding either new sludge or NH4Cl(5–6 g/L) to provide the same initial conditions for all the runs.

The volume of biogas and its composition were determined dai-ly and samples were collected from effluents of both methanizerand acidifier at least five times per week.

2.4. Chemical analysis

Various parameters were measured during the experiments: to-tal and soluble COD, TS, VS, TSS, VSS, TOC, TKN, alkalinity, total anddissolved phosphorus were determined according to the standardmethods of the American Publics Health Association (APHA,1995). The dark red purple colour intensity was determined bymeasuring the sample absorbance at 550 nm using a spectropho-tometer (PYE-Unicam, United Kingdom). All these analysis werecarried out in triplicate.

2.4.1. VFA analysisVFA extracts were prepared as follows. Samples of OMW were

centrifuged at 10,000 rpm for 20 min to remove suspended solidsand subsequently acidified with 17% H3PO4 to pH 2.0 and extractedthree times with diethyl ether (v/v) at ambient temperature. Thethree organic fractions of extract of each sample were combinedand dried with anhydrous Na2SO4 for 30–60 min. All samples ex-tracts were stored at 4 �C. Afterwards, VFA concentrations weredetermined using a gas chromatograph (HP-Agilent, USA)equipped with a flame ionisation detector (FID). The column wasa capillary FFAP (30 m length, 0.25 mm internal diameter and0.25 lm film thickness). Nitrogen was used as the carrier gas.Injector and detector temperatures were 250 �C and 300 �C,

low rate (L/d) Influent pH (*) Influent composition (*)

OMW (ml) Water (ml) OMSW (g)

.3 6.0 ± 0.3 1300 ± 10 0.0 73 ± 1

.3 6.0 ± 0.3 650 ± 10 650 ± 10 73 ± 1

.75 6.0 ± 0.3 750 ± 0.1 0.0 42 ± 1

.75 6.0 ± 0.3 375 ± 5 375 ± 5 42 ± 1

cates.

Table 5Experimental conditions of the methanizer at different OLRs.

Run HRT(day)

InfluentTCOD(g COD/L) (*)

OLR(g COD/L/d) (*)

Operationaltime (day)

Flowrate(L/d)

InfluentpH (*)

1 36 165 ± 4 4.59 ± 0.11 72 0.5 7.5 ± 0.32 36 82 ± 4 2.28 ± 0.11 72 0.5 7.5 ± 0.33 24 165 ± 4 6.87 ± 0.17 70 0.75 7.5 ± 0.34 24 82 ± 4 3.42 ± 0.17 70 0.75 7.5 ± 0.35 18 165 ± 4 9.17 ± 0.22 50 1 7.5 ± 0.36 18 82 ± 4 4.56 ± 0.22 50 1 7.5 ± 0.3

* Each value is an average of three replicates. ± shows standard deviations amongreplicates.

B. Fezzani, R. Ben Cheikh / Bioresource Technology 101 (2010) 1628–1634 1631

respectively. The temperature of the oven was programmed to risefrom 100 to 240 �C during the analysis of VFA .

2.4.2. Ammonium nitrogen analysisTotal and dissolved ammonium nitrogen salts were determined

according to Nessler methods using the reagents of aqua Merckkits (Reagents MERCK, Germany).

To each 5 ml of diluted sample (1:10 or 1:50) was added in se-quence three drops (or 1 ml) of potassium sodium tartrate solu-tion, Nessler’s reagent (potassium iodomercurate II) and sodiumhydroxide solution. After 30 min the yellow-orange colour wasmeasured at 425 nm using a spectrophotometer (PYE-Unicam,United Kingdom) and ammonium nitrogen concentration (in mg/L) was determined using standards.

Fig. 2. Gas meter.

2.4.3. Phenol analysisPhenolic compounds were determined according to Folin–Cio-

calteu method. Samples were acidified with HCl to pH 2 and ex-tracted three times with ethyl acetate (v/v) at ambienttemperature. The three organic fractions were combined and driedwith anhydrous Na2SO4 for 30–40 min. Then, the extract was re-dissolved with a mixture of methanol/water (60/40, by v/v). 2 mlof Folin–Ciocalteu reagent and 5 ml of sodium carbonate solution(20% w/v) were added, in sequence, to each 50 ml of sample. After60 min the blue colour formed was measured at 730 nm using aspectrophotometer (PYE-Unicam, United Kingdom). Results wereexpressed in terms of phenol equivalent.

2.4.4. Biogas analysisThe volume of biogas was measured using a gas-meter (Ritter-

Bochum Langendreer, Germany) as shown in Fig. 2. Methane per-centage was measured using a chemical method that consisted ofdissolving the CO2 gas contained in 100 ml biogas into NaOH solu-tion (270 g/L). H2S and NH3 percentages were measured using Drä-ger tubes (Dräger, Germany).

2.5. Statistical analysis

The statistical analysis of the data and the results in this study(analysis of average values, variance and standards deviation) wereperformed using Excel 2003 (Microsoft, USA).

3. Results and discussion

3.1. VFA concentrations at different OLRs

VFAs were produced in the 1st reactor conceived for the acidi-fication process. Their main components were acetic, propionic,butyric and caproic acids. The evolution with time of these compo-nents at different HRTs and influent concentrations are shown inFig. 3. Their average values (expressed in g COD/L) at the steady-state period of each HRT are shown in Table 6. As can be seen, indi-vidual VFA concentrations were increased with the increase ofboth influent substrate concentration and HRT. The highest VFAconcentrations were obtained with the highest feed concentration(TCOD = 196 g COD/L) and at the longest HRT (24 days) that corre-sponded to an OLR of 8.17 g COD/L/d. We note also from Table 6and Fig. 3 that the compositions of VFA at different OLRs weremarked by the presence of acetic acid as a major product rangingfrom 26% to 50% of TVFA, then came butyric acid in the range of27–35% of TVFA and finally propionic and caproic acids with 17–22% and 12–22% of TVFA, respectively. Also, we have noted thatvaleric acid represented only approximately 1% of TVFA.

3.2. Biogas production

The daily biogas production observed at different HRTs for thetwo effluent substrate concentrations from the acidifier are illus-trated in Fig. 4. At a HRT of 18 days, we observe an intense produc-tion of biogas during the first 25 days followed by a gradual dropfrom 27 to 30th day then a sharp decrease of biogas productionto reach values below 13 L/day at the 50th day due to inhibitionby accumulation of VFA. At HRTs of 24 and 36 days we observe asharp increase of biogas production during the first 25 days fol-lowed by a slight decrease until reaching a steady-state of biogasproduction at the 30th day. The steady-state results of main efflu-ent characteristics, biogas productivity and composition obtainedfrom the methanizer at different OLRs are summarised in Table 7.

As indicated the best biogas productivity (54.26 L/L OMW fed)with 83% of methane content was achieved at a HRT of 36 days

Fig. 3. Variation of VFA with time: acetic acid (d), propionic acid (h), butyric acid(4) and caproic acid (e) in the acidification reactor operated at different HRTs (14and 24 days) and different OMW SCOD concentrations: 133 g COD/L (A) and196 g COD/L (B).

1632 B. Fezzani, R. Ben Cheikh / Bioresource Technology 101 (2010) 1628–1634

for the highest effluent substrate concentration from the acidifiercorresponding to an OLR of 4.59 g COD/L/d. For both acidifier efflu-ents, studied at HRTs of 24 and 36 days, the pH remained within anoptimal working range (7.2–7.55) and the total volatile fatty acids(TVFA) concentration was below 3.7 g COD/L. However, for both

Table 6Steady state average performance of the acidifier at different ORLs.

Parameters Units Run 1

Input TCOD g COD/L 196 ± 5Input SCOD g COD/L 82 ± 3OLR g COD/L/d 14 ± 0.36HRT days 14Output TCOD g/L 150 ± 4Output SCOD g/L 105 ± 2SCOD removal % �28 ± 0.55TS g/L 98 ± 3Acetic acid g COD/L 10.70 ± 0.032Propionic acid g COD/L 3.79 ± 0.075Butyric acid g COD/L 5.96 ± 0.11Caproic acid g COD/L 1.49 ± 0.072TVFA g COD/L 21.94 ± 0.07Alkalinity g CaCO3/L 4.45 ± 0.35NHþ4 —N g N/L 0.35 ± 0.07TKN g N/L 1.72 ± 0.2TPO�4 —P g/L 1.28 ± 0.03Soluble phenol g C6H6O/L 7.85 ± 0.05Phenol removal % �5 ± 0.5pH – 5.95 ± 0.1Colour (550 nm) – 15.7 ± 1

Values are the averages of determinations taken at steady-state period of each HRT. ± s

acidifier effluents studied at a HRT of 18 days, the pH of the meth-anizer effluent dropped below 6.3 and TVFA concentration in-creased to 22.17 g COD/L. This accumulation of VFA in themethanizer effluent at this HRT was mainly due to the fact thatmethanogens could not convert to methane all the acetic acid inthe acidifier effluent. The excess of VFA built up in the digestercaused a drop in pH and inhibition of the methanogenesis process.Comparing the results of methane productivity of the overall two-phase AD system, as presented in Table 8, with those obtainedfrom a one-phase reactor in our previous study (Fezzani and BenCheikh, 2007) for a feed concentration of 133 ± 3 g COD/L and aHRT of 48 days, methane productivity was increased from 15 L/L OMW fed for a one-phase AD reactor to approximately 32 L/L OMW fed for two-phase AD reactors. This increase of methaneproductivity was due to enhanced characteristics of effluents pro-duced by the acidifiers. In fact, these effluents contained high levelsof VFAs which were easily metabolised and converted into CH4 andCO2 in the methanizers at short residence time compared to one-phase AD that requires a long residence time to convert complexsubstrates present in the original influents to VFAs and subse-quently to methane and carbon dioxide. Furthermore, as indicatedin Table 6, the increase of both alkalinity and TKN of acidifier efflu-ents (up to 5.7 g CaCO3/L and 1.95 g N/L for an OMW of 82 g COD/Land up to 4.9 g CaCO3/L and 1.3 g N/L for an OMW of 41 g COD/L,respectively) made them more resistant to pH instability and pre-vented them from the risk of ammonium nitrogen shortage by themethanogens (archaea).

3.3. Biogas composition

The average composition of the biogas produced during thesteady-state period at different OLRs is shown in Table 7. Themethane percentage was increased with an increase of HRT. Thebest methane percentage of 84% was obtained with the lowestOLR (2.28 ± 0.15 g COD/L) which corresponded to a HRT of 36 daysand an acidifier effluent concentration of 56 g COD/L. Indeed, atlong HRT there was increase of contact between archaea and sub-strates. Since low feed concentration was characterised by lowconcentration of inhibitor compounds such as phenol compoundsand TVFA, that may explain why the methanogenic process wasenhanced. Furthermore, the observed methane rates were higherthan those observed in one-phase AD reactors (Fezzani and Ben

Run 2 Run 3 Run 4

133 ± 3 196 ± 5 133 ± 341 ± 3 82 ± 3 41 ± 39.5 ± 0.21 8.17 ± 0.36 5.54 ± 0.1214 24 2475 ± 4 165 ± 4 82 ± 450 ± 2 135 ± 3 56 ± 3�16 ± 0.4 �65 ± 0.35 �86 ± 0.758 ± 4 110 ± 5 70 ± 44.99 ± 0.011 15.18 ± 0.53 7.07 ± 0.0393.47 ± 0.015 6.99 ± 0.22 5.49 ± 0.095.51 ± 0.09 13.56 ± 0.64 8.05 ± 0.291.91 ± 0.024 4.53 ± 0.55 5.61 ± 0.03815.87 ± 0.035 40.26 ± 0.48 26.22 ± 0.112.95 ± 0.3 5.77 ± 0.3 4.9 ± 0.250.27 ± 0.05 0.25 ± 0.03 0.15 ± 0.041.15 ± 0.15 1.95 ± 0.25 1.3 ± 0.10.75 ± 0.04 1.35 ± 0.05 0.88 ± 0.034.12 ± 0.04 8.5 ± 0.07 5.7 ± 0.03�10 ± 0.6 �14 ± 0.3 �52 ± 0.755.8 ± 0.1 6.1 ± 0.1 6.05 ± 0.19.2 ± 1 19.5 ± 1.5 11.5 ± 2

hows standard deviations.

Fig. 4. Biogas production at different HRTs: 36 days (A), 24 days (B) and 18 days (C)and different acidifier effluents TCOD concentrations: 82 g COD/L (h), 165 g COD/L(4).

Table 7Steady state average performance of the methanizer at different OLRs.

Parameters Units Run 1 Run 2

Input SCOD g COD/L 165 ± 4 82 ± 4Input SCOD g COD/L 135 ± 3 56 ± 3OLR g COD/L/d 4.59 ± 0.11 2.28 ± 0.11HRT Days 36 36Output TCOD g/L 37.2 ± 0.13 24.3 ± 0.21Output SCOD g/L 15.4 ± 0.16 11.5 ± 0.11SCOD removal % 89 ± 0.95 80 ± 0.94Soluble phenol g C6H6O/L 2.15 ± 0.2 1.10 ± 0.2Phenol removal % 75 ± 0.1 81 ± 0.15Colour (550 nm) – 5.02 ± 0.2 2.4 ± 0.1Colour removal % 75 ± 0.1 79 ± 0.2pH – 7.25 ± 0.2 7.52 ± 0.1TVFA g COD/L 1.2 ± 0.14 0.9 ± 0.65Alkalinity g CaCO3/L 6.2 ± 0.07 4.85 ± 0.1NHþ4 —N g N/L 0.95 ± 0.1 0.85 ± 0.1TKN g N/L 1.55 ± 0.15 1.1 ± 0.13TPO�4 —P g/L 1.25 ± 0.1 0.95 ± 0.1Biogas flow rate L/d 27.13 ± 0.74 21.32 ± 1.02Biogas productivity L/L OMW fed 54.26 ± 1.48 42.64 ± 2.04CH4 content % 83 ± 0.086 84 ± 0.075H2S content ppm 150 ± 0.20 140 ± 0.18

Values are the averages of determinations taken at steady-state period of each HRT. ± s

B. Fezzani, R. Ben Cheikh / Bioresource Technology 101 (2010) 1628–1634 1633

Cheikh, 2007). It should also be noted that H2S percentages weredecreased with the increase of HRT while NH3 was not present inthe biogas.

3.4. COD removal efficiency

Effluent COD removal efficiencies by the two-phase AD systemfor different OLRs are summarised in Table 8. The best SCOD re-moval efficiency of 82% was achieved with an OLR of8.17 ± 0.36 g COD/L/d in the acidogenic reactor and with an OLRof 4.59 ± 0.11 g COD/L/d in the methanogenic reactor. Again thisresult was higher than those observed with a conventional one-phase digester treating the same mixture of OMW with OMSWas mentioned in our previous study (Fezzani and Ben Cheikh,2007) and also higher than some reports from the literature (Mar-ques, 2001; Angelidaki et al., 2002). However, as shown in Table 6,the acidogenic stage did not show any reduction in COD, in con-trast total, and soluble COD of all acidifier effluents were increased.The increase of soluble COD of acidifier effluents was due to thehydrolysis of particulate organic compounds of OMSW. However,the increase of TCOD of acidifier effluents was due to the previousincrease of soluble COD.

3.5. Phenol and dark red colour removal efficiencies

The observed phenol removal from the acidifier, the methanizerand the overall two-phase AD system are given in Tables 6–8. Ascan be seen, the percentage reduction of phenol in the two-phaseAD system was ranging in 70–78% which was higher than observedin one-phase AD reactor (50–70% of phenol reduction) by Marques(2001) who studied the mesophilic anaerobic co-digestion of OMWand piggery effluent. Moreover, all effluents rejected from themethanizer operated at HRTs of 36 and 24 days exhibited a brightyellow colour with a reduction of dark red purple colour intensityup to 79% of the initial colour of acidifier effluents and up to 55% ofthe initial colour of OMW. This means that polyphenolic com-pounds of high molecular weight responsible for the dark red pur-ple colour (anthocyanins and tannins) (Hamdi, 1992) weredegraded completely into polyphenolic compounds of low molec-ular weight (responsible for the yellow colour) due to optimised

Run 3 Run 4 Run 5 Run 6

165 ± 4 82 ± 4 165 ± 4 82 ± 4135 ± 3 56 ± 3 135 ± 3 56 ± 36.87 ± 0.17 3.42 ± 0.17 9.17 ± 0.22 4.56 ± 0.2224 24 18 1845.4 ± 0.27 27.3 ± 0.2 90 ± 0.56 58 ± 0.6517.8 ± 0.17 13.7 ± 0.15 78 ± 0.8 41 ± 0.687 ± 0.3 76 ± 0.2 43 ± 0.5 27 ± 0.62.45 ± 0.2 1.50 ± 0.2 7.80 ± 0.2 5.10 ± 0.271 ± 0.2 74 ± 0.1 8 ± 0.25 11 ± 0.26.05 ± 0.1 3.1 ± 0.12 13.26 ± 0.76 7.5 ± 0.869 ± 0.1 73 ± 0.4 32 ± 0.7 35 ± 0.67.55 ± 0.2 7.46 ± 0.1 6.2 ± 0.2 6.34 ± 0.23.6 ± 0.15 2.46 ± 0.15 22.17 ± 0.8 14.63 ± 0.755.2 ± 0.17 4.1 ± 0.2 7.2 ± 0.3 6.25 ± 0.260.83 ± 0.05 0.77 ± 0.03 0.45 ± 0.1 0.35 ± 0.10.95 ± 0.1 0.87 ± 0.07 1.15 ± 0.04 0.65 ± 0.041.15 ± 0.1 0.88 ± 0.05 1.2 ± 0.05 0.8 ± 0.0737.66 ± 0.85 29.1 ± 0.8 9.5 ± 2.5 13 ± 4.550.22 ± 1.13 38.8 ± 1.07 9.5 ± 2.5 13 ± 4.580 ± 0.052 82 ± 0.042 62 ± 0.22 56 ± 0.12120 ± 0.12 100 ± 0.25 200 ± 0.3 18 ± 0.35

hows standard deviations.

Table 8Steady state average performance of the overall two-phase AD system under the best OLRs.

Parameters Units Run 1 Run 2 Run 3 Run 4

OLR (acidifier) g COD/L/d 8.17 ± 0.36 5.54 ± 0.12 8.17 ± 0.36 5.54 ± 0.12OLR (methanizer) g COD/L/d 4.59 ± 0.11 2.28 ± 0.11 6.87 ± 0.17 3.42 ± 0.17Overall SCOD removal efficiency % 82 ± 0.95 74 ± 0.2 79 ± 0.3 76 ± 0.2Overall phenol removal efficiency % 72 ± 0.1 78 ± 0.15 68 ± 0.2 70 ± 0.1Overall colour removal efficiency % 37 ± 0.67 55 ± 0.34 24 ± 0.67 42 ± 0.3Overall methane productivity L/L OMW fed 30.03 ± 0.82 23.88 ± 1.14 40.17 ± 0.9 31.81 ± 0.88Over all methane yield m3/kg TCOD in 0.346 ± 0.24 0.534 ± 0.57 0.309 ± 0.18 0.474 ± 0.29

1634 B. Fezzani, R. Ben Cheikh / Bioresource Technology 101 (2010) 1628–1634

values of COD:N ratios of influents loaded to the methanizer. Thisresult has proven the ability of two-phase system to remove darkred colour by degradation of the highly polymerised phenolic frac-tion from OMW compared to one-phase anaerobic digestion sys-tem as mentioned in other studies. Indeed, Marques (2001)demonstrated that some highly polymerised phenolic compoundswere not degraded and no significant decrease in OMW black col-our was achieved. However, the concentrations of soluble phenolcompounds and the intensity of dark red colour of acidifier efflu-ents were increased dramatically up to 8.5 g C6H6O/L and up to140% of initial colour of OMW, respectively. These substantial re-sults could be attributed to the hydrolysis of OMSW which contrib-uted significant amounts of soluble phenol compounds and solubletannins and anthocyanins (see Table 6).

4. Conclusions

We conclude from this study that OMW could be degradedwith high performance by co-digestion with OMSW using two-phase AD reactors operated at mesophilic temperature withouthigh dilution and without addition of an expensive chemicalnitrogen substrate. The optimal values of HRTs were 24 days forthe first stage and 36 days for the second stage. The best overallmethane productivity of 40.17 ± 0.9 L/L OMW fed was obtainedwith an OMW TCOD level of 196 ± 5 g COD/L co-digested withOMSW at a HRT of 24 days in both the first and the second stage.Whereas, the best overall and SCOD removal efficiency of82 ± 0.95% was achieved with an OMW TCOD level of196 ± 5 g COD/L co-digested with OMSW at a HRT of 24 days inthe first stage and of 36 days in the second stage. Furthermore,phenol and colour removal efficiencies of two-phase AD systemwere ranging in 70–78% and in 24–55%, respectively. In addition,highly polymerised phenolic fraction responsible of the dark redpurple colour in OMW (tannins and anthocyanins) was com-pletely degraded by two-phase AD operated at HRTs of 24 and36 days. Nevertheless and despite these benefits, the polyphenoliccompounds of low molecular weight (responsible of the yellowcolour) were still present in all effluents rejected from the two-phase AD reactors. Also, inhibitions of the methanogenesis pro-cesses by accumulation of VFA were observed in the methane pro-duction reactors operated at HRTs 618 days.

Acknowledgements

The author wish to express his gratitude and thanks to Pr.Abdelhafidh Nafti from Chemical Department at High School ofFood industrial of Tunis for his kind and excellent help with someexperimental work of this study.

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