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32 (2008) 155 161

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Potential for methane production from typical Mediterranean agro-industrial by-products

M.S. Fountoulakis, S. Drakopoulou, S. Terzakis, E. Georgaki, T. Manios
Laboratory of Solid Waste & Wastewater Management, School of Agricultural Technology, Technological Educational Institute of Crete, GR-71004 Iraklio, Crete, Greece

art i cle info

Article history: Received 17 July 2007 Received in revised form 3 September 2007 Accepted 15 September 2007 Available online 24 October 2007 Keywords: Olive mill Winery Slaughterhouse Anaerobic digestion Co-digestion Methane Bio-energy

ab st rac t
This work examines the potential for methane production from anaerobic co-digestion of olive mill wastewater and wine-grape residues with slaughterhouse wastewater. Continuous (mesophilic) and batch (mesophilic and thermophilic) experiments were studied, both with the separate types of by-products and with mixtures. Methane yields from olive oil wastewater, winery residues and slaughterhouse wastewater were 108, 147 and 297 L CH4 kg1 COD fed respectively. Co-digestion with 50% olive oil wastewater and 50% slaughterhouse wastewater or 50% winery residues gave a methane yield of 184 and 214 L CH4 kg1 COD, respectively. Furthermore, the methane yield was 188 L CH4 kg1 COD added, co-digesting a mixture of 50% winery residues and slaughterhouse wastewater. Finally, the same mixtures under thermophilic conditions gave methane yields of 282, 301 and 219 L CH4 kg1 COD, respectively. These results suggest that methane can be produced very efciently by co-digesting olive oil wastewater, wine-grape residues and slaughterhouse wastewater. & 2007 Elsevier Ltd. All rights reserved.



The olive and grape agro-industrial sectors are of major economic importance in all Mediterranean countries. These agro-industrial activities generate large amounts of by-products, which are totally unexploited and in some cases dangerous for the environment. After 2000, Greece has produced an annual yield of approximately 377.75 kt of olive oil and 348.43 kt of must [1]. This corresponds to 2.266 Mt of olive oil wastewater and 135.50 kt of wine-grape residues (WGR). Another important agro-industrial sector in the Mediterranean area is livestock farming. Greece produces 315 kt of meat annually, corresponding to 4.185 Mt of slaughterhouse wastewater (SW) [1]. The disposal of olive mill and SW is one of the major environmental problems of the olive oil and meat industries. Winery residues are not a signicant environmental problem, although they currently remain an unused organic material.
Corresponding author. Tel./fax: +30 2810 379456.

Anaerobic digestion of biomass is a well-known biological process producing biogas and biofertilizer. A signicant number of biogas plants have been built, mainly in Northern Europe, and now the concept is spreading all over the world. Biogas plants treat various types of organic residues including sewage sludge, food industry residues and manure. Agricultural industries mostly have a specic, seasonal product, while other industries vary their production during the year. The amounts of organic residues generated may not be sufcient to make digestion cost-effective. However, the establishment of a centralized facility co-digesting a number of organic residues would ensure the viability of the process. Co-digestion of different types of organic by-products has been increasingly applied in order to improve plant protability [27]. In most cases biogas yields were improved due to positive synergistic effect of using co-substrate in co-digestion. The easier handling of mixed wastes, the use of

E-mail address: mfountoul@teemail.gr (M.S. Fountoulakis). 0961-9534/$ - see front matter & 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biombioe.2007.09.002


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common access facilities and the known effect of economy scale are some of the advantages of co-digestion [7]. However, it is not clear whether some by-products might have adverse effects when added to a stable digester or used in conjunction with another type of residues. The concept of co-digestion and a centralized facility for processing organic residuals is not new. Mata-Alvarez et al. [8] review all the co-digestions efforts up to 2000. In Denmark, around 20 centralized digestion plants co-digesting manure and industrial organic wastes are operating [9]. Most industrial co-digestion plants treat the organic fraction of municipal solid wastes plus some other organic wastes such as sewage sludge [10,11]. Furthermore, there were efforts in laboratories codigesting several other organic residuals such as sisal pulp and sh wastes [3], cattle slurry and chicken manure [12], whey and olive pulp [7] barley and kitchen waste [6]. The anaerobic digestion of olive oil wastewater [1315], SW [1618] and WGR [19,20] separately has been investigated extensively in the past. Methane production from olive mill wastewater (OMW) and winery wastewater has been found to correlate with chemical oxygen demand (COD) removal at a ratio of 0.300.35 m3 kg1 COD removed [14,20]. On the other hand, methane yields from the treatment of SW are in the range of 0.120.32 m3 kg1 COD fed [16]. Previous studies on the anaerobic co-digestion of OMW have found improved yields. Angelidaki et al. [21] showed that the high buffering capacity contained in manure, together with the content of several essential nutrients, make it possible to degrade olive mill efuents without previous dilution, without addition of external alkalinity and without addition of external nitrogen source. Another study attempted to optimize biogas production from OMW by codigesting with diluted poultry manure at mesophilic conditions. The reactor operated with no problem reaching rapidly stable conditions and producing 1 1 [22]. biogas at a rate of 1.53 L L R d To our knowledge, this paper is the rst work focussing on the co-digestion of these organic residues, present in large volumes across the whole Mediterranean region. The examination of a centrally located anaerobic digestion facility intended to receive slaughterhouse, olive mill and winery wastewater is therefore very interesting. The objectives of this paper are (a) to compare the anaerobic digestion of OMW, SW and wine grape residues separately, as well as the co-digestion of these substrates and (b) to determine the biochemical methane potential of several mixtures of these substrates in mesophilic and thermophilic conditions.

milled WGR were extracted with 2 L of tap water for 0.5 h at 50 1C. SW was supplied from the drainage system of the largest slaughterhouse in Crete (Agia Barbara). All the samples were stored at 4 1C.


Continuous experiments

Four digesters with a working volume of 1 L were constructed using glass asks. The digesters were sealed with rubber stoppers containing an inuent/efuent port to allow injection of by-products. A water bath was used to maintain the temperature of the digesters at 35 1C. The asks were connected to PVC tubes lled with water acidied to pH 3. Biogas was collected by displacement of water. The reactors were operated in a draw-and-ll mode (on a daily basis) with a retention time of 20 days. Initially, the reactors were inoculated with anaerobic sludge originating from the municipal Sewage Treatment Plant of the city of Iraklio. The feed in the reactors was: OMW (R1), SW (R2), winery residues (R3) and mixture OMW:SW at a ratio of 1:1 (R4). The digesters were operated using this feed for 100 days. The feed in the reactor R1 was then changed to OMW:WGR at a ratio of 1:1 and in reactor R3 to WGR:SW at a ratio of 1:1. The performance of the digesters (biogas production and composition in CH4, pH, total suspended solids (TSS) and dissolved COD) in each case was monitored at close time intervals.


Batch experiments

Batch experiments were carried out in duplicates both at 35 and 55 1C. The seed in the rst case (35 1C) was obtained from the analog reactor from the continuous experiments. In the second case (55 1C), the seed originated from a 1 L thermophilic (55 1C) digester, fed daily with a mixture of WGR:SW (1:1) with a hydraulic retention time of 20 days. Tests were initiated by transferring inoculum under anaerobic conditions to 160-mL serum bottle test reactors. The test reactors were ushed with an oxygen-free gas mixture (7030%, nitrogencarbon dioxide) before and after transferring. Then the serum bottles were sealed immediately using rubber septa and aluminum crimp caps. The nal volume was 120 mL, comprising 100 mL inoculum and 20 mL of sample. Gas production was measured manually with syringes.


Analytical methods


Materials and methods


OMW was obtained from an olive oil processing factory using three-phase decanter centrifugation (Stavrakia, Region of Crete, Greece). Note that the overwhelming majority of olive oil mills in Greece use the three-phase system. WGR were collected from a winery enterprise in the city of Iraklio, Crete. Immediately after collection, by-products were milled by a laboratory grinder to an average particle size of 23 mm and stored in the freezer at 4 1C. At timed intervals, 200 g of

The pH was measured by an electrode (Crison, GLP 21), while total (TSS) and volatile suspended solids, dissolved COD, Kjeldahl nitrogen (TKN) and total phosphorus (TP) were determined according to APHA (1995) [23]. Total and dissolved (after centrifugation and ltration of OMW) phenolic compounds were determined spectrophotometrically according to the FolinCiocalteu method [24]. Determination of ammonium was carried out using Merck Spectroquants Photometric Test Kits. Gas samples were collected in gas-tight syringes and transferred to the gas chromatograph by sealing the needle with a butyl rubber stopper. Twenty microliters were injected into a gas chromatograph (Agilent 6890N GC System) for analysis of methane and carbon dioxide. A thermal conductivity detector


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and a capillary column (GS Carbonplot, 30 m 0.32 mm, 3 mm) were used. The column was operated isothermally at 80 1C and the detector port was operated at 150 1C. Helium was used as the carrier gas at a ow rate of 15 mL min1.


Results and discussion

By-products characterization

As a rst step, it was important to determine the major physicochemical characteristics of each wastewater. The results are presented in Table 1. There are signicant differences in the composition of wastewaters. OMWs have a higher organic content (81.20 g L1 as COD) than the other two (15.88 and 4.08 g L1 as COD for winery and SW, respectively). Previous studies reported COD values of OMW between 45 and 170 g L1 [13,14]. The specic amounts of wastewater and the chemical composition vary widely between slaughterhouses, depending on the degree of further processing of the slaughtered animals. Tritt et al. [17] reported COD level in SW between 0.5 and 10.5 g L1. The COD/nitrogen ratio was very low for both OMW and WGR extract compared to SW. Similar results were obtained in earlier studies [13,17,21]. OMW and WGR as shown for pH values are signicantly acidic, so pH values in the feed were adjusted to 7 by adding sodium hydroxide. OMWs were analyzed further to determine total and dissolved phenolic compounds. The concentration of total and dissolved phenols was found to be 9.7 and 7.8 g L1 respectively.


Continuous experiments

The reactors responded differently to each substrate. Fig. 1 shows the characteristics of the reactors during the period of operation. A decrease in efuent pH was noticed in the reactor (R1) fed with OMW from 7.3 to 6.2 within 20 days. It was therefore decided to add sodium hydroxide to this reactor in order to maintain the pH at optimum levels for anaerobic digestion. However, no decrease of pH was observed in the reactor fed with a mixture of OMW and

Table 1 Characteristics of wastewater

Constituent Olive mill wastewater Winery residues extract 3.8 1760 1580 15,880 100 52.5 2.1 Slaughterhouse wastewater

pH TSS (mg l1) VSS (mg l1) COD (mg l1) TKN (mg l1) TP (mg l1) NH4-N (mg l1)

5.4 24,720 23,170 81,200 375 220 162

7.3 1008 955 4080 102.5 1.1 9

slaughtering wastewater. Reactors fed with WGR and SW remained stable in all cases. As shown in Fig. 2, a signicant decrease of biogas production in the digester fed with OMW was observed due to low pH and probably also to the buildup of volatile fatty acids. After the adjustment of pH in the reactor, biogas production improved although biogas yield was still low. The high content of polyphenols and high COD/N ratio in the OMW made the reactor unstable. Beccari et al. [25] stated that polyphenols are the most bio-recalcitrant compounds in OMW since only 2030% of polyphenols were degraded in methanogenic conditions. Furthermore, Sorlini et al. [26] carried out a study of the effect of polyphenols in OMW on its biodegradation and identied them as being responsible for the inhibition of the anaerobic process. Mixing SW with OMW both dilutes the organic content keeping toxic substances such as polyphenols below a required level and corrects the COD/N ratio. The digester fed with SW showed a high increase in the biogas production rate, from 200 to 500 ml L1 d1 within 15 days, although biogas production decreased and stabilized at much lower levels after 30 days. Lipids represent an important fraction of the particulate organic charge in SW [27]. Long-chain fatty acids are the main intermediates, and their accumulation had signicant effects on the anaerobic digestion process [28,29]. A temporary increase of biogas production was noticed for the rst 23 days in the digester fed with WGR; however, 10 days later the biogas decreased. The accumulation of phenolic compounds is a possible reason for the reduction of biogas yield [30]. The composition of biogas in methane in all cases uctuated between 62% and 69%. The main characteristics of the reactors in each steady state and the corresponding biogas production and methane yield are presented in Table 2. The digesters were operated at an HRT of 20 days in all cases and the reactor performance was very stable. The use of SW as co-substrate improves methane yield during the anaerobic digestion of OMW and WGR. The methane yield in the reactors was 170, 163 and 191 L CH4 kg1 COD added for digested OMW:SW, OMW:WGR and WGR:SW mixtures respectively. Angelidaki et al. [21] report biogas production of approximately 1250 mL L1 d1 codigesting OMW with manure at a ratio of 50:50 under thermophilic conditions with an organic loading rate of 7.8 g COD L1 d1. This corresponds to a biogas yield of 160 L CH4 kg1 COD added. On the other hand, other research [22] has shown a biogas production rate of 210 mL L1 d1 digested diluted poultry manure and olive mill efuent at a ratio of 50:50 under mesophilic conditions and a hydraulic retention time of 20 days. The mass of CH4 produced was calculated using the density of CH4 0.72 kg m3, at standard conditions. The corresponding energy yield is 6.13, 5.86 and 6.87 MJ kg1 COD added, for digested OMW:SW, OMW:WGR and WGR:SW respectively (assuming that the energy yield from methane is 50.12 MJ kg1).


Batch experiments

The cumulative methane yield during the batch experiments for the determination of the methane potential of the co-digestion of different agro-industrial by-products is shown


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50 40 30 d-COD & TSS concentation (g/l) 20 10




7.5 7.0

6.5 6.0 5.5

inoculumn addition 0 50 40 30 20 10 0 -20 0 20 40 60 80 100 120 140 160 0 Time (d) 20 40 60 80 100

5.0 7.5 7.0 6.5 6.0 5.5 5.0

Fig. 1 Concentration of total suspended solids (n), d-COD ($) and pH value ( ) during operation of reactors.


1000 800 600 biogas production (ml/l/d) 400 200

inoculumn addition OMW OMW+WGR



0 1200 1000 800 600 400 200 0 0 20 40 60 80 100 120 140 160 0 10 20 30 40 50 60 70 80 90 100 110 Time (d)


Fig. 2 Biogas production during anaerobic digestion of agro-industrial wastewaters.

in Fig. 3. The methane production yield was assumed to have a rst-order rate of decay, according to the following equation: B B0 1 ekt , (1)

where B is the cumulative methane yield at time t. B0 was assumed to equal the nal B. k was estimated taking the reciprocal of the time from the start of the BMP assay until B equalled 0.632B0 [31,32].



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Table 2 Biogas production rate, methane yield and the main characteristics of the reactors fed with various substrates at steady state Biogas production (ml l1 d1)
OMW SW WGR OMW+SW OMW+WGR SW+WGR 676798 93717 182721 566757 534756 176710

Methane yield (m3 kg1 COD added)

0.10870.016 0.29770.081 0.14770.011 0.17070.018 0.16370.014 0.19170.010

d-COD (g l1)
20.272.4 2.170.7 1.170.6 6.471.0 14.172.3 1.070.4

TSS (g l1)
19.472.3 15.172.0 8.672.8 17.671.3 14.872.3 5.471.2

6.970.1 7.470.2 7.270.1 7.370.2 6.970.1 7.170.1

0.30 methane yield (l CH4 / g COD added)

0.25 0.20


0.10 0.05

0.00 -5 0 5 10 15 20 25 30 35 Time (d) 40 45 50 55 60 65

Fig. 3 BMP test of different agro-industrial wastewater mixtures in mesophilic (lled symbols) and thermophilic (open symbols) conditions.

Maximum methane yields were calculated to be 184, 214 and 188 L CH4 kg1 COD added, for mixtures of OMW:SW, OMW:WGR and SW:WGR, respectively (Table 3). To our knowledge, no previous studies have calculated methane yields for these mixtures, only as separate substrates. Manjunath et al. [33] evaluated the biochemical methane potential of SW and found a methane yield of 200 L CH4 kg1 COD fed. Gunaseelan has reported a ultimate methane yield from Vitis vinifera (grape vine) of 420 L CH4 kg1 VS fed. Finally, Erguder et al. [15] found in batch reactors that 1 L of OMW produced 57.1 L methane gas, corresponding to approximately 240 L CH4 kg1 COD fed. The corresponding energy yield is 9.22, 10.73 and 9.42 MJ kg1 COD added, respectively. The ultimate methane yields of the co-substrates are higher than those estimated from the continuous reactors for two OMW mixtures. A higher hydraulic retention time applied in the reactors, or a pre-treatment process to increase the bioavailability of the solid organic matter, could precede the anaerobic co-digestion of OMW. The methane yields of the thermophilic co-digestion were 1435% higher than those of the mesophilic one (Table 3).

Similar results have been reported in a previous study, which found an increase in methane yields from mesophilic to thermophilic conditions between 19.2% and 29.3% for digested mixtures of food residues and sewage sludge [34]. Thermophilic digestion, intrinsically, has higher degrading capability and methanogenic activity [35]. Furthermore, most of the thermophilic conditions provided a more balanced fermentation system in biogas production [34]. The maximum recoverable energy was calculated to be 14.14, 15.09 and 10.98 MJ kg1 COD added co-digesting OMW:SW, OMW:WGR and SW:WGR at a ratio of 1:1 respectively.



Agro-industrial residues are potential renewable energy resources. In this paper, we investigated the methane potential of industrial olive, wine grape and meat byproducts. The anaerobic co-digestion of olive mill, winery and slaughterhouse by-products is a feasible process. The addition of SW to a reactor digesting OMW or WGR increased methane yield and stabilized the whole process. The results


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Table 3 Biochemical methane potentials (BMP) during co-digestion of agro-industrial wastewaters in mesophilic and thermophilic conditions Sample Temperature (1C) Ultimate methane yield (l g1 COD added)
0.184 0.282 0.214 0.301 0.188 0.219

Methane production rate constant (1 d1)

0.109 0.045 0.117 0.054 0.161 0.170


35 55 35 55 35 55



indicate that co-digestion in a simple one-stage reactor with 50% OMW and 50% SW, 50% OMW and 50% WGR, 50% WGR and 50% SW gave a methane yield of 170, 163 and 191 L CH4 kg1 COD added, respectively. This was a methane yield increase of 2336% compared to that obtained from the digestion of pure OMW and WGR. The ultimate methane yield of co-digesting OMW:SW, OMW:WGR and WGR:SW under mesophilic conditions was estimated to be 184, 214 and 188 L CH4 kg1 COD added, respectively. Co-digestion of OMW:SW, OMW:WGR and WGR:SW under thermophilic conditions produced an increase of 34.5%, 28.9% and 14.1% respectively. The use of SW as co-substrate in the anaerobic digestion of OMW and WGR has many advantages: improvement of the methane yield, supplying the system with missing nutrients and solution of the seasonal production problem of the other substrates. The results of this experiment suggest that the codigestion of these organic residues is very promising.

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