biogas production from anaerobic co-digestion of agroindustrial wastewaters under mesophilic...

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Desalination 248 (2009) 891–906

Biogas production from anaerobic co-digestion ofagroindustrial wastewaters under mesophilic

conditions in a two-stage process

Margarita A. Dareioti, Spyros N. Dokianakis, Katerina Stamatelatou,Constantina Zafiri, Michael Kornaros*

Department of Chemical Engineering, University of Patras, 1 Karatheodori st., GR 26500 Patras, GreeceTel:/Fax: +30-2610-997418; email: [email protected]

Received 25 July 2008; accepted 30 October 2008

Abstract

Co-digestion of organic wastes is a technology that is increasingly being applied for simultaneous treatment ofseveral solid and liquid organic wastes, in which the content of nutrients can thereby be balanced, and the negativeeffect of toxic compounds on the digestion process may be decreased giving an increased gas yield from the bio-mass. Moreover, co-digestion may contribute to a more efficient use of anaerobic digestion (AD) reactors andcost-sharing by processing multiple waste streams in a single facility.

In this study, efficient biodegradation of a mixture containing olive mill wastewater (OMW), liquid cow man-ure (LCM) and cheese whey (CW) using a two-stage anaerobic process was examined. Two continuously stirredtank reactors (CSTRs) were used under mesophilic conditions (358C) in order to enhance acidogenesis and meth-anogenesis. The overall process was designed with a hydraulic retention time (HRT) of 19 days. The acidogenicreactor was initially fed with an influent mixture composed of 55% OMW, 40% CW and 5% LCM. After the 87thday of operation, the mixture was changed to 90% of CW and 10% of LCM based on the actual availability ofwastes produced by the respective agroindustries. The average removal of dissolved chemical oxygen demand(COD) was 75.5% and 85.2% for the two examined scenarios at OLR of 5.5 ± 0.36 and 4.5 ± 0.30 g total COD/Lreactor/d, while the methane production rate at the steady state reached 1.35 ± 0.11 and 1.33 ± 0.15 L CH4/Lreac-tor/day respectively.

Keywords: Agroindustrial wastewaters; Anaerobic digestion; Cheese whey; Liquid cow manure; Olive millwastewater

*Corresponding author.Presented at the 2nd Conference on Small and Decen-tralized Water and Wastewater Treatment Plants(SWAT), Skiathos Island, Greece, May 2–4, 2008

0011-9164/09/$– See front matter © 2009 Published by Elsevier B.V.doi:10.1016/j.desal.2008.10.010

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1. Introduction

Some agroindustries such as olive oil mills,cheese factories and dairy farms represent a con-siderable share of the Mediterranean countrieseconomy. Industries processing agricultural rawmaterials such as various fruits, vegetables,meat, milk and so on, generate millions of tonsof wastewaters and large amounts of by-products,which are totally unexploited and in some casesdangerous for the environment. These productionfacilities are usually scattered throughout thecountryside and the raw materials processed areproduced at a seasonal rate resulting thus to waste-waters varying significantly during the year inboth quantity and characteristics.

The by-products of olive oil production such asolive mill wastewaters (OMW) and olive cakepose a serious environmental risk, especiallyin the Mediterranean, Aegean and Marmararegions that account for approximately 95% ofthe worldwide olive oil production [1]. OMWcontains polyphenols and sugars, volatile acids,polyalcohols and nitrogenous compounds [2].The total concentration of phenols whichcontribute to a high toxicity and antibacterialactivity [3] can reach up to 10 g/L [4]. Thechemical oxygen demand (COD) ranges from 25to 162 g O2/L and the biological oxygen demand(BOD5) from 9 to 100 g O2/L [5].

Cheese factories generate wastewaters ofwhich whey is the most important waste streamproduced with a high organic content (up to70 g COD/L), which is highly biodegradable,and low alkalinity (50 meq/L) [6]. Cheese whey(CW) contains a significant amount of carbohy-drates (4–5%), mainly lactose, proteins notexceeding 1%, fats at about 0.4–0.5%, lacticacid less than 1% and salts that may range from1% up to 3% [7]. Dairy farms produce animalmanure, in both liquid and semi-liquid forms,depending on the amount of fresh water usedfor daily operations. The potential pollutantsfrom decomposing livestock manure include

pathogens, nutrients, methane and ammoniaemissions [8].

Anaerobic digestion process is a particularlyattractive treatment solution for high strengthwastewaters due to the operational economy andgeneration of biogas from a renewable source(biomass) with pollution decreasing at the sametime. In addition, in the specific case of agroin-dustrial wastewaters treatment, the remainingstabilized slurry after digestion may be used asa fertilizer [9]. Nowadays, it is becoming impera-tive that we develop sustainable energy supplysystems covering industrial and domestic energydemands using renewable sources. A significantnumber of biogas plants have already been built,mainly in Northern Europe, and now the conceptis spreading all over the world. Biogas plants treatvarious types of organic residues including sew-age sludge, food industry residues and manure.Several studies have shown that, generally, thesensitivity and the performance of the anaerobicdigestion (AD) process may be improved by com-bining several waste streams. Co-digestion ofdifferent types of organic by-products has beenincreasingly applied in order to improve plantprofitability and overcome a number of problemssuch as nutrient imbalance, rapid acidificationand the presence of inhibiting compounds,among other factors [10–15]. Other advantagesof this technology are potential improvement ofmethane yield [16] due to the supply ofadditional nutrients from the co-digestates andmore efficient use of equipment and cost-sharingby processing multiple waste streams in a singlefacility, while at the same time it may secure amuch more stable year round operation.

This article focuses on three representatives,with high organic content and seasonally pro-duced agroindustrial wastewaters, for Greeceand other Mediterranean countries: cheese whey(production period: January–June), olive millwastewaters (production period: October–March)and liquid cow manure (production period:whole year). The aim of this work was to examine,

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in the lab scale, the capability of a centrallylocated anaerobic digestion facility to co-treatdifferent wastewater streams in order to secure astable year-round operation. To overcome thelimitations of conventional digestion methods,increase the robustness of the system, facilitate abetter application of control and optimize theoverall AD process. A two-stage AD configura-tion was used in this study to avert the imbalancebetween the processes of acidogenesis andmethanogenesis. Such a two-stage AD approachhas been previously successfully applied to, forexample, municipal solid wastes [16–17], cropresidues [18], agroindustrial residues [19], marketwastes [20–21] and food wastes [22–23]. It isbased on physically separating into two intercon-nected reactors, the two distinctly different groupsof bacteria (acidogens and methanogens), andmaximizing their growth by maintaining optimumconditions in each tank for each particular groupof bacteria. The first group, the acidogenicbacteria, is grown in the acidogenic reactorwhere pH is naturally low and the residencetime is maintained between 1 and 4 days. The sec-ond group, the methanogenic bacteria, is grown inthe methanogenic reactor where pH is naturallymuch higher and where residence time can bebetween 10 and 20 days, depending on the waste-water characteristics. The acidogenic bacteria willnot thrive in the methanogenic reactor as most ofits feed materials are used in the acidogenic; themethanogenic bacteria cannot thrive in the acido-genic reactor as the retention time is too short andthe pH is too low.

2. Materials and methods

2.1. Agroindustrial wastewaters

The OMW used in this study was obtainedfrom a local olive oil mill using a three-phase cen-trifugation decanter located in Patras (WesternGreece). The co-substrate (cheese whey) was pro-vided from a cheese factory located in the same

region producing mainly the white cheese‘‘feta’’ with daily production of 30 m3 of waste-waters. The LCM used for the tests was collectedfrom a dairy farm breeding 700 cows. This facilitywas fully automated and the generated wastewaterwas collected in an anaerobic lagoon after beingcentrifuged in order to separate the liquid fromthe solid cow manure. The sample from LCMused in our experiments was collected fresh afterthe centrifuge and before entering the lagoon.The LCM sample was stored immediately aftersampling in the freezer at�188C until subsequentuse throughout the experimentation period. Themean composition of raw OMW, CW and LCMis summarized in Table 1.

2.2. Experimental setup

Experiments were carried out in two CSTRreactors, one used for acidogenesis and the otherfor methanogenesis. The two reactors were cylin-drical in shape, made entirely of stainless steel(INOX 316), having double wall and working

Table 1

Composition of olive mill wastewater (OMW), liquid

cow manure (LCM) and cheese whey (CW)

Parameter OMW(g/L)

LCM(g/L)

CW(g/L)

pH 5.0 7.02 6.3TSS 37.0 20.6 9.0VSS 34.5 12.9 8.0TS 83.3 59.3 63.8VS 54.9 40.1 49.6Total COD 131.0 53.4 72.1Dissolved COD 67.1 18.7 53.5BOD5 41.0 20.0 36.0Total Carbohydrates 26.2 4.6 40.9Dissolved Carbohydrates 21.7 0.7 35.7TKN 0.7 3.4 0.9Ammonium Nitrogen 0.1 1.7 0.1Total Phosphorus 0.4 0.3 0.3Dissolved Phosphorus 0.2 0.002 0.2Total Phenols 6.8 0.9 0.1Alkalinity 1.5 13.5 0.8

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volume of 750 ml and 4 L respectively. Theywere operated at a total retention time of19 days and constant temperature of 35 ± 0.28Cvia a thermocouple controller. Agitation wasensured by a geared motor drive unit which wasinstalled on the top of each reactor. The feedstockwas stored in a tank placed in a refrigerator tomaintain its temperature constant at 48C and250 ml/d were fed to the acidogenic reactor viaa precise peristaltic pump. The effluent fromthis reactor was used for feeding the methano-genic one. Biogas was measured automaticallyby a tailor-made device operating via a combina-tion of an engine oil-filled U-tube, an electron –valve and a counter. The measurement wasbased on counting the number of displacementsof constant oil volume by the produced biogas.

2.3. Chemical analysis

pH was measured by an electrode (Orion3-Star), while total and volatile suspended sol-ids (TS and VS, respectively), dissolved andtotal COD, TKN, ammonium nitrogen, totaland ortho-phosphates and alkalinity weredetermined according to Standard Methods[24]. For the determination of carbohydrates,a colored sugar derivative was producedthrough the addition of L-tryptophan, sulfuricand boric acids, which was subsequentlymeasured colorimetrically at 520 nm [25].Total and dissolved (after centrifugation andfiltration of OMW) phenolic compounds weredetermined spectrophotometrically accordingto the Folin–Ciocalteu method [26].

For the quantification of volatile fatty acids(VFAs), samples were removed from each testreactor at preset time intervals (three times aweek). From the sample, 1 ml was immediatelyacidified with 30 ml of 20% H2SO4 and centri-fuged (43000 rpm) for 15 min to remove bio-mass. The supernatant was filtered though0.22mm nylon filter, transferred to 2 ml septum-capped vials and analyzed on a gas chromato-

graph (VARIAN CP-30) equipped with a flameionization detector and a capillary column(Agilent Technologies, Inc., 30 m� 0.53 mm).The oven was programmed from 1058C to1608C at a rate of 158C/min, and subsequentlyto 2358C (held for 3 min) at a rate of 208C/min.Helium was used as the carrier gas at 15 ml/min, the injector temperature was set at 1758Cand the detectors at 2258C and 2008C [27].

Gas samples were collected in gas-tightsyringes and transferred to the gas chromato-graph by sealing the needle with a butyl rubberstopper. For methane and carbon dioxide analy-ses, 20 ml were injected into the gas chromato-graph. A thermal conductivity detector (TCD)and a packed column (Poropak Q, 80/100-mesh)were used. The column was operated isother-mally at 808C and the detector port was operatedat 1808C. Nitrogen was used as the carrier gas at aflow rate 10 ml/min [27].

2.4. Reactor start-up and operation

For start-up, the acidogenic reactor was filledup with 750 ml of feed, which consisted of55%, 40% and 5% of OMW, CW and LCM,respectively, and was operated anaerobically ata batch mode for 72 h in order to activate theindigenous microflora. The operation was subse-quently switched to continuous mode at the des-ignated HRT (3 days). Prior to cultivation, thereactor was flushed with argon gas for 10 minto ensure anaerobic conditions. The reactor wasoperated under these conditions for 87 days.According to Henze and Harremoes [28], aratio of COD/N = 400/7 is required for a bal-anced carbon to nitrogen feed. Based on thephysicochemical characteristics of the individ-ual waste streams and their mixture, 0.26 g/Lof urea (NH2CONH2) were added to the feedfor the first 87 days of operation in order toensure surplus in nitrogen concentration. Thecomposition of the feed was changed from the88th day to 90% CW and 10% LCM. In this


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scenario, no nitrogen source was added, sincethe 400/7 ratio of COD/N [28] in the feed wassatisfied due to the nitrogen content in theLCM. The nitrogen availability for the anaerobicbacteria was also verified throughout the exper-imentation period by measuring the ammoniumnitrogen concentration in the effluent from themethanogenic reactor. This transition from amixture of OMW+CW+LCM to CW+LCMalone is typical and anticipated in Greece andother Mediterranean countries due to the sea-sonal type of operation of these agroindustries.After the end of March, the operation of olivemills is ceased and only cheese-making facto-ries and dairy farms are still in operation. Forexample, a recent (2008) survey carried outin a Greek Ionian island identified 16 olivemills, 14 cheese-making factories and 1 dairyfarm producing 37, 26 and 3 m3 of wastewaters,respectively, on an average, daily basis. Thecomposition of the feeding mixture in eachscenario as well as the particular sequence ofscenarios followed in this study were selectedaiming to investigate, in the lab scale, theperformance of a centrally located anaerobicdigestion facility operating with the waste mix-tures actually available in this island throughoutthe year.

The methanogenic reactor was seeded in the6th day with 4L anaerobic digested sludgetaken from the municipal wastewater treatmentplant of Patras (Greece) and flushed with argongas for 10 min. Following this step, the effluentof the acidogenic reactor was fed to the methano-genic according to the operating HRT (16 days).Alkalinity was kept in satisfactory levels byadding 14 g NaHCO3/L in the feed of the metha-nogenic reactor throughout the experimenta-tion period. The OLR in the methanogenicreactor in the first scenario was 5.5 ± 0.36 gtotal COD/Lreactor/d (2.8 ± 0.17 g dissolvedCOD/Lreactor/d) and in the second 4.5 ± 0.30 gtotal COD/Lreactor/d (3.5 ± 0.17 g dissolvedCOD/ Lreactor/d) respectively.

3. Results

3.1. Wastewaters characterization

There are significant differences in the com-position of wastewaters according to Table 1.OMW presents the higher organic content(131 g/L as total COD) than the other two(72.1 and 53.4 g/L CW and LCM, respectively).OMW and CW have low nitrogen content incontrast with LCM, so the COD/N ratio is verylow for both wastewaters. Phenols concentrationin OMW is 6.8 g/L as it was expected [29].

3.2. Continuous experiments – acidogenicreactor

No dissolved COD removal was observedfor the 170 days of operation of the acidogenicreactor for both scenarios tested (Fig. 1). How-ever, in terms of solids hydrolysis, the reactorresponded differently to each scenario (differentsubstrate). TS and VS concentrations presenteda minor decrease, 10.3% and 14.9% respec-tively, between influent and effluent during thesecond scenario (90% CW and 10% LCM) dueto hydrolysis, which was not observed duringthe first phase of operation (Fig. 2). This canbe attributed to both the insufficient retentiontime of waste in the acidogenic reactor and thedifferent substrate composition in the feed(high concentration of OMW). A decrease inthe effluent pH comparing with the influent(from 5.8 ± 0.16 to 4.1 ± 0.19) was noticed inthe reactor (data not shown) until the 87th dayof operation. From the 88th day (2nd scenario),the value for the pH in the influent was6.7 ± 0.58 and decreased to 3.5 ± 0.27 in theeffluent. The reduction of the pH value wasexpected due to VFAs production by theacidogenic bacteria (Fig. 3). For example, theconcentration of acetic acid increased from732 ± 75 (mean value) to 3302 ± 245 mg/L forthe first scenario and 966 ± 159 to 4913 ± 118mg/L for the second scenario, respectively.


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The concentrations of other VFAs like propionic,butyric, isobutyric, valeric and isovalericacids remained practically constant at about164 ± 134 mg propionic acid/L (first scenario)and 194 ± 40 mg propionic acid/L (second sce-nario) and less than 50 mg/L (for each one ofthe other acids) throughout the operation ofacidogenic reactor regardless of the type of sce-nario tested. It can be easily observed thatVFAs concentration in the effluent increased lin-early with the time of operation, mainly due to thelinear increase of acetic acid concentration. Thiseffect can be attributed to an increase in the activ-ity and/or the concentration of the acidogenicbacteria. Biogas production in the acidogenicreactor was also observed (Fig. 4). For the firstscenario, the rate over the entire experimentwas oscillatory with an average of 0.46 ± 0.21L/Lreactor/d in which the percentage of hydrogenin the gas phase at the steady state was 27%. In

the second scenario, no hydrogen productionwas observed which may be attributed to thelack of OMW in the influent composition andto low pH and high VFAs concentration. Theaverage rate of biogas production for this sce-nario was 0.66 ± 0.19 L/Lreactor/d with a compo-sition of *90% in CO2. Production of carbondioxide without accompanying methane produc-tion in the hydrolytic/acidogenic stage is asign of rapid fermentation [30]. It should benoted that no methane was detected throughoutthe experimentation period. Phenol concentra-tion decreased by 18% from 2.14 ± 0.10 to1.75 ± 0.33 g/L during the first scenario and 20%from 0.20 ± 0.02 to 0.16 ± 0.01 g/L during the sec-ond scenario. The concentration of total carbohy-drates decreased from 29.1 ± 1.8 to 16.2 ± 1.7 g/L(44.4% decrease) until the 87th day of operationand from 33.1 ± 3.2 to 13.7 ± 1.4 g/L (58.6%decrease) until the 165th day, respectively.

Time (d)

55% OMW 40% CW 5% LCM

90% CW 10% LCM

Influent

Effluentd-

CO

D (

g/L)

100

80

60

40

20

0

0 30 60 90 120 150 180

Fig. 1. Concentration of dissolved COD during the operation of acidogenic reactor.


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3.3. Continuous experiments – methanogenicreactor

A methane bioreactor was used for treatingthe acidified effluent of the first stage in orderto assess the rate and extent of methanogenesisby co-digesting two different waste mixtures(scenarios). The d-COD removal was 75.5%and 85.2% (mean values) during the first andsecond scenarios, respectively (Fig. 5). Thetotal COD removal at the steady states of bothscenarios was 64% and 79%, respectively. Theremoval of COD in conjunction with gas produc-tion in the anaerobic digester provided evidenceof microbial activity, particularly methanogenicbacteria. A relatively short period of around10 days was required to achieve the acclimatiza-tion and stable activity of methanogenic bacteriaas measured by COD degradation and methaneevolution. The TS removal remained around

41% and 30% for the two scenarios from73.9 ± 6.2 to 43.5 ± 5.2 g/L and 61.2 ± 5.5 to42.6 ± 4.1, respectively. On the other hand, theremoval efficiency in VS concentration for theexamined scenarios was 59% and 56% from46.2 ± 5.2 to 18.9 ± 2.1 g/L and 32.3 ± 3.5 to14.3 ± 4.7 g/L, respectively (Fig. 6). As shownin Fig. 7, the biogas production rate presenteda high increase until the 30th day of operationfrom 0.72 (at the end of 1st day of operation)to 1.92 L/Lreactor/day. Between the 31st dayand 87th day, the rate was rather stable at1.82 L/Lreactor/day (mean value). Althoughthe influent composition was changed after the87th day, the biogas production rate was notseriously affected (1.68 L/Lreactor/day) untilthe 111th day of operation, one and a halfHRT later. After this day, an increase in therate from 1.68 to 2.17 L/Lreactor/day wasobserved. A temporary decrease of biogas

55% OMW 40% CW 5% LCM

90% CW 10% LCM

TS Influent

VS Influent

TS Effluent

VS Effluent

Time (d)

TV,

VS

(g/

L)

140

120

100

80

60

40

20

0

0 30 60 90 120 150 180

Fig. 2. Concentration of total solids (TS) and volatile solids (VS) during the operation of acidogenic reactor.


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55% OMW 40% CW 5% LCM

90% CW 10% LCM

Influent

Effluent

Time (d)

Acidogenic reactor

Ace

tic a

cid

(mg/

L)

8000

6000

4000

2000

0

0 30 60 90 120 150 180

Fig. 3. Concentration of acetic acid in the influent and the effluent of acidogenic reactor.

55% OMW 40% CW 5% LCM

90% CW 10% LCM

Time (d)

Bio

gas

(L/L

reac

tor/d

)

1.8

1.5

1.2

0.9

0.6

0.3

0.0

0 30 60 90 120 150 180

Fig. 4. Biogas production rate in the acidogenic reactor.


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production was noticed during the 142nd dayand 155th day; however, 9 days later the biogasrate increased at much higher levels reaching2.31 L/Lreactor/day. The composition of meth-ane in the biogas (Fig. 7) fluctuated between59.7% and 79.3% for the first scenario and59.8% and 66.9 % for the second scenario withmean values of 73.5 ± 4.7% and 63.8 ± 2.0%,respectively. The methane production rate atthe steady state reached 1.35 ± 0.11 and1.33 ± 0.15 L CH4/Lreactor/day for the twoexamined scenarios. The methane yield in thereactor was 243 and 305 L CH4/Kg CODadded for digesting the OMW:CW:LCM andthe CW:LCM mixture, respectively. An increasein effluent pH was noticed in the reactor from6.5 to 7.9 (data not shown) until the 36th dayof operation, although the influent pH wasdecreasing versus time from 6.8 to 5.4 due toVFAs production from the acidogenic reactor.

This value which is at the desired (optimum) lev-els for anaerobic digestion remained stable atthese levels for all the experimentation period.However, the influent value of the pH was con-tinuously decreasing during the second experi-mentation period and stabilized at 4.7 on the113th day, without affecting the effluent pH(data not shown). This stability could be attrib-uted to the addition of NaHCO3 in the influentat a concentration of 14 g NaHCO3/L. The addi-tion was taking place from the first day of theexperimentation period. The total bicarbonatealkalinity after the addition of NaHCO3

increased to 8800 mg of CaCO3/L and keptrising during operation until the value of16,750 in the 144th day of operation. The differ-ence in the value of the pH between the effluentof the acidogenic reactor and influent of the meth-anogenic is due to this addition. The influent ofthe methanogenic reactor was rich in VFAs, as

55% OMW 40% CW 5% LCM

90% CW 10% LCM

Time (d)

0 30 60 90 120 150 180

Influent

Effluentd-

CO

D (

g/L)

100

80

60

40

20

0

Fig. 5. Concentration of dissolved COD during the operation of methanogenic reactor.


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anticipated due to the pretreatment achieved inthe acidogenic reactor (Fig. 8). During the first20 days of the experiment, an accumulation ofpropionic acid was observed in the effluent ofthe methanogenic reactor, which was surpassedsuccessfully after the 40th day of operation.The mean value of total VFAs concentration inthe effluent for the two working scenarios was1767 ± 791 and 1281 ± 478 mg/L, respectively.It should be mentioned that the total VFAs con-centration in the influent of the reactor was4025 ± 1892 and 6152 ± 1478 mg/L (mean val-ues). The removal of total carbohydrates in glu-cose equivalents was 97% (data not shown) in thewhole process (both reactors) since the concen-tration of carbohydrates in the influent was29.1 ± 1.8 and 33.1 ± 1.4 g/L for the two scenariosand decreased to 0.8 ± 0.2 and 1.1 ± 0.3 g/L,respectively. Phenol concentration decreased by53% from 1.75 ± 0.33 to 0.83 ± 0.19 g/L duringthe first scenario and 50% from 0.16 ± 0.03 to

0.08 ± 0.02 g/L for the second scenario. Themean ammonium nitrogen concentration meas-ured in the effluent of the methanogenic reactorduring the two scenarios tested were0.18 ± 0.02 g/L and 1.1 ± 0.08 g/L, respectively,verifying nitrogen availability for the anaerobicbacteria.

4. Discussion

The high organic load and the presence ofinhibitory compounds in the OMW mandate itsmixing with other industrial wastewaters inorder to digest them successfully and eliminatethe environmental risks of no treatment at all.A number of different types of anaerobic reactorsand methods have been investigated for thetreatment of OMW in which dilution, nutrientaddition and alkalinity adjustment were alsorequired [31–32]. In a recent study [12], otherresearchers used an aerobic pre-treatment

0 30 60 120 150 1800

20

40

60

80

100

120

140TS Influent

VS Influent

TS Effluent

VS Effluent

90% CW10% LCM

55% OMW40% CW5% LCM

TS

, VS

(g/

L)

Time (d)

90

Fig. 6. Concentration of total solids (TS) and volatile solids (VS) during the operation of methanogenic reactor.


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stage with mycelium C. tropicalis in order toimprove the process efficiency by reducing thecomponents (contained in the OMW and CW)that are inhibitory to methanogenesis. Thesecond stage of this process was mesophilicanaerobic digestion in which the maximumOLR was 3.0 g COD/L/d, achieving an averageCOD removal of 83% similar to our results. Itshould be mentioned that in this study [12]the start-up period was much longer (3 months)in which feed, diluted with tap water, wasused as influent. Other studies report that theco-digestion of OMW with piggery effluentusing two identical upflow anaerobic filtersshowed COD removal of about 76% [33–34].In another study [35], authors reported a biogasproduction of approximately 1250 mL/L/day inan anaerobic co-digesting process treatingOMW with manure at a ratio of 50:50 underthermophilic conditions with an organic loadingrate of 7.8 g COD/L/day, which corresponds to amethane yield of 160 L CH4/Kg COD. They alsoshowed that the amount of nitrogen needed to

obtain a stable degradation of OMW can be pro-vided by cattle manure, swine manure or piggeryeffluent during co-degradation. Other research-ers [14] state that the co-digestion of OMWwith slaughterhouse wastewaters and wine-grape residues results to a methane yield of170, 163 and 191 L CH4/Kg COD added, respec-tively. Our case proved to be more efficient thanthese studies although we did not use any pre-treatment or high-digestion temperature (ther-mophilic conditions). In a recent publication,other researchers [36] have shown a biogas pro-duction rate of 210 mL/L/day digested dilutedpoultry manure and olive mill effluent at aratio of 50:50 under mesophilic conditions anda hydraulic retention time of 20 days. Our meth-ane yield rates were higher than the onesreported. Another study [27] in which treatmentof CW with a two-stage process took placereports a yield of 22.23 L of CH4/L of treatedinfluent which is very similar to the one derivedfrom the second scenario (21.28 L of CH4/L oftreated influent) that contained 90% CW.

55% OMW 40% CW 5% LCM

90% CW 10% LCM

Time (d)0 30 60 90 120 150 180

4

3

2

1

0

100

80

60

40

20

0

Bio

gas

(L/L

reac

tor/d

)

CH

4 (%

in b

ioga

s)

Fig. 7. Biogas production rate and methane percentage in the methanogenic reactor.


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55% OMW 40% CW 5% LCM

90% CW 10% LCM

Time (d)

Influent(a)

Acetic

Propionic

Isobutyric

Butyric

Isovaleric

Valeric

0 30 60 90 120 150 180

15000

12000

9000

6000

3000

0

VFA

s (m

g/L)

Fig. 8. Concentration of volatile fatty acids (VFAs) in (a) the influent and (b) the effluent of methanogenic reactor.

55% OMW 40% CW 5% LCM

90% CW 10% LCM

Time (d)

Effluent

Acetic

Propionic

Isobutyric

Butyric

Isovaleric

Valeric

(b)

0 30 60 90 120 150 180

4000

3000

2000

1000

0

VFA

s (m

g/L)


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Similar results were achieved by otherresearchers [37] for the anaerobic treatmentof CW (23.40 L of CH4/L of treated influent).The value of the pH in the methanogenic reac-tor was stabilized from the beginning of theprocess without any fluctuations at all, duringthe experimentation period, even though theinfluent pH was constantly decreasing at thesame time. The stability of the system againstthe influent pH could be attributed to theexcess of alkalinity that was produced duringthe anaerobic digestion process (increasedfrom 8800 to 16,750 mg of CaCO3/L). Thisfact indicates that the addition of NaHCO3 inthe influent of the second scenario of ourexperimentation should be reconsidered or atleast gradually reduced.

It should also be mentioned that in our casethe presence of phenols (because of the OMW)did not seem to affect or inhibit, in any way,the process of anaerobic digestion. On the con-trary, a 61.2% biodegradation of phenols wasobserved. This result, however, is in contrastwith other researchers [38] who have statedthat phenols and especially polyphenols are themost bio-recalcitrant compounds in OMWsince only 20–30% of polyphenols was degradedin methanogenic conditions in their experimentsand therefore identified them as being responsi-ble for the inhibition of anaerobic process [39].

5. Conclusions

The overall objective of this article was toproduce a sustainable fuel source from agroin-dustrial wastewaters, which can be integratedinto the existing energy infrastructure in themedium term, while in the longer term can pro-vide a safe and economical means of supplyingthe needs of a developing hydrogen and biogasfuel economy. It has been shown in this workthat co-digestion of OMW, CW and LCM in atwo-stage AD system consisting of a separateacidogenic and methanogenic reactors is an effi-

cient method for treating these wastes with aconcomitant production of biogas. The indige-nous microorganisms of OMW and CW provedto be capable of acidogenesis and there was noneed for using other sources of inoculum duringthe acidogenic reactor startup. The ultimatemethane yield of co-digesting OMW, CW andLCM under mesophilic conditions (358C) at atotal operating HRT of 19 days was estimatedto be 243 and 305 L CH4/Kg COD for the exam-ined scenarios, which is very satisfactory com-paring to other projects. By using thesuggested process, a reduction of the volatilesolid contents between 56% and 59%, dependingon the scenario, can be expected to result. Theuse of CW and LCM as co-substrates in theanaerobic digestion of OMW has many advan-tages such as stability of the process, improve-ment of the methane yield and the feasibilityof the process due to the seasonal productionof these kinds of wastewaters, minimization ofnutrients addition and elimination of the needfor prior treatment.

Acknowledgment

The authors wish to thank the CommunityInitiative INTERREG IIIA Greece, Italy(2000–2006) for the financial support of thiswork under Grant No. I3101004 (Acronym:AGROENERGY).

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