anaerobic digestion challenge of raw olive mill wastewater
TRANSCRIPT
Bioresource Technology 102 (2011) 10810–10818
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Bioresource Technology
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Anaerobic digestion challenge of raw olive mill wastewater
M.A. Sampaio, M.R. Gonçalves, I.P. Marques ⇑Bioenergy Unit, National Laboratory of Energy and Geology I.P. (LNEG), Estrada Paço do Lumiar 22, 1649-038 Lisboa, Portugal
a r t i c l e i n f o a b s t r a c t
Article history:Received 30 May 2011Received in revised form 26 August 2011Accepted 1 September 2011Available online 10 September 2011
Keywords:Raw olive mill effluentBiogasAnaerobic hybrid digesterPhenolic compoundsOrganic shocks operation
0960-8524/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.biortech.2011.09.001
⇑ Corresponding author. Tel.: +351 210924600; faxE-mail addresses: [email protected] (M
[email protected] (M.R. Gonçalves), [email protected] (I.
Olive mill wastewater (OMW) was digested in its original composition (100% v/v) in an anaerobic hybrid.High concentrations (54–55 kg COD m�3), acid pH (5.0) and lack of alkalinity and nitrogen are someOMW adverse characteristics. Loads of 8 kg COD m�3 d�1 provided 3.7–3.8 m3 biogas m�3 d�1 (63–64%CH4) and 81–82% COD removal. An effluent with basic pH (8.1) and high alkalinity was obtained. A goodperformance was also observed with weekly load shocks (2.7–4.1, 8.4–10.4 kg COD m�3 d�1) by introduc-ing piggery effluent and OMW alternately. Biogas of 3.0–3.4 m3 m�3 d�1 (63–69% CH4) was reached.
Developed biomass (350 days) was neither affected by raw OMW nor by organic shocks. Through theeffluents complementarity concept, a stable process able of degrading the original OMW alone wasobtained. Unlike what is referred, OMW is an energy resource through anaerobiosis without additionalexpenses to correct it or decrease its concentration/toxicity.
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1. Introduction
Olive oil production is expanding worldwide as a result of itshealth-promoting effects. Most countries make use of the three-phase centrifugation system, from which large quantities of a strongreddish-brown liquid called olive mill wastewater are obtained(Morillo et al., 2009; McNamara et al., 2008). This effluent hasbecome a serious environmental problem as a result of the oliveoil increasing production and industrialization of the extractionprocess that generates larger amounts of OMW (Kapellakis et al.,2006). Moreover, the scattered and seasonal nature of olive oilproduction did not contribute to find a solution in order to properlymanage the resulting effluent (McNamara et al., 2008). OMW cannotbe 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 which make it unsuitable for furtheruse as fertilizer or as irrigation water (Niaounakis and Halvadakis,2006).
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, infiltration 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.
ll rights reserved.
: +351 217127195..A. Sampaio), marta.goncal
P. Marques).
OMW has been the subject of many waste treatment studiesinvolving chemical and physical treatment (coagulation/floccula-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, significant 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 benefit/price ratio and overcomethis 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 (45–220 g COD L�1), this effluent is classified among thestrongest industrial liquid wastes that corresponds to concentra-tions 20–4400 times higher than the ordinary urban wastewater(Azbar et al., 2009; Xing et al., 2000) and, consequently, it repre-sents a significant 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
Table 1Characterization of the effluents used in the hybrid feed.
OMW PE
pH 4.96(0.08) 6.99(0.01)
Partial alkalinity (kg CaCO3 m�3) �0 4.85(0.14)
Total alkalinity (kg CaCO3 m�3) 2.40(0.07) 8.63(0.39)
COD (kg O2 m�3) 55.28(2.3) 30.71(0.00)
CODS (kg O2 m�3) 50.81(0.57) 12.12(0.12)
NH3 (kg N m�3) �0 1.83(0.04)
Total N (kg N m�3) 0.21(0.02) 2.35(0.53)
TSS (kg m�3) 3.18(0.07) 19.60(1.65)
TS (kg m�3) 28.23(0.36) 23.31(0.05)
VSS (kg m�3) 0.53(0.00) 4.80(0.38)
VS (kg m�3) 15.85(3.22) 15.70(0.01)
VFA (kg acetic acid m�3) 2.64(0.38) 3.43(0.27)
TPh (kg caffeic acid m�3) 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)
OMW, olive mill wastewater; PE, piggery effluent; �0, below detection limit.Values are the averages of determinations. Values in brackets show standarddeviations.
M.A. Sampaio et al. / Bioresource Technology 102 (2011) 10810–10818 10811
substrate 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-benefit 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, moreflexible and cheaper. So, the concept of OMW complementaryeffluent 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 effluent during the experimental period. The firstresults have showed to be possible to treat anaerobically the rawOMW using another effluent and digesting them simultaneously(Marques et al., 1997, 1998).It was also proved that the piggeryeffluent can work as a good complementary effluent 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 OMW and manure were the best co-digestion option (Angelidakiand Ahring, 1997). More recently, a combination of OMW anddiluted poultry manure was degraded in a cylindrical down flowanaerobic 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 m�3 d�1 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. Efflu-ent was successfully degraded and a methane production rate ofabout 1.35 m3 m�3 d�1 was obtained using a HTR of 19 days. Othermixture (75% OMW plus 25% pig slurry) was pre-treated byCandida tropicalis and digested in a fixed bed reactor (HTR of11 days) to give 1.61 m3 m�3 d�1 of biogas (Martinez-Garciaet al., 2009). The up-flow fixed bed digester (anaerobic filter), 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 effluent (v/v) and about 6 days of HTR, a production rateof 1.31 m3 CH4 m�3 d�1 was registered. Following the work per-formed and aiming to make the process even simpler and cheaper,it was decided to test other up-flow digester type. An anaerobichybrid digester was used instead of the anaerobic filter (Gonçalveset 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 m�3 d�1 (Gonçalveset 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 effluents that are gener-ated (7–30 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(OMW and the complementary effluent), the other goal of thisstudy is to evaluate the conditions of process stability when so dif-ferent effluents are alternately introduced.
2. Methods
2.1. Substrates: agro-livestock and industrial effluents
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 year�1. Piggery effluentwas 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
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(TNHþ4 -N) concentrations were determined according to StandardMethods (APHA, 1998). The proportion of ammonium concentra-tions and free ammonia (NHþ4 versus NH3) were estimated accord-ing to Eq. (1) (El-Mashad et al., 2004), where T is the absolutetemperature (273–373 K).
NH3 � N ¼ ðTHNþ4 � NÞ � 1þ 10�pH=10� 0:1075þ 2725=Tð Þð Þ� �� �
ð1Þ
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 quantified viaMerck Nitrogen cell tests (10–150 mg NL�1). 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 modifiedFolin–Ciocalteu 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 flame ionization detector and a2 m � 2 mm Carbopack B-DA/4% Carbowax 20 M (80–120 mesh)column. Nitrogen was used as carrier gas (30 mL mn�1). Tempera-ture of the column, injector and detector was 170, 175 and 250 �C,respectively. Total VFA concentrations were expressed as acetic
10812 M.A. Sampaio et al. / Bioresource Technology 102 (2011) 10810–10818
acid. Soluble samples were 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 mn�1).
2.3. Reactor operation
Experiments were carried out by using an up-flow anaerobichybrid digester that was previously used and described elsewhere(Gonçalves 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 influent con-sisted of a blend of the raw OMW and its complementary substrate(piggery effluent, PE) obtained by an increase of OMW contentalong the experimental time (Marques, 2001). Gas productionwas evaluated by a wet gas meter and corrected to standard con-ditions for pressure and temperature (1 atm, 0 �C). Volume ofdigested flow was registered every day in order to determine thehydraulic retention time (HRT) of the assay. Influent and effluentsamples 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 first 14 days of operation,the hybrid was fed with piggery effluent and a HRT of aboutsix days was set. Afterwards, the digester influent 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 0–14 0A1 15–82 53
83–9495–136
A2 137–168 69A3 169–232 83
B 233–287 100
Stop 288–299 –C0 300–313 100C1 314–315 0
315–320 100C2 321–322 0
322–327 100C3 328–329 0
329–334 100C4 335–336 0
336–341 100C5 342–343 0
343–350 100
Raw OMW and PE, mixture feed (Phase A), Raw OMW feed (Phase B) and Raw OMW orOMW, Olive Mill Wastewater; PE, piggery effluent; HRT, Hydraulic Retention Time; OLRValues are the averages of determinations. Values in brackets show standard deviations
(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 effluent 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, five 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 efficiently convertedinto biogas. The hybrid stability and its capacity in convertingthe potential toxic matters of the influent are documented by theremoval ability and methane production of the digester biomassover the 350 days of experiment.
3.1. Phase A. Complementary substrates trial: raw OMW and PE,mixture feed
Operating under organic loading rates (OLR) of 5.2 kg CODm�3 d�1 (Phase A2, Table 2) and 7.4–9.0 kg COD m�3 d�1 (PhaseA3), ranges of total COD removal of 75% and 80–83% and biogas vol-umes of 2.7 and 2.8–3.6 m3 m�3 d�1, containing 67% and 66–67%CH4, were obtained, respectively. The increase of the influent con-centration till 57 kg COD m�3 (Phase A3) did not cause instabilityneither decrease of the hybrid performance. The methane yieldof 0.429 m3 CH4 kg�1 COD removal (Phase A2) evolved to0.317–0.358 m3 CH4 kg�1 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 m�3 d�1)
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..
y = 0,3519x + 0,5881R² = 0,6414
y = -0,4157x + 67,902R² = 0,0647
0
10
20
30
40
50
60
70
80
0
1
2
3
4
5
6
7
8
9
10
4 5 6 7 8 9 10
CH
4(%
)
Bio
gas
(m3 m
-3d-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 (Gonçalves et al., submitted for publication).
M.A. Sampaio et al. / Bioresource Technology 102 (2011) 10810–10818 10813
The main results of the OMW digestion obtained in differentyears and provided from different mills (Gonçalves 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
Table 3Anaerobic digestion of olive mill wastewater: operation methodologies using substrates m
No. Reactor,temp. (�C)
Effluents(% v/v)
Additionalactions
HRT (d) OLR (kg COD
1 AF, 35 OMW-83 None 6–7 7.7–9.6PE-17
2 CSTR, 55 OMW-75 None 13 7.8Manure-25
3 AF, 35 OMW-91 None 6–7 5.0–5.7PEdig.-9
4 AF, 35 OMW-91 None 6–7 6.6–8.0PE-9OMW-83 8.3–10PEdig.-17
5 – ,35 OMW-28 pHadjustments
18 4.84diluted poultrymanure
6 Fixed-bedreactor, 37
OMW-75,cheese whey-25
- sterilization- treatment:
C. tropicalis (a)- 26 g NaOH L�1
addition- NaHCO3
addition
- 3.0
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 ureaL�1
feed)addition,acidogenicreactor
- 14 g NaHCO3
L�1 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 6–7 7.4PE-17
12 AH, 37 OMW-100 None 6–7 8.01
AF, anaerobic filter; 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 d�1/18 L = 1.61 L L reactor�1 d�1 (data from last four days of operation).
it is possible to infer that the increase of OLR (5.2–9.0 kg CODm�3 d�1) promotes the production of the biogas volume(2.4–3.6 m3 m�3 d�1) and maintains its quality along the time ofoperation. Methane concentrations (62–67%) were preserved in a
ixtures.
m�3 d�1) Biogas methane (m3 m�3 d�1) CODremoval(%)
References
3.6–4.0 70–77 Marques et al. (1997)2.3–2.6– – Angelidaki and Ahring
(1997)1.551.7–2.1 73–75 Marques et al. (1998)1.1–1.42.1 63.2 Marques (2001)1.33.42 73.62.21.53 – Gelegenis et al. (2007)0.99
1.25 83 Martinez-Garcia et al.(2007)
1.61b 85 Martinez-Garcia et al.(2009)
–1.35
75.5 (% CODs) Dareioti et al. (2009)
–0.91
63.2 Dareioti et al. (2010)
3.16 78.6 Gonçalves et al.,submitted forpublication
1.96
3.18 81.3 Present work2.123.742.36
81.5 Present work
; OMW, olive mill wastewater; PE, Piggery effluent, PEdig., Piggery effluent digestedoxygen demand; CODs, soluble COD,
b
c
Phase B: 100% OMW Phase C: organic pulsesStop
0
10
20
30
40
50
60
70
80
90
0
2
4
6
8
10
12
230 240 250 260 270 280 290 300 310 320 330 340 350 360
CH
4(%
)
OR
L (k
g C
OD
m-3
d-1);
Bio
gas (
m3
m-3
d-1)
Time (d)
OLR Biogas CH4
0102030405060708090100
0123456789
10
230 240 250 260 270 280 290 300 310 320 330 340 350 360
VFA
rem
(%)
pH /
FVA
(g L
-1)
Time (d)
pH in pH ef VFA ef VFA rem
0102030405060708090100
0
2
4
6
8
10
230 240 250 260 270 280 290 300 310 320 330 340 350 360
TPh
rem
(%)
TPh
in (
gL-1
)
Time (d)
TPh in TPh rem
(a)
(b)
(c)
Fig. 2. Hybrid digester behaviour: Phase B and C (a) gas production, (b) pH and VFA, (c) TPh (phenol compounds), in, influent; ef, effluent; rem, removal.
10814 M.A. Sampaio et al. / Bioresource Technology 102 (2011) 10810–10818
narrow range of the anaerobic process usual values. Regarding thedigester removal ability, the increase of phenols concentration ininfluent (2.1–2.3 to 2.9–3.1 kg TPh m�3) did not cause a relevantalteration on unit capacity (58.9–61.1% and 56.6–59.2% TPhremoval, respectively). Concerning the total COD, the more concen-trated influent (49–57 kg m�3) corresponded to the highest CODremovals recorded (80–83%).
Several authors have presented studies on the use of differenteffluents to digest OMW anaerobically. Table 3 summarizes themain data obtained from some operation methodologies that com-bine OMW with other effluents. This strategy has been used bydiverse authors but it was usually associated to several otherphases of operation mainly related to the influent preparation tothe anaerobic step. Pre-treatment ( C. tropicalis: Martinez-Garciaet al., 2007, 2009); pH adjustments (Gelegenis et al., 2007); ureaand alkali (14 g NaHCO3 L�1) additions (Dareioti et al., 2009) aresome examples of the undertaken actions. Comparatively, thiswork group has been operated with the lower HRT and the highest
volume of the raw OMW in the influent (83/91%) and, conse-quently, highest loading rates (5–10 kg COD m�3 d�1) were testedand higher volumes of gas (1.7–4.0 m3 biogas m�3 d�1; 1.1–2.6 m3
CH4 m�3 d�1) 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 only with the original OMW (Phase B:233–287 days), OLRs of about 8 kg COD m�3 d�1 provided a totalCOD removal of 81–82% and a biogas volume of 3.7–3.8 m3 m�3 d�1
(63–64% CH4). The digester performance is presented in Fig. 2 andTables 4 and 5 (Phase B). The acid pH (4.9–5.1), the high concentra-tions of COD (54–55 kg m�3), VFA (2.8–4.0 kg m�3) and phenoliccompounds (3.0–3.3 kg TPh m�3) 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 OMWthat were registered along this assay. Even under these unfavourableconditions, the hybrid feed was accepted and degraded by the
Table 4Hybrid data: COD removal and gas production.
Phase Total COD Soluble COD Biogas (m3 m�3 d�1) CH4 (%) Y (m3 CH4 kg�1 COD)
Inf. (kg m�3) Rem. (%) Inf. (kg m�3) Rem. (%)
B 55.4(1.7) 82.0(1.0) 44.7(3.2) 80.7(0.8) 3.78(0.26) 63.7(1.2) 0.361(0.054)
54.2(2.4) 81.0(1.7) 48.2(3.4) 80.6(2.2) 3.70(0.16) 62.5(1.2) 0.377(0.067)
Stop – – – – – – –C0 67.7(3.57) 81.0(0.6) 53.2(0.13) 76.7(0.3) 3.35(0.08) 63.1(1.0) 0.249(0.017)
C1 22.0(1.3) 74.2(0.7) 14.5(0.3) 74.4(0.4) 3.22(0.56) 63.2(-) 0.332(0.004)
57.8(0.0) 44.4(0.0)
C2 17.9(0.9) 80.8(0.3) 13.0(0.3) 77.9(1.0) 3.39(0.65) 62.5(-) 0.350(0.006)
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) 3.43(0.88) 63.7(-) 0.371(0.023)
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) 3.15(0.85) 68.7(-) 0336(0.006)
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) 3.03(1.01) 62.9(-) 0.326(0.013)
57.8(1.7) 48.4(1.2)
(–) Single determination; Values are the averages of determinations taken at steady-state period. Values in brackets shows standard deviations. Y, methane yield.
Table 5Hybrid mean data: alkalinity and nitrogen contents.
Phase Substrate Partial alkalinity (kg CaCO3 m�3) Total alkalinity (kg CaCO3 m�3) NHþ4 (kg N m�3) NH3 (kg N m�3)a Total N (kg N m�3)
in ef in ef in ef in ef in ef
B OMW �0 5.03(0.41) 2.42(0.06) 6.32(0.23) 0.01(0.01) 0.13(0.06) – – 0.20(0.01) 0.42(0.08)
OMW �0 5.05(0.11) 2.46(0.05) 6.20(1.17) �0 0.02(0.02) – – 0.19(0.01) 0.31(0.04)
Stop – – – – – – – – – – –C0 OMW �0 4.85(0.02) 2.37(0.11) 6.20(0.18) �0 �0 – – 0.20(0.02) 0.24(0.02)
C1 PE 3.13(0.18) 4.88(0.04) 6.48(0.11) 6.33(1) 1.74(1) 0.10(1) 0.081(�) 0.007(�) 1.99(0.51) 0.37(1)
OMW �0 2.37(0.11) �0 – 0.20(0.02)
C2 PE 3.40(0.19) 5.19(0.55) 6.50(1) 6.71(0.48) 1.69(0.02) 0.13(0.00) 0.070(�) 0.008(�) 1.83(1) 0.42(1)
OMW �0 2.25(1) �0 – –C3 PE – 4.71(0.05) – 5.69(0.12) 1.60(0.04) 0.11(0.01) 0.139(�) 0.003(�) 2.25(1) 0.40(1)
OMW �0 – �0 – –C4 PE 3.96(0.02) 4.70(0.35) 6.36(0.02) 5.61(0.09) 1.55(0.05) 0.12(0.01) 0.130(�) 0.005(�) – 0.43(0.01)
OMW �0 2.04(0.19) �0 – –C5 PE 3.94(0.37) 4.75(0.07) 5.36(0.16) 5.55(0.11) 1.32(0.01) 0.11(0.01) 0.150(�) 0.004(�) – 0.39(0.02)
OMW �0 – �0 – –
In, influent; ef, effluent; �0, below detection limit; (1) single determination; (�) standard deviation that was not calculated.a Calculated from Eq. (1).
Table 6Conversion capacity of the hybrid digester: Phases A, B and C.
Phase OLR (kg COD m�3 d�1) COD rem (%) Biogas (m3 m�3 d�1) CH4 (m3 m�3 d�1) Y (m3 CH4 kg�1 COD)
Aa 8.20 81.3 3.18 2.12 0.34B–C0 8.83 81.4 3.61 2.28 0.33C1–C5
b 7.72 79.0 3.25 2.08 0.34
a 83% OMW + 17% PE (v/v).b 83% OMW + 17% PE (time feed).
M.A. Sampaio et al. / Bioresource Technology 102 (2011) 10810–10818 10815
developed biomass as documented by the comfortable methaneyield reached (Y = 0.361–0.377 m3 CH4 kg�1 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 OMW alone, nitrogen addi-tion was needed and a COD:N ratio of 61:1 to 42:1 was necessary forthe optimal degradation process (Angelidaki et al., 2002). Effec-tively, in this case, the influent was just composed by OMW thathas not undergone any alteration and presents characteristicspotentially adverse to a successful development of anaerobicprocess.
Concerning the effluent, pH became basic (8.1) and alkalinityincreased to about 5.0 (PA) and 6.3 kg CaCO3 m�3 (TA). Total
phenolic compounds removal of 46% was reached, being theremained fraction (1.4–1.5 kg m�3) probably due to the presenceof polymerized phenolic matter since no colour clarification wasnoticed. Instead, a slight increase of the colour absorbance valueswas registered in digester effluent. Absorbance data of 24–27 and30–31 were recorded in the influent and effluent, respectively.VFA were mostly consumed in the system and the VFA removalof 96–98% resulted into an effluent of 0.08–0.1 kg m�3 (Fig. 2b),being the acetic acid the main component (89% total VFA).Regard-ing the solids, influent concentrations of 34.5 TS kg m�3 (±3.2) and1.04 VSS kg m�3 (±0.36) (269–314 days), correspond to effluentamounts of 17.5 TS kg m�3 (±0.99) and 0.43 VSS kg m�3 (±0.24),respectively. These data indicate that the reactor was not subjected
10816 M.A. Sampaio et al. / Bioresource Technology 102 (2011) 10810–10818
to 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 B–C0) reveals that the OLR increase to 8.8 kg CODm�3 d�1 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 figures reinforce the findingalready made on the quality of the hybrid functioning regardingits great ability to degrade the raw OMW without the addition ofanother effluent.
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 (15–22 and 53–58 kg COD m�3, respectively) haveoriginated OLR alterations of three-four times (2.7–4.1 and8.4–8.9 kg COD m�3 d�1). The hybrid replied positively to theseconditions by providing gas volumes of 3.0–3.4 m3 m�3 d�1
(63–69% CH4) and methane yields of 0.326–0.371 m3 CH4 kg�1
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 C1–C5) it is noticed that the degree of influenttreatment and the reached gas production in both trials are identi-cal (3.2 m3 biogas m�3 d�1 and 2.1 m3 CH4 m�3 d�1, 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 effluents have been supplied to the sys-tem in both operational situations. Data indicates that theoperation mode, feeding the digester with effluents mixture oreffluents 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 (Gonçalves 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 originalOMW that was also used in the earlier period. It is interesting to notethe response of the system. A rapid increase in gas production wasobserved in 6 days (Fig. 2a) and removals of 81% COD total(68 kg m�3), 79% COD soluble (53 kg m�3) and 99% VFA (5.2 kg m�3)
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 m�3 d�1 (influent of 31 kg COD m�3, 15–82 days) to18.5 kg COD m�3 d�1 (influent of 112.9 kg COD m�3, 83–94 days: Ta-ble 2). Additionally, in the final phase of this period, the influent waschanged to a new stock of OMW and PE and an OLR of 5.6 kg CODm�3 d�1 (95–136 days) was applied. As a result, the removal capac-ity of the unit (77–83% COD, 83–136 days) was not accompanied byits ability in converting the organic matter into gas. Despite the goodquality of the biogas obtained (70–72% CH4) indicating an activemethanogenic population, a low methane yield (0.181–0.186 m3
CH4 kg�1 COD removal) was reached through these periods(83–136 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 influent (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 (Gonçalves et al., 2009, submitted for publication). Theadaptation capacity to different OMW stocks has also been verifiedby 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 and OMW alternately (Phases C1–C5, 314–350 days).The conversion efficiency data and the working process stabilityobtained indicate that the microbial 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 m�3 d�1), 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 low methane yield anda high VFA level in the effluent (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 25–150 mg NH3-N L�1 have been reported asinhibitory for mesophilic treatment. Levels up to 1.1 g NH3 L�1
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(233–313 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 (72–150 mg L�1, 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 effluents that arealternately applied.
Another relevant aspect is the similarity of average data of thegas production and treatment efficiency of the anaerobic process
M.A. Sampaio et al. / Bioresource Technology 102 (2011) 10810–10818 10817
to carry out with a feed mixture of 83% OMW v/v or feed pulses ofindividual substrates (Phase A versus Phases C1–C5, Table 6). Thus,given that both feed models provide analogous results, the pulsesprocedure has the advantage of suppressing the effluents 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 influent 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 additionaleffluent 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 significant 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 effluent 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 effluents 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 effluent 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 influent. 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/ortoxicity. The digested flow with a basic pH and high alkalinity anddevoid 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. Conclusions
The high energy potential of OMW (54–55 kg COD m�3) isdirectly recoverable through anaerobic digestion, without requiring
any prior correction and decrease of its concentration and/or toxicityand making the process simpler, more flexible and cheaper.
The hybrid worked under stable conditions when successiveshocks of loads (2.7–4.1, 8.3–10.7 kg COD m�3 d�1) were appliedby feeding alternately with different effluents.
The strategy developed, on the basis of complementaryeffluents 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.
Acknowledgements
The authors acknowledge the financial support of the ‘‘Fun-daçãopara a Ciência e a Tecnologia’’, FCT/MCTES, through the Pro-ject PTDC/ENR/69755/2006 and also through the grant given toMarta Gonçalves SFRH/BD/40746/2007.
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