Bioresource Technology 123 (2012) 507–513

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Bioresource Technology

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Enhanced methane production from pig manure anaerobic digestion using fishand biodiesel wastes as co-substrates

Leticia Regueiro a, Marta Carballa a,⇑, Juan A. Álvarez b, Juan M. Lema a

a Department of Chemical Engineering, School of Engineering, University of Santiago de Compostela, Rúa Lope Gómez de Marzoa s/n, 15782 Santiago de Compostela, Spainb AIMEN Technological Center, C/Relva, 27 A – Torneiros, 36410 Porriño Pontevedra, Spain

h i g h l i g h t s

" Fish and biodiesel waste were used as co-substrates in pig manure anaerobic digestion." Both co-substrates improved methane yield but caused VFA and ammonium accumulation." Shorter HRT and FW < 10% in the feeding allow to control ammonium inhibition." Biodiesel waste co-digestion requires feeding shares < 6% and/or fed-batch operation." The poorer the co-digester operation, the higher the Methanosarcina/Methanosaeta ratio.

a r t i c l e i n f o

Article history:Received 22 May 2012Received in revised form 26 July 2012Accepted 28 July 2012Available online 7 August 2012

Keywords:Agroindustrial wastesAmmoniaCo-digestionMethanosarcinaVolatile fatty acids

0960-8524/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.biortech.2012.07.109

⇑ Corresponding author. Tel.: +34 881 816020; fax:E-mail addresses: leticia.regueiro.abelleira@gmai

carballa@usc.es (M. Carballa), jaalvarez@aimen.es (J.A(J.M. Lema).

1 PM, pig manure;2 FW, fish waste;3 BW, biodiesel waste;4 w/w, wet weight basis;5 PM, pig manure;

a b s t r a c t

Co-digestion of pig manure (PM1) with fish (FW2) and biodiesel waste (BW3) was evaluated and comparedwith sole PM digestion. Results indicated that co-digestion of PM with FW and/or BW is possible as long asammonium and volatile fatty acids remained under inhibitory levels by adjusting the operating conditions,such as feed composition, organic loading rate (OLR) and hydraulic retention time (HRT). PM and FW co-digestion (90:10 and 95:5, w/w4) was possible at OLR of 1–1.5 g COD/L d, resulting in biogas productionrates of 0.4–0.6 L/L d and COD removal efficiencies of 65–70%. Regarding BW, good results (biogas produc-tion of 0.9 L/L d and COD elimination of 85%) were achieved with less than 5% feeding rate. Overall, oper-ating at the same OLR, the biogas production and methane content in the co-digester was higher than inthe only PM digester.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

In the last years, anaerobic digestion of animal wastes has beenpromoted in order to avoid the uncontrolled emissions of CH4 dur-ing storage (Novak and Fiorelli, 2010). Pig manure (PM5) can be anexcellent base substrate for anaerobic digestion due to its inherentbuffering capacity and high content of a wide range of nutrients re-quired for the development of anaerobic microorganisms. However,PM has a low biogas yield, around 20–30 m3/ton (Angelidaki andEllegaard, 2003), and high ammonium concentrations (2–3 g N–

ll rights reserved.

+34 881 816702.l.com (L. Regueiro), marta.. Álvarez), juan.lema@usc.es

NH4+/L). Consequently, PM is preferably co-digested with high car-

bon content wastes, on one hand, to improve the C/N ratio (Hartmanand Ahring, 2006), and on the other hand, to increase the biogas pro-duction, essential for the plant’s economy. It has been shown thatbioenergy production in farm biogas plants could be enhanced by80–400% by using organic wastes and by-products as co-substrates(Braun and Wellinger, 2003; Weiland, 2010). Despite the well-known reported co-digestion benefits, such as optimum humidityand C/N ratio or inhibitory substances dilution (Mata-Álvarezet al., 2000), it is not clear whether some substrates have adverse im-pact when they are co-digested with another waste (Callaghan et al.,2002). Therefore, it is critical to obtain an optimal mixture of theavailable co-substrates as well as the optimum operating conditionswhich allow high biogas yields without compromising the stabilityof the process (Alvarez et al., 2010).

Fish and shellfish canning industry is an important sector inGalicia (NW of Spain), with around 65% of the total Spanishproduction and representing 45% of the Galician factories and67% of the jobs (Garcia et al., 2003). This sector generates different

508 L. Regueiro et al. / Bioresource Technology 123 (2012) 507–513

solid wastes with a wide range of characteristics depending on theraw material processed (tuna, mussel, sardine, mackerel, etc.). Ingeneral, fish wastes (FW6) are protein-rich substrates, although theyalso contain important lipids. Protein-rich materials have a fastbiomethanation, but their degradation products (ammonium) caninhibit the process as well (Chen et al., 2008). Ammonium inhibitionis directly related to the concentration of the undissociated form(NH3) or free ammonia (FA7), thus becoming more important at highpH levels. The inhibitory FA concentration varies depending onoperational parameters such as origin of inoculum, substrate, pHand temperature (Alvarez and Liden, 2008). The reported FAinhibitory concentrations for mesophilic conditions range from 25to 140 mg N-FA/L, whereas during the thermophilic digestion ofcattle manure, higher values (around 390–700 mg N-FA/L) weretolerated after an initial acclimation period (Guerrero et al., 1997).

Biodiesel fuels have recently drawn much attention given thatthey have various advantages over petroleum-based fuels (Itoet al., 2005). The biodiesel production is carried out by catalyzedtransesterification with alcohol (usually methanol). Beside desiredmethylesters, this process also generates few other products,including crude glycerol or biodiesel waste (BW8), oil-pressed cakesand washing water. BW is easily separated from the aqueous phaseand it is composed mainly of glycerol. In many occasions, it alsocontains a significant fraction of lipids due to inefficient separationsystems. It is estimated that 1 kg of crude glycerol is generated per9 kg of biodiesel produced (Dasari et al., 2005). The importantincrease in the biodiesel production of the last years has resultedin crude glycerol surplus that implied a dramatic 10-fold decreasein biodiesel waste prices (Yazdani and Gonzalez, 2007), andconsequently, crude glycerol is often regarded as a waste streamwith an associated disposal cost. This biodiesel residue is readilydigestible and can be easily stored over a long period, making it anideal co-substrate for anaerobic digestion (Ma et al., 2008), whereasits digestion as sole substrate is not viable as no nitrogen would beavailable for microbial populations (Robra et al., 2010). Lipid-richmaterials are known to have high methane production potentials(Hansen et al., 1999), but their degradation products, the long-chainfatty acids (LCFA), are known to be inhibitors of methanogenicmicroorganisms (Pereira et al., 2004). Besides, operationalinstabilities related to sludge flotation and washout are also reported(Jeganathan et al., 2006).

The aim of this work was to evaluate the use of fish waste andbiodiesel waste as co-substrates to enhance the mesophilicanaerobic digestion of pig manure at laboratory scale. The effectof feeding mixture on process performance and microbialcommunity composition was investigated. The results obtainedin the co-digestion systems were compared with those obtainedin a reactor treating only pig manure.

2. Methods

2.1. Wastes and inoculum

PM was taken from a sewer of a 150-pig fattener and sowfarm, which collects both feces and urine. PM samples werehomogenized, sieved to 2 mm and stored at 4 �C until use tominimize decomposition. Different batches of PM were usedthroughout the experiment (>200 days) due to the impossibilityof storing the total amount required.

FW was delivered by a canning industry and it consisted ofheads, tails, bones and viscera of tuna fish. FW was homogenized

6 FW, fish waste;7 FA, free ammonia;8 BW, biodiesel waste;

by grinding and stored at �20 �C. BW was taken from a biodieselfactory and was stored at 4 �C without pre-treatment. It containedmainly glycerol produced in the transesterification. One batch ofFW and BW was sufficient for the complete study.

Anaerobic granular biomass from an internal circulation reactortreating brewery wastewater was used as inoculum, with an initialin-reactor biomass concentration of about 10 g VSS/L.

2.2. Anaerobic reactors

Experiments were carried out in three continuous stirred tankreactors (160 rpm, Heidolph RZR 2041), two co-digesters and oneonly-PM digester, constructed in methacrylate and with a workingvolume of 9.2 L, approximately. Reactors were operated at 35 �C byhot water recirculation. The applied feedstock mixtures wereprepared every 2–3 days, diluted with tap water according to theapplied organic loading rate (OLR), and stored at 4 �C prior touse. The digesters were fed manually after an equivalent volumeof digester mixed liquor was removed. Temperature, pH, stirringspeed and biogas production were monitored on-line. Otherphysico-chemical parameters (solids, chemical oxygen demand(COD), alkalinity, volatile fatty acids (VFA) and ammonium) weremeasured twice per week.

2.3. Operational strategy

The operation of the two co-digesters was identical. They werestarted-up with a mixture of PM-FW-BW (84:5:11 in wet weight(w/w) basis), which was the optimum mixture obtained fromlinear programming (Alvarez et al., 2010), at an OLR of 0.5 gCOD/L d and a hydraulic retention time (HRT) of 40 d. After thestart-up (day 0–19), the operation of the co-digesters can bedivided in three periods according to the feeding blend, HRT andOLR applied. In period I (days 20–59), the reactors were fed witha mixture of PM and FW (90:10, w/w) at OLR of 1 g COD/L d andHRT of 35 d. In period II (days 60–115), the percentage of FWwas decreased to 5% and the OLR and HRT were increased to1.5 g COD/L d and decreased to 30 d, respectively. In addition, threepulses of BW (5 g COD/L) were added to the reactors on days 80, 90and 100. In the last period (period III, days 116–200), FW wasreplaced by BW in order to prevent ammonia inhibition, the OLRwas increased to 2 g COD/L d and the HRT was reduced to 25 d.In all periods, the feeding mixture was diluted with tap water toattain the proper OLR.

The start up of the only-PM digester with OLR of around 0.5COD/L d and HRT of 20 d took 36 days. In period I (days 37–89),the OLR was increased up to 1 g COD/L d and the HRT was slightlylowered to 18 d in order to reach the target OLR, since the COD ofPM was lower in this period. The OLR was further increased to 1.5and 2 g COD/L d and the HRT was decreased to 15 and 12 d inperiods II (days 90–104) and III (days 105–150), respectively. Sincedifferent batches of PM were used along the experiment (withdifferent COD concentrations), dilution with tap water was notalways required to attain the established OLR.

2.4. Analytical methods

pH, COD, total solids (TS), volatile solids (VS), total suspendedsolids (TSS), volatile suspended solids (VSS), total Kjeldahl nitrogen(N-TKN), ammonium (N–NH4

+), TA (total alkalinity) and PA(partial alkalinity) were performed following standard methods(APHA, 1995). Biogas production was measured online by Rittermilligascounters (Dr. Ing. Ritter Apparatebau GmbH, Bochum,Germany) and biogas composition was analyzed by gaschromatography (HP, 5890 Series II). VFA (acetic, propionic,i-butyric, n-butyric, i-valeric and n-valeric) were analyzed by gas

L. Regueiro et al. / Bioresource Technology 123 (2012) 507–513 509

chromatography (HP, 5890A) equipped with a Flame IonizationDetector (HP, 7673A). Total lipids content was determinedusing the standard Soxhlet method (APHA, 1995) and proteinconcentration was calculated from the organic nitrogen content.Carbohydrates were estimated as the remaining fraction of VS afterproteins and lipids were subtracted. FA concentration wascalculated using the NH4

+–NH3 equilibrium constant (Ka),N–NH4

+ concentration (g/L), pH and temperature (T, �C), accordingto equations 1 and 2 (Cuetos et al., 2008):

NH3 ¼NHþ4

1þ 10ðpKa�pHÞ ð1Þ

pKa ¼ 4 � 10�8T3 þ 9 � 10�5T2 � 0:0356Tþ 10:072 ð2Þ

2.5. Fluorescent in situ hybridization (FISH)

Active bacterial and archaeal populations were identified andsemi-quantified by the FISH technique. Fresh biomass sampleswere collected from the reactors on days 70, 90, 110 and 200,disrupted and fixed with 4% paraformaldehyde solution accordingto the procedure described by Amann et al. (1995). Hybridizationwas performed at 46 �C for 90 min adjusting the formamideconcentrations for each probe. The probes used were: Eub338mix(Bacteria), Arc915 (Archaea), Ms821 (Methanosarcina), Mx825(Methanosaeta). All the details of each probe (formamidepercentage, sequence and target organism) can be found in theprobeBase database (http://www.microbial-ecology.net/probebase/).All probes were 50 labeled with the fluorochromes FITC and Cy3.Fluorescence signals were recorded with an acquisition system(Coolsnap, Roper Sicientific Photometrics) coupled to an Axioskop2 epifluorescence microscope (Zeiss, Germany). DAIME program(Daims et al., 2006) was used to make the semi-quantitativecounting with at least six photos taken per 20 lL of fixed sample(108–109 cells per mL).

3. Results and discussion

3.1. Wastes characterization

Table 1 shows the physico-chemical characterization of thesubstrates (PM, FW and BW) used in this study. The threesubstrates had neutral pH values (between 6.9 and 7.3). PM hadlow TS (17 g/kg), VS (12 g/kg) and COD (30 g/kg) levels comparedto FW (370 g TS/kg, 270 g VS/kg and 410 g COD/kg) and BW

Table 1Physico-chemical characteristics (average values) of pig manure, fish waste andbiodiesel waste.

Parameter Pig manurea Fish waste Biodiesel waste

pH 6.9 ± 0.2 7.1 7.3Density (kg/L) 1.0 ± 0.0 1.1 1.0TS (g/kg) 17.3 ± 4.5 369 950VS (g/kg) 11.7 ± 5.3 270 938CODtotal (g O2/kg) 29 ± 12 410 1390CODsoluble (g O2/kg) 15.3 ± 7.1 – –N-TKN (g N/kg) 3.3 ± 0.6 34 0.2N–NH4

+ (g N/kg) 3.1 ± 0.4 0.7 0Total alkalinity (g CaCO3/kg) 7.7 ± 1.3 0.4 0Proteins (g/kg) 1.1 ± 0.2 206 1.2Lipids (g/kg) 1.5 ± 0.3 28 77.3Carbohydrates (g/kg) 9.2 ± 3.8 36 922COD/N ratio 8.9 ± 1.3 12.2 7315

TS, total solids; VS, volatile solids; COD, chemical oxygen demand; N-TKN, totalKjeldahl nitrogen; N–NH4

+, ammonium.a Standard deviations are only shown for pig manure since several batches of this

substrate were used along the experiment.

(950 g TS/kg, 940 g VS/kg and 1390 g COD/kg), but high alkalinity(7.7 g/kg). As expected, the highest nitrogen concentration wasfound in FW (34 g N-TKN/kg), while it was negligible in BW. Incontrast, BW had a high content of lipids (77.3 g/kg) and of easilybiodegradable carbohydrates (almost 925 g/kg). In summary, PMprovides moisture and alkalinity, FW provides mainly nitrogenfrom proteins and also lipids but in a minor proportion (mainlyfrom fishbone fiber and cartilage) and BW provides easilydegradable COD (carbohydrates) and lipids.

3.2. Continuous pig manure anaerobic co-digestion

Two co-digesters were operated for 200 days at identicalconditions (duplicates). The performance of the two co-digesterswas very comparable, and therefore, only data from one co-digester are shown in figures and tables.

pH and biomass concentrations remained at 7.2–8.2 and5–7 g VSS/L, respectively, during the whole experiment (data notshown). Fig. 1 shows the performance of one co-digester in termsof OLR and biogas production (Fig. 1A), COD concentrations ininfluent and effluent (Fig. 1B), VFA levels (Fig. 1C) and ammoniumand FA concentrations (Fig. 1D).

At the end of the start-up period (day 19), the biogas productionreached 0.22 L/L d (Fig. 1A), with methane content of 50%, and theCOD removal efficiency was around 61%. During this period, aceticacid was consumed, but propionic acid remained in values around800 mg/L (Fig. 1C). This was probably due to the relatively highvolumetric percentage of BW in the feeding mixture (11%), sinceBW is considered as rapidly biodegradable organic substrate,which generates large amounts of VFA (Astals et al., 2011). Severalauthors (Amon et al., 2006; Astals et al., 2011) have shown that4–6% (w/w) of BW in the feeding is considered to be the limitingconcentration to maintain a stable anaerobic digestion process.Moreover, the propionic to acetic acid ratio at the end of thestart-up was about 3.8 and it has been reported that a propionicto acetic acid ratio greater than 1.4 is a hint of immediate digesterfailure (Pullammanappallil et al., 2001). Thus, in period I, BW waseliminated from the feeding mixture.

In the first days of period I (days 20–30), when the co-digesterwas fed with a mixture of PM and FW (90:10, w/w), the VFAcontent decreased to less than 0.2 g/L, despite the OLR doublingi.e. from 0.5 to 1 g COD/L d. This fact suggests that the eliminationof BW from the feeding mixture had a satisfactory effect on processstability. Consequently, the biogas production and the CODremoval increased to 0.43 L/L d and 70%, respectively. But fromday 30 on, ammonium concentrations started rising (Fig. 1D) as aresult of the digestion of the proteins present in FW and the highammonium levels in PM (3 g N–NH4

+/kg, Table 1). Althoughno accumulation of VFA was observed (Fig. 1C), total CODconcentrations in the effluent increased from 9 to 11.5 g/L(Fig. 1B) and biogas production decreased to 0.25 L/L d (Fig. 1A)by the end of this period. To attenuate the ammonium accumula-tion, the percentage of FW in the feeding mixture and the HRTwere decreased in period II in order to promote ammonium washout. As expected, ammonium levels decreased during the first daysof period II, but from day 80 on, they increased again (Fig. 1D). Thechange in the PM stock (with higher ammonium concentrations)combined with the higher OLR applied in this period (1.5 gCOD/L d, Table 2) provoked this increase in the ammonium levelsin the reactors.

In period II, three pulses of BW (5 g COD/L) were performed ondays 80, 90 and 100, following the strategy proposed by Cavaleiroet al. (2009) to achieve efficient methane yields from lipid-richsubstrates. This strategy consists of the combination of lipid-richsubstrate feeding periods (LCFA accumulation) with non-feedingperiods (degradation of accumulated LCFA). These authors

Fig. 1. Organic loading rate (OLR) and biogas production (A), total influent (CODin) and effluent (CODef) chemical oxygen demand (BW pulses in period II were not consideredin influent COD (CODin)) (B), volatile fatty acids (VFA) concentration (C) and ammonium and free ammonia (FA) concentrations (D) in one co-digester. Arrows in Fig. 1Aindicate BW pulses of 5 g COD/L on days 80, 90 and 100.

Table 2Operational parameters of the lab-scale anaerobic co-digester and only-pig manure digester during the different operational periods (values shown corresponded to steady-stateconditions of each period).

Type reactor Period PM-FW-BW (% w/w)a Duration (days) OLR (g COD/L d) HRT (d) pH CH4 (%) COD removal (%) Biogas (L/L d)

Co-digester Start-up 84–5-11 0–19 0.5 40 7.4 ± 0.2 50 ± 2 60.8 ± 0.3 0.22 ± 0.01Period I 90–10-0 20–59 1.0 35 7.6 ± 0.3 57 ± 3 69.6 ± 1.2 0.43 ± 0.04Period II 95–5-0 60–115 1.5 30 8.0 ± 0.2 59 ± 2 65.7 ± 0.8 0.59 ± 0.05Period III 95–0-5 116–200 2.0 25 7.7 ± 0.2 60 ± 3 78.5 ± 0.7 0.91 ± 0.06

Digester Start-up 0–36 0.6 20 7.6 ± 0.2 56 ± 4 40.2 ± 0.4 0.15 ± 0.02Period I 37–89 1.0 18 7.9 ± 0.2 52 ± 7 37.5 ± 0.9 0.25 ± 0.03Period II PM 90–104 1.5 15 8.0 ± 0.2 52 ± 5 40.1 ± 2.0 0.40 ± 0.01Period III 105–150 2.0 10 8.1 ± 0.1 55 ± 6 52.6 ± 3.7 0.67 ± 0.10

a w/w: wet weight.

510 L. Regueiro et al. / Bioresource Technology 123 (2012) 507–513

reported that at least two cycles of accumulation-degradation ofLCFA were necessary before continuous operation The positiveeffect of BW pulses on biogas production was clear (Fig. 1A), but

the % of pulse COD converted into methane in relation to themaximum theoretical methane production (taking into account0.35 m3 CH4/kg CODconverted) decreased with each consecutive

L. Regueiro et al. / Bioresource Technology 123 (2012) 507–513 511

pulse (52.1%, 26.8% and 24.2%), probably as a result of the increasein VFA levels after the first pulse addition (Fig. 1C). The majorcomponents of the VFAs were acetic and propionic acid, whichreached values up to 2.5 g/L and 2.0 g/L, respectively. The increasedVFA levels also resulted in higher effluent COD concentrations(Fig. 1B), and consequently, the COD removal efficiency decreasedfrom 70% (before pulses) to 66% (after pulses). By the end of periodII, VFA and the COD levels lowered, but did not reach the originalvalues (Fig. 1B and C), indicating that organic compounds relatedto BW, such as alcohols or LCFA, were accumulated into theco-digesters. In contrast, the biogas production was not inhibited,suggesting that the degradation of PM:FW mixture was notaffected and just the COD from pulses was accumulated.

By the end of period II, the ammonium levels were stillhigh (2 g N–NH4

+/L), which combined with high pH values (around7.8, data not shown) resulted in FA levels around 260 mg N–NH3/L,which might be inhibitory for anaerobic communities (Chen et al.,2008). Furthermore, FA levels were much more dependent on pHvariations than on the ammonium concentration, because betweendays 106 and 112, FA increased from 125 to 260 mg N–NH3/L,when the pH varied from 7.8 to 8.1, despite the decrease ofthe ammonium levels from 2.2 to 2 g N–NH4

+/L (Fig. 1D).Consequently, FW was replaced by BW in period III. In this case,the percentage of BW in the mixture (5%) did not exceed the 6%that Amon et al. (2006) set as maximum for a good performance.Besides, HRT was decreased to 25 d in order to promoteammonium washout. These changes led to a drop in ammoniumand FA concentrations, which stabilized at 1300 mg N–NH4

+/Land 100 mg N–NH3/L, respectively, by the end of the experiment(Fig. 1D). In period III, biogas production increased as a conse-quence of the higher OLR applied (Fig. 1A). However, from day130, it went down due to the accumulation of propionic acid(Fig. 1C), resulting on 0.3 L/L d on day 150. To surpass this inhibi-tion, the co-digesters were not fed between days 151 and 160 andpropionic acid was consumed giving rise to a recovery in biogasproduction rates. After this event, the co-digesters operated stablyfor 40 days, with biogas production rates of 0.9 L/L d and CODremoval efficiencies of almost 80%, which emphasizes the highbiodegradability of BW.

The microbiology of the co-digesters in the last two operationalperiods was studied with the FISH technique (Supplementarymaterial (SM), Fig. SM 1). A rough estimation of the relative sharesof each main population indicated that half of the active popula-tion was Archaea and the other half was Bacteria. This ratio didnot vary, even when the feeding mixture changed as happenedbetween days 100 and 120. The relative abundance of archaealpopulations (Methanosaeta and Methanosarcina) present wascorrelated with the co-digester performance, particularly withVFA concentrations. Before the pulses (day 70), the predominantarchaeal population was Methanosaeta (Fig. SM-1A), demonstratedby the presence of the characteristic shape of this population intubular sheath. Methanosaeta are well known as the mostabundant acetoclastic methanogens in bioreactors with low VFAand ammonium levels (Karakashev et al., 2008), as occurred beforethe pulses. After the three pulses (day 90), this populationdisappeared almost completely and the niche was filled byMethanosarcina, which displayed the individual coccoid cells shapeforming aggregates similar to a bunch of grapes (Fig. SM-1B).Methanosarcina is able to use both the acetoclastic and thehydrogenotrophic pathways, thus being more tolerant to methano-genesis inhibitors compared to Methanosaeta (Liu et al., 2011). Itspresence in anaerobic reactors is often associated with thedeterioration of reactor performance (Blume et al., 2010; Horiet al., 2006). Consequently, the appearance of this archaeal popula-tion is probably related to the VFA accumulation that occurredbetween days 85 and 110 (Fig. 1C). On day 120 (Fig. SM-1C), when

the co-digester was recovering from inhibition (VFA concentrationwas decreasing), the two methanogenic populations coexisted inthe reactor. At the end of the experiment (day 200), when theco-digester showed a good stable performance, the methanogenicpopulation was again mainly Methanosaeta (Fig. SM-1D). Thesevariations in Methanosaeta and Methanosarcina populations weresemi-quantified by using the probes Mx825 and Ms821, respec-tively. Methanosaeta fractions (with respect to total archaealpopulation) were 95%, 0%, 45% and almost 100% on days 70, 90,110 and 200, respectively, while those of Methanosarcina were0%, 100%, 40% and 0%, respectively.

3.3. Continuous pig manure anaerobic digestion

pH and biomass concentrations remained at 7.4–8.3 and 5–8g VSS/L, respectively, during the whole experiment (data notshown). Fig. 2 shows the results of the operation of the reactortreating PM as a sole substrate. During the start-up (day 0–36), bio-gas production stayed at around 0.15 L/L d (Fig. 2A) with methanecontent of 56%, leading to average COD removal efficiencies of 40%(Fig. 2B). VFA concentrations were negligible (Fig. 2C) and ammo-nium levels remained below 1.2 g N–NH4

+/L (Fig. 2D). The increasein the OLR from 0.6 to 1 g COD/L�d in period I derived in greaterbiogas production rates (0.25 L/L d, methane content of 52%), butthe COD removal efficiencies remained at lower values (around38%). The accumulation of ammonium observed at the end of thestart-up continued and stabilized at around 3 g N–NH4

+/L(Fig. 2D). The latter combined with the accumulation of VFA be-tween days 65 and 80 (Fig. 2C) provoked a drop on the biogas pro-duction, and consequently, on the COD removal efficiencies(Fig. 2B). Although the reactor recovered quite quickly from VFAaccumulation, ammonium concentrations were still high(Fig. 2D), and therefore, the HRT was decreased to 15 days in per-iod II. This modification combined with a higher COD content inPM yielded an increase in the OLR to 1.5 g COD/L d. Consequently,biogas production and COD removal efficiencies increased to 0.40L/L d and 40%, respectively. Since ammonium levels did not de-crease, the HRT was further lowered to 10 days on day 115 (periodIII). Despite the fluctuations in the OLR (1.6–2.2 g COD/L d, Fig. 2A)caused by variations in COD content of PM, biogas productionremained quite constant at 0.7 L/L d (Fig. 2A). By the end of theexperiment, VFA concentrations were negligible (Fig. 2C), butammonium and FA stayed at around 2.5 g N–NH4

+/L and250 mg N–NH3/L, respectively (Fig. 2D).

3.4. Improved pig manure anaerobic digestion by using FW and BW

Table 2 shows a comparison of the results obtained in theco-digester and in the only-PM digester at steady-state conditionsduring each operational period. Operating at the same OLR, thebiogas production and methane content in the co-digester werehigher than in the only-PM digester. More specifically, biogasproduction was improved by 47%, 72%, 48% and 36% at OLR of0.5, 1, 1.5 and 2 g COD/L d, respectively. Moreover, the CODconverted into methane was much higher in the co-digestionprocess, reaching values near to 79% (period III), while in thedigestion process the maximum value was 53% (period III).

An additional advantage of the co-digestion process was theammonium and FA control in the reactors. The highest valuesachieved in the co-digester (2 g N–NH4

+/L and 250 mg N–NH3/L)were around 1.5-fold lower than those in the only-PM digester(3.2 g N–NH4

+/L and 320 mg N–NH3/L). Although anaerobicmicroorganisms can adapt to relatively high FA levels, somestudies suggested that microorganisms adapted to the anaerobicdigestion of PM were inhibited at FA concentrations around0.7–1.1 g N–NH3/L (Nielsen and Angelidaki, 2008).

Fig. 2. Organic loading rate (OLR) and biogas production (A), total influent (CODin) and effluent (CODef) chemical oxygen demand (B), volatile fatty acids (VFA) concentration(C) and ammonium and free ammonia (FA) concentrations (D) in the pig manure anaerobic reactor.

512 L. Regueiro et al. / Bioresource Technology 123 (2012) 507–513

The main problems associated to FW and BW were ammoniumand VFA accumulation, respectively. The first can be surpassed byworking at low OLR as in period II (around 1.5 g COD/L d) and/or atshorter HRT to promote ammonium washout, and the second onecan be solved by adapting the feeding strategy of BW. In this study,two alternatives were evaluated: pulses in period II and continuousoperation in period III. Not good results were obtained with thepulses, probably due to their frequency and COD concentration,while the continuous operation with BW percentages below 6%in the feeding mixture derived a stable performance with goodbiogas production (0.91 L/L d), which is consistent with resultsobtained by other authors (Astals et al., 2012; Robra et al., 2010).

4. Conclusions

Anaerobic co-digestion of PM with FW or BW evidenced anupgrade with respect to sole PM digestion, not only in terms ofincreased biogas production, but also on process stability. A

protein-rich substrate (FW) was more problematic to co-digestwith PM due to their similar characteristics (high nitrogencontent), but good results were achieved by keeping the FW inthe feeding mixture below 10%. The lipid-rich substrate (BW)helped to decrease ammonium concentrations in the reactor, butprovoked VFA accumulation events, which could be solved byadjusting the BW fraction in the feeding mixture (<6%) or applyinga fed-batch strategy.

Acknowledgements

This research was supported by the Spanish Ministry ofEconomy and Competitiveness, through NOVEDAR_Consolider(CSD2007-00055) and COMDIGEST (CTM2010-17196) projects,and by the Xunta de Galicia through GRC2010/37 project and thepostdoctoral contract to Dr. Marta Carballa (Isidro Parga Pondal,IPP-08-37). The authors are also grateful to Laura Otero andPatricia Veiga for their experimental work.

L. Regueiro et al. / Bioresource Technology 123 (2012) 507–513 513

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.biortech.2012.07.109.

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