A

hwfacaTuf©

K

1

aws

h(wpoaau

1d

Biochemical Engineering Journal 40 (2008) 99–106

Anaerobic digestion of solid slaughterhouse waste (SHW) at laboratoryscale: Influence of co-digestion with the organic fraction

of municipal solid waste (OFMSW)

Marıa Jose Cuetos a, Xiomar Gomez a, Marta Otero b, Antonio Moran a,∗a Institute of Natural Resources (IRENA), Avda. de Portugal 41, University of Leon, 24071 Leon, Spain

b Centre for Environment and Marine Studies (CESAM), Department of Chemistry, University of Aveiro,Campus Universitario de Santiago, 3810-193 Aveiro, Portugal

Received 26 September 2006; received in revised form 21 November 2007; accepted 25 November 2007

bstract

Mesophilic anaerobic digestion of slaughterhouse waste (SHW) and its co-digestion with the organic fraction of municipal solid waste (OFMSW)ave been evaluated. These processes were carried out in a laboratory plant semi-continuously operated and two set-ups were run. The first set-up,ith a hydraulic retention time (HRT) of 25 days and organic loading rate (OLR) of 1.70 kg VS m−3 day−1 for digestion, and 3.70 kg VS m−3 day−1

or co-digestion, was not successful. The second set-up was initiated with an HRT of 50 days and an OLR of 0.9 kg VS m−3 day−1 for digestionnd 1.85 kg VS m−3 day−1 for co-digestion. Under these conditions, once the sludge had been acclimated to a medium with a high fat and ammoniaontent, it was possible to decrease the HRT while progressively increasing the OLR to the values used in the first set-up until an HRT of 25 days

−3 −1

nd OLRs of 1.70 and 3.70 kg VS m day , for digestion and co-digestion, respectively (the same conditions of the digesters failures previously).hese digesters showed a highly stable performance, volatile fatty acids (VFAs) were not detected and long chain fatty acids (LCFAs) werendetected or only trace levels were measured in the analyzed effluent. Fat removal reached values of up to 83%. Anaerobic digestion was thusound to be a suitable technology for efficiently treating lipid and protein waste.

2007 Elsevier B.V. All rights reserved.

id slau

eapaad

ddtT(

eywords: Anaerobic processes; Biogas; Co-digestion; Process inhibition; Sol

. Introduction

Anaerobic digestion of organic matter has been reported aswidely used technology in the efficient treatment of organicaste and the simultaneous production of a renewable energy

ource through the use of biogas [1–3].Slaughterhouses generate meat and products marketed for

uman consumption, pollutant solid waste and other by-productsskins, fats, and bones), as well as substantial volumes ofastewater as a result of cleaning operations. Animal by-roducts are all bodies or parts of animals and products of animalrigin not intended for human consumption, either because they

re not fit for human consumption or there is no market for thems foodstuff [4]. Consumer demand for meats with a low unsat-rated fat content in order to reduce cholesterol has led to the

∗ Corresponding author. Tel.: +34 987 291841; fax: +34 987 291839.E-mail address: amoran@unileon.es (A. Moran).

ebmbacL

369-703X/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.bej.2007.11.019

ghterhouse waste; Waste treatment

xpansion of the poultry industry and hence to an increase in lipidnd protein waste. This waste causes important environmentalroblems as a result of organic pollution and microbial loads,nd the increasing problems of its removal must be addressed asresult of legislative constraints and the cost of treatment andisposal.

Slaughterhouse waste is an ideal substrate for anaerobicigestion and elimination of more than 90% chemical oxygenemand (COD) can be attained [5]. Lipids represent an impor-ant fraction of the organic charge in slaughterhouse waste [6].hey consist mainly of triglycerides and long chain fatty acids

LCFAs). Triglycerides may be hydrolyzed to LCFA and glyc-rol. Accumulation of LCFA may inhibit anaerobic digestion,ecause they are toxic for acetogens and methanogens, the twoain groups involved in LCFA degradation [7–9]. Another inhi-

ition mechanism is the result of the adsorption of the surfacective acids onto the cell wall [10,11], thus affecting the pro-esses of transportation and protection. The unionized form ofCFA adsorbs initially to the microbial cell surface and is then

1 ngine

tcapLl

ocnaottmdbap

mtnlcpita

iifehblc

a(orocwti

2

2

fTp

wf−aampt

awdpfsda

wadTdiSos

wrTittdcsaADsle

ooSvac

2

00 M.J. Cuetos et al. / Biochemical E

aken up into the cell [9]. LCFA are beta-oxidized to acetate,arbon dioxide and hydrogen in anaerobic digestion [12] whichre then converted to biogas. What normally occurs during therocess is rapid hydrolysis/acidogenesis, with accumulation ofCFA and volatile fatty acids (VFAs), removal of LCFA, fol-

owed by removal of VFA and methane production [7,13].Inhibition of the anaerobic digestion of waste with a high

rganic content is usually also caused by high ammonia con-entrations [1,14], produced in the degradation of proteins fromitrogen-rich slaughterhouse waste [15]. Poultry by-productsre rich in nitrogen because they may contain high proportionsf non-digester material, especially if the birds are fed prioro slaughtering [16]. Two different mechanisms are attributedo ammonia inhibition of methanogens. According to the first

echanism, activities of methane synthesizing enzymes areirectly inhibited by free ammonia. In the second, hydropho-ic free ammonia molecules diffuse passively into the cell andre rapidly converted to ammonium owing to the intracellularH conditions [17].

In studies carried out on this type of waste, co-digestion ofixtures is employed to stabilize the feed to the reactor [18,19],

hus improving the C/N ratio and decreasing the concentration ofitrogen, which in certain cases may produce inhibition prob-ems. The use of a co-substrate with a low nitrogen and lipidontent increases the production of biogas. Alternatively, two-hase digestion systems may be employed [15,20], with whichmprovements are achieved in process efficiency, in the reduc-ion of solids, the removal of chemical oxygen demand (COD)nd the conversion of biogas.

It should not be forgotten that municipal solid waste (MSW)s another type of residue that is affronting more restrictive leg-slation with respect to landfill disposal of the biodegradableraction [21,22]. Co-digestion of the mixtures of waste with oth-rs presenting a lower nitrogen and lipid content may result inigh methane yields due to the fact that the characteristics ofoth types of waste are complementary, thus reducing prob-ems associated with the accumulation of intermediate volatileompounds and high ammonia concentrations [23].

The aim of the present study was to carry out the biometh-nization (anaerobic digestion) of both slaughterhouse wasteSHW) and mixtures of solid slaughterhouse waste with therganic fraction of municipal solid waste (OFMSW) at labo-atory scale in mesophilic semi-continuously fed digesters. Theperational conditions needed to achieve a stable digestion pro-ess were determined. At the same time, the results obtainedere compared, assessing the contribution of the organic frac-

ion of municipal solid waste to the slaughterhouse waste richn lipids.

. Materials and methods

.1. Experimental set-up

The inoculum used for starting up the digesters was obtainedrom the wastewater treatment plant of the city of Leon (Spain).he slaughterhouse waste was collected at the Huevos Leonoultry slaughterhouse in the same city. The entrails together

2

i(

ering Journal 40 (2008) 99–106

ith the contents of the stomach and intestines were collectedrom eviscerated poultry. They were ground and frozen at18 ◦C until subsequent use. Given the difficulty of obtainingrepresentative sample of OFMSW, a simulated OFMSW withparticle size of less than 3 mm was used as substrate. Thisixture contained fruit and vegetable waste and followed the

roportions reported in previous studies [22,24]. This waste washen stored at 4 ◦C until used.

Two feeds were prepared for the study being periodicallynalyzed to always ensure the same solids content. A SHW feedas prepared by diluting the sample with distilled water to theesired feed load; the SHW:H2O ratio being 1:5 in weight. Theercentages of total solids (TS) and volatile solids (VS) of thiseed were 4.7 and 4.3%, respectively. A combined mixture withlaughterhouse waste was prepared with an SHW:OFMSW co-igestion ratio of 1:5 in weight. The mix as prepared had a TSnd VS percentages of 10 and 9.3%, respectively.

The digestion of SHW and its co-digestion with OFMSWas carried out in two completely mixed stirred digesters, withworking volume of 3 L and thermostatized at 34 ± 1 ◦C. Bothigesters had a side inlet via which the systems were fed daily.wo set-ups were implemented in order to run experiments underifferent operational conditions. In the nomenclature used todentify digestion systems letter A refers to the digester treatingHW, and letter B to the one treating the mixture. A number (1r 2) was added according to conditions imposed in the first orecond set-up.

In the first set-up of two reactors Digesters A1 and B1ere operated at an OLR of 1.7 and 3.7 kg VS feed m−3 day−1,

espectively, both at a hydraulic retention time (HRT) of 25 days.he start-up of these digesters was performed applying this feed-

ng rate from day 1 of experimentation to reactors loaded withhe inoculum previously described. In the second set-up the ini-ial HRT of the digesters was 50 days. This HRT was selectedue to the difficulty of treating waste with a high lipid and proteinontent in accordance with other successful anaerobic digestiontudies of SHW, where the HRT are not as low as in the case ofnaerobic digestion of other organic wastes [25,26]. Digester2 operated at a loading of 0.90 kg VS feed m−3 day−1, andigester B2 at a loading of 1.85 kg VS feed m−3 day−1. Sub-

equently, the HRT was successively reduced and the organicoading was gradually increased in accordance with the param-ters shown in Table 1.

For each experimental condition, the reactors were continu-usly operated for two consecutive HRTs to ensure a situationf steady state before changing their operational conditions.teady state was defined as the situation where the coefficient ofariation for daily gas production was less than 10% [27,28]nd this coincides with a stable effluent volatile fatty acidsoncentration.

.2. Analysis

.2.1. Routine analysesThe following parameters were monitored periodically dur-

ng the digestion process: pH, total solids (TS), volatile solidsVS), alkalinity, chemical oxygen demand (COD), ammonia,

M.J. Cuetos et al. / Biochemical Engineering Journal 40 (2008) 99–106 101

Table 1Digester operational parameters in the second reactor set-up carried out

Digester Description Days HRT (days) Loading rate (kg VS feed m−3 day−1)

A2 SHW digestion 0–100 50 0.90A2 SHW digestion 101–175 36 1.16A2 SHW digestion 176–225 25 1.70B 0B 5B 5

yota

aM

E

F

p

wgT

Hha

w

Gi8ut(hoa

st(ga1dwSpfi

2

Btm[mbC

lK[

cem3fit(iamtwwllsSigma.

The main properties of the original waste are given in Table 2.At it may be seen the used SHW and OFMSW wastes havedifferent properties. While SHW has lower organic carbon (%),

Table 2Characteristics of the original waste and the inoculum used

Parameters OFMSW SHW Inoculum

Total organic carbon (%) 42.7 ± 0.7 23.1 ± 0.4 23.6 ± 0.7Organic matter (%) 73.4 ± 0.9 39.6 ± 0.9 31.3 ± 0.7Total nitrogen (%) 1.3 ± 0.4 6.2 ± 0.3 4.2 ± 0.4

2 SHW + OFMSW co-digestion 0–102 SHW + OFMSW co-digestion 101–172 SHW + OFMSW co-digestion 176–22

ield and composition of the biogas produced and concentrationf volatile fatty acids (VFAs). All these variables were measuredwice a week, except for ammonia, which was monitored onceweek, and gas production, which was daily measured.

The analyses of pH, total and volatile solids (TS and VS),lkalinity and ammonia were carried out according to Standardethods [29].The concentration of free ammonia was calculated based on

qs. (1) and (2) [1,17]:

A = TAN

1 + 10(pKa−pH) (1)

Ka = 0.09018 + 2729.92

T + 273.15(2)

here FA is the free ammonia, TAN is the total ammonia nitro-en, pKa is the dissociation constant for the ammonium ion andis the temperature in ◦C.The chemical oxygen demand (COD) was determined using a

anna Instruments Series C99 multi-parameter photometer. Theomogenized sample was digested in the presence of dichromatet 150 ◦C for 2 h in a Hanna C9800 reactor.

Daily gas production was measured using a reversible deviceith liquid displacement with a wet-tip counter.Biogas composition was analyzed using a Varian CP-3800

C gas chromatograph equipped with a thermal conductiv-ty detector (TCD). A 4 m long column packed with Hayesep0/100 Mesh followed by a 1 m long Molecular Sieve col-mn 13× 80/100 Mesh (1.0 m × 1/8 in. × 2.0 m) were usedo separate methane (CH4), carbon dioxide (CO2), nitrogenN2), hydrogen (H2) and oxygen (O2). The carrier gas waselium and the columns operated at 331 kPa at a temperaturef 50 ◦C. Standard 234 from Supelco was used to calibrate thepparatus.

Volatile fatty acids (VFAs) were determined on theame gas chromatograph, using a flame ionization detec-or (FID) equipped with a Nukol capillary column30 m × 0.25 mm × 0.25 �m) from Supelco. The carrieras was helium. Injector and detector temperatures were 220nd 250 ◦C, respectively. The oven temperature was set at

◦ ◦

50 C for 3 min and thereafter increased to 180 C. Theetection limit for VFA analysis was 5.0 mg L−1. The systemas calibrated with a mixture of standard volatile acids fromupelco (for the analysis of fatty acids C2–C7). Samples werereviously centrifuged (10 min, 3500 × g) and the supernatantltrated through 0.45 �m cellulose filters.

CTTTV

N

50 1.8536 2.5625 3.70

.2.2. Other analyses performedThe original waste and digestates were characterized.

iodegradability analysis was carried out, total nitrogen concen-rations were determined by the Kjeldahl method [30], organicatter was analyzed according to the Walkey-Black method

30], and organic carbon was determined considering an organicatter content/organic carbon ratio of 1.7241. The organic car-

on was subsequently divided by the total nitrogen to obtain the/N ratio.

The total lipid content was determined using a Standard Soxh-et method [29], and the protein content was calculated from thejeldahl-N content using a conversion factor of 6.25 (for meat)

31].Gas chromatography was used for the analysis of the long

hain fatty acids (LCFAs). Samples for LCFA analysis werextracted as described by Fernandez et al. [32]. Samples wereixed with n-heptane, the solution was then centrifuged for

0 min at 3500 × g and filtrated through 0.45 �m celluloselters. The sample was injected into a Perkin-Elmer AutoSys-

em XL chromatograph equipped with an HP Innowax column30 m × 0.25 mm × 0.25 �m). The carrier gas was helium. Thenitial oven temperature of 120 ◦C was maintained for 1 min,nd then increased to 250 ◦C, with a ramp of 8 ◦C min−1,aintaining this temperature for 7 min. Injector and detector

emperatures were 250 and 275 ◦C, respectively. The systemas calibrated using a mixture of LCFA from individual acidsith concentrations in the range of 0–100 mg L−1. The detection

imit for LCFA analysis was 5.0 mg L−1. The acids ana-yzed were lauric (C12:0), myristic (C14:0), palmitic (C16:0),tearic (C18:0), oleic (C18:1) and linoleic (C18:2), all from

/N ratio 32.1 ± 1.2 3.7 ± 0.4 5.5 ± 0.4otal fat (%) 0.7 ± 0.4 40.5 ± 0.5 NAotal protein (%) 8.3 ± 0.7 38.9 ± 0.9 NAS (%) 6.4 ± 0.2 28.3 ± 0.3 4.1 ± 0.4S (%) 6.0 ± 0.2 26.0 ± 0.5 2.9 ± 0.4

A: not analyzed.

1 ngine

o(h

3

tlda[

3

dtoftrt

ptimstab

sap

r

aimi[fIia

irrAiftdoflfid

Fr

02 M.J. Cuetos et al. / Biochemical E

rganic matter (%) and C/N than OFMSW, its total nitrogen%), fat (%), protein (%) and both TS (%) and VS (%) are muchigher.

. Results and discussion

Anaerobic digestion of SHW and anaerobic co-digestion ofhe mixtures of SHW with the OFMSW were carried out ataboratory scale at 34 ± 1 ◦C. Two set-ups were operated underifferent conditions to study the effect of the variation in HRTnd organic loading, key parameters in the digestion of SHW9,33].

.1. First reactor set-up

Gas production commenced immediately after loading theigesters, but after a few days of functioning (around day 10 ofhe study in Digester A1, and day 15 in Digester B1), the volumef biogas decreased progressively (Fig. 1). This decrease wasollowed by a drop in methane production from values of 65%o values of below 45% in both reactors. From this point on, aeduction in pH was observed decreasing from values of 7 to 7.5o below 6.5.

Fig. 1 shows that there is a significant decrease in daily gasroduction in correlation with an increase in VFA concentra-ion in the effluents. Acetic acid was the most abundant VFAn Digester A1, mainly causing inhibition of acetate consuming

ethanogens. Furthermore, the concentrations of LCFA mea-

ured in the effluents after dismantling the digesters were high;he total LCFA content of Digester 1 was 4300 mg L−1 andlmost double for the co-digestion reactor (Digester B1). Inoth cases, oleic was the most abundant acid, which alone con-

r2tc

ig. 1. Daily biogas and VS concentration in Digester A1 (A) and Digester B1 (B), aeactor set-up.

ering Journal 40 (2008) 99–106

tituted over 50% of the analyzed LCFA, followed by stearicnd linoleic acids, the lowest concentrations corresponding toalmitic, lauric and myristic acids (data not shown).

The data obtained greatly exceeded the concentrationseported as toxic by other authors [34–37].

Therefore, the low methane production rates may bettributed to the accumulation of VFA, intermediary compoundsn the metabolic pathway of methane fermentation, which cause

icrobial stress if present at high concentrations, thus result-ng in a decrease of pH and leading to failure of the digesters38,39]. VFA, along with LCFA, are potential inhibitors of theormation of methane in reactors, stopping the overall process.n view of the results obtained, and after 30 days of function-ng, it was decided to dismantle the failed reactors, without anyttempt to recover them.

VS removal remained around 70% in Digester A1 and 65%n Digester B1. However, during the period of time in which theeactors were working the VS concentrations measured in theeactors (Fig. 1) decreased from 15.0 to 6.0 g L−1 in Digester1 and from 13.0 to 5.5 g L−1 in Digester B1. Despite mechan-

cal stirring of the systems, it was not possible to prevent theormation of foam on the top of the reactors, thus demonstratinghe high tendency of slaughterhouse waste to a non-uniformistribution in the digesters. This agrees with the results ofther authors, who state that lipids have a tendency to formoating aggregates and foam that may cause problems of strati-cation [13,40]. The value of measured solids in the dismantledigesters, after homogenization of the floating layer with the

est of the content of the reactor, increased significantly to0.0 and 30.9 g L−1, respectively. Despite failure of the reac-ors, final fat removal reached values of 50 and 65% in eachase.

nd VFA concentrations in Digester A1 (C) and Digester B1 (D) during the first

ngine

cviooaitid

3

iAiHwct

f

tath

to

dcurt

itravvtraibtt

ao

TS

P

BCCTVVpATFV

L

CFSM

N

M.J. Cuetos et al. / Biochemical E

In both digesters, the final free ammonia concentration asonsequence of protein mineralization was 10.6 mg L−1. Thisalue is quite lower than those considered as inhibitory in stud-es published to date [41], in which free ammonia concentrationf 150–200 mg L−1 mark the limit of being inhibitory. More-ver, other authors [5,42] have reported that concentrations ofmmonium of up to 5–8 g L−1 may be tolerated by microorgan-sms if the pH is sufficiently high. These findings indicate thathe VFA and LCFA accumulation, and not the presence of Nn the system, was the fundamental cause of inhibition of theigestion process in the present study.

.2. Second reactor set-up

The short HRT selected for the start-up of the systems resultedn overloading and subsequent failure of the digestion process.

new approach was chosen in order to shorten the HRT, start-ng off from a greater value and progressively decreasing theRT while increasing the organic loading. This methodologyas an attempt to adapt anaerobic microorganisms to the new

onditions by pre-exposing them to non-inhibitory concentra-ions.

The main results corresponding to the steady-state periods,or different HRT and loading, are presented in Table 3.

To avoid problems of system instability, a new set of reac-

ors was assembled reducing the organic loading and employingn HRT of 50 days. The reactors were kept under the opera-ional conditions reported in Section 2 during two consecutiveydraulic retention times. Under these conditions, operation of

adio

able 3teady-state operational parameters for the periods studied of Digesters A2 and B2

arameters SHW

50 days 36 days 25

iogas (L day−1) 2.5 ± 0.1 3.4 ± 0.1 4.3H4 (%) 64.3 ± 2.2 65.6 ± 0.3 66.O2 (%) 35.7 ± 2.2 34.4 ± 0.3 33.S (g L−1) 16.1 ± 2.3 12.7 ± 1.9 13.S (g L−1) 10.1 ± 0.5 8.7 ± 0.8 8.7S removal (%) 76.7 ± 1.7 79.2 ± 2.1 79.H 7.6 ± 0.2 7.7 ± 0.2 7.8lkalinity (mg L−1) 6342.9 ± 258.2 8800.0 ± 400.0 934otal ammonia (mg L−1) 2143.3 ± 88.4 3022.2 ± 149.4 321ree ammonia (mg L−1) 115.0 ± 12.7 203.3 ± 48.8 270FA (mg L−1) ND ND ND

CFA (mg L−1)Lauric ND ND NDMyristic ND ND 5.4Palmitic ND 6.13 19.Linoleic 9.16 5.83 12.Oleic 12.98 ND 24.Stearic ND ND 13.

OD (mg L−1) 18798.6 ± 998.7 17116.0 ± 966.6 171at removal (%) 77.4 ± 1.3 61.1 ± 1.0 61.GP biogas (m3 kg−1 VS feed) 1.0 ± 0.2 1.0 ± 0.2 0.8ethane yield (m3 kg−1 VS feed) 0.6 ± 0.1 0.7 ± 0.2 0.6

D: not detected.

ering Journal 40 (2008) 99–106 103

he reactors was highly stable, a fact confirmed by the presencef stable pH, alkalinity and COD profiles (Fig. 2).

Levels of VFA of below 2.5 and 1.0 g L−1, respectively, wereetected at the commencement of digestion of the SHW ando-digestion with the OFMSW (Fig. 2). A short time after start-p, and without having completed one operational hydraulicetention time, no values of VFA were detected in either of thewo reactors (Fig. 3).

The average values of total ammonia during the steady staten the digesters with an HRT of 50 days (Table 3) were belowhe toxic limits reported in the literature [41,42]. However, theelease of ammonia from the proteins provoked an increase inlkalinity concentration of the system, leading to an averagealue for the period higher than 5000 mg L−1, the recommendedalue for standard rate sewage sludge digesters [43]. Due tohe high protein concentrations in the waste, pH values in theeactors were expected to be slightly higher than those reporteds suitable for the development of methanogenic microorgan-sms with another type organic substrate; pH values remainedetween 7.5 and 8.0. Therefore, as a result of the increase in pH,he free ammonia concentration is high, but not inhibitory forhe development of microorganisms.

Only trace concentrations (<13 mg L−1) of linoleic and oleiccids were detected (Table 3). At the same time, no accumulationf fats was detected in the reactors, fat removal of 77% was

chieved in Digester A2 and 81% in Digester B2, while VSestruction was 77 and 81%, respectively. Solids concentrationn the reactors remained constant, without any formation of foamr floating layers during functioning.

SHW + OFMSW

days 50 days 36 days 25 days

± 0.1 3.7 ± 0.1 5.6 ± 0.1 8.6 ± 0.12 ± 0.5 59.4 ± 4.1 61.9 ± 1.4 64.5 ± 2.68 ± 0.5 40.6 ± 4.1 38.1 ± 1.4 35.4 ± 2.65 ± 1.8 27.2 ± 0.7 21.8 ± 1.4 23.1 ± 2.2± 0.7 18.1 ± 0.1 16.1 ± 0.5 16.2 ± 0.50 ± 0.9 80.6 ± 1.0 82.6 ± 1.6 82.6 ± 1.6± 0.1 7.7 ± 0.1 7.8 ± 0.1 7.9 ± 0.12.8 ± 200.0 8120.0 ± 109.5 10600.0 ± 178.9 12166.7 ± 150.60.8 ± 103.9 2106.8 ± 59.2 3830.2 ± 73.8 4099.7 ± 53.9.2 ± 24.6 121.9 ± 4.8 237.9 ± 26.5 337.4 ± 24.9

ND ND ND

ND ND ND2 ND ND ND78 ND ND 6.0905 7.5 ND 8.2185 12.38 6.41 13.7569 ND ND 9.63

56.3 ± 758.6 36139.2 ± 153.4 26395.8 ± 502.9 27603.0 ± 472.61 ± 0.5 81.4 ± 0.4 81.4 ± 0.7 82.5 ± 0.5± 0.1 0.7 ± 0.1 0.7 ± 0.1 0.8 ± 0.1± 0.1 0.4 ± 0.1 0.5 ± 0.1 0.5 ± 0.1

104 M.J. Cuetos et al. / Biochemical Engine

Fs

wTftsiwasst

clmsc8

tHu3t

iiViot

wtfrrmoi

fbSa1boiVrtt[

ptwos

tmt

ig. 2. pH, COD, alkalinity and ammonium in Digesters A2 and B2 during theecond reactor set-up.

As the performance of these reactors was stable, their HRTsere reduced to 36 days, while gradually increasing in the OLR.he OLR was set at 1.16 kg VS feed m−3 day−1 for the reactor

ed with SHW (Digester A2) and 2.56 kg VS feed m−3 day−1 forhe reactor fed with SHW and OFMSW (Digester B2). Fig. 3hows that, at the beginning of this new period consisting of anncrease in organic loading and a decrease in HRT, the systemsere characterized by increases in the values of alkalinity and

mmonia as well as a decrease in COD values until reaching theteady state. Once a period equivalent to an HRT had elapsed, theystems were capable of adjusting to the new established condi-ions. Although higher values for the parameters were observed

i[mf

ering Journal 40 (2008) 99–106

ompared to the results obtained from the previous period (alka-inity, ammonia and COD), the systems were able to reach and

aintain stable profiles until the end of the period. The hightability of the reactors was confirmed by the lack of VFA con-entrations (Fig. 2) and traces of LCFA concentrations, of belowmg L−1, detected in the effluents.

After two HRTs under these conditions, a further reduc-ion of the HRT to a value of 25 days was carried out.ence, the same loading at which the reactors in the first set-p (A1 and B1) had been inhibited was repeated (1.70 and.70 kg VS feed m−3 day−1 for Digesters A2 and B2, respec-ively).

As in the previous case, with the change of HRT and load-ng the system needed a period of time to adapt to the newlymposed conditions until reaching steady profiles. Once more,FA concentrations were imperceptible. LCFA concentrations

n the effluents at the end of the period were very low (Table 3),leic acid being the most abundant, with a maximum concen-ration of less than 25 mg L−1.

On decreasing HRT and increasing the organic loading, itas observed an increase in the volume of gas produced and in

he percentage of methane in the analyzed biogas, and a highat removal, as a consequence of lipid and protein content of theeactors. In all circumstances, the pH value of the systems alwaysemained between 7 and 8. VFA and LCFA concentrations wereuch lower than the concentrations reported in other studies

f slaughterhouse waste, in which their accumulation cause thenhibition of the anaerobic process [9,40].

Daily biogas yield increased in line with increased loading.Methane yield ranged between 0.6 and 0.7 m3 kg−1 VS

eed in the anaerobic digestion of slaughterhouse waste, andetween 0.4 and 0.5 m3 kg−1 VS feed with the mixture ofHW and OFMSW. Specific gas production (SGP) in thenaerobic digestion of SHW likewise ranged between 0.8 and.0 m3 kg−1 VS feed and in the co-digestion with OFMSWetween 0.7 and 0.8 m3 kg−1 VS feed for the different peri-ds studied. These values are higher than those reportedn the literature, which are not higher than 0.6–0.7 m3 kg−1

S feed for co-digestion of other substrates [22,44,45]. Thiselatively high SGP is a result of the efficient degrada-ion of the slaughterhouse waste and as a consequence ofhe high theoretical methane potential of lipids and proteins46–48].

The high values of methane yield achieved and the high pro-ortion of methane in the biogas produced in the process (upo 66 and 65% in digestion and co-digestion, respectively), asell as the lack of volatile intermediaries during the steady statef the implemented treatments, indicate that fat removal in thelaughterhouse waste was carried out efficiently.

Anaerobic digestion was carried out thanks to the acclima-ization of the bacterial consortium to an ammonia-rich

edium [26,49], the sludge was progressively acclimated tohe new conditions, high fats and LCFA concentrations. This

s agreement with the research carried out by other authors10,13,32], and prove that the adaptation of methanogenicicroorganisms to different LCFA concentrations is

easible.

M.J. Cuetos et al. / Biochemical Engineering Journal 40 (2008) 99–106 105

F 2 (B)s

4

sto

osoTwl(east1tat

f

tctoaamc

ao

A

eos

R

ig. 3. Daily biogas and VS concentration in Digester A2 (A) and Digester Becond reactor set-up.

. Conclusions

Anaerobic digestion of slaughterhouse waste (SHW) mayeem a complex task due to the high lipid and protein content ofhe waste. However, the methanization was successfully carriedut in semi-continuously fed digesters at 34 ◦C.

Anaerobic digestion of SHW and its co-digestion with therganic fraction of municipal solid waste (OFMSW) was notuccessful in initial assays working with an HRT of 25 days andrganic loading of 1.70 and 3.70 kg VS m−3 day−1, respectively.hese systems were working without a period of adaptation andere not able to overcome the disturbance of the initial shock

oad. This led to the accumulation of volatile intermediariesLCFA and VFA) and failure of the digestion process. How-ver, it was possible to carry out anaerobic digestion of SHWnd co-digestion of mixtures of SHW with OFMSW by progres-ively decreasing the HRT from 50 to 25 days while increasinghe organic loading from 0.9 and 1.85 kg VS m−3 day−1 to.70 and 3.70 kg VS m−3 day−1, respectively. In consequence,he adaptability of anaerobic microorganisms to a fat-and freemmonia-rich medium was observed by pre-exposing the cul-ures to non-inhibitory concentrations.

Total fat removal was 61% for the SHW digestion and 83%or the co-digestion of the mixture of SHW with OFMSW.

The addition of OFMSW to the co-digestion system con-ributed to a significant increase in the daily biogas yield wheno-digesting with SHW along with an increase of VS in the reac-ors. The biogas yield of the co-digestion systems doubled thatf the SHW digestion system (i.e. 8.6 L day−1 cf. 4.3 L day−1

t 25 days of HRT). The presence of a co-substrate slightlylleviates the concentration of volatile intermediaries at the com-encement of the treatment. However, total and free ammonia

oncentrations are higher during co-digestion, differences which

, and VFA concentrations in Digester A2 (C) and Digester B2 (D) during the

re more pronounced the lower the HRT and the higher therganic loading as a result of the contribution of the OFMSW.

cknowledgements

This research was made possible through the projects: refer-nce no. 2005/48 supported by Technological Agrarian Institutef Castilla y Leon and reference no. ENE 2005-08881-C02-01upported by the Ministry of Education and FEDER funds.

eferences

[1] K.H. Hansen, I. Angelidaki, B.K. Ahring, Anaerobic digestion of swinemanure: inhibition by ammonia, Water Res. 38 (1998) 5–12.

[2] F.J. Callaghan, D.A.J. Wase, K. Thayanithy, C.F. Forster, Continuous co-digestion of cattle slurry with fruit and vegetable wastes and chickenmanure, Biomass Bioenergy 27 (2002) 71–77.

[3] L. de Baere, The role of anaerobic digestion in the treatment of MSW: state-of-the-art, in: 10th World Congress of Anaerobic Digestion, Proceedings,vol. 1, 2004, pp. 395–400.

[4] Regulation (EC) no. 1774/2002, Hazard potential of animal by-products,Official Journal of the European Communities L 273, Luxemburg, 2002.

[5] H. Siegrist, W. Hunzinker, H. Hofer, Anaerobic digestion of slaughterhousewaste with UF-membrane separation and recycling of permeate after freeammonia stripping, Water Sci. Technol. 52 (1–2) (2005) 531–536.

[6] L. Masse, D.I. Masse, K.J. Kennedy, Effect of hydrolysis pretreatment onfat degradation during anaerobic digestion of slaughterhouse wastewater,Process Biochem. 38 (2003) 1365–1372.

[7] K. Hanaki, T. Matsuo, M. Nagase, Mechanism of inhibition caused bylong-chain fatty acids in anaerobic digestion process, Biotechnol. Bioeng.23 (1981) 1591–1610.

[8] C.-S. Hwu, B. Donlon, G. Lettinga, Comparative toxicity of long-chain

fatty acid to anaerobic sludges from various origins, Water Sci. Technol.34 (5–6) (1996) 351–358.

[9] E. Salminen, J. Rintala, Semi-continuous anaerobic digestion of solid poul-try slaughterhouse waste: effect of hydraulic retention time and loading,Water Res. 36 (2002) 3175–3182.

1 ngine

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

06 M.J. Cuetos et al. / Biochemical E

10] M.M. Alves, J.A. Mota, R.M. Alvares, M.A. Pereira, M. Mota, Effectsof lipids and oleic acid on biomass development in anaerobic fixed-bedreactors. Part II. Oleic acid toxicity and biodegradability, Water Res. 35 (1)(2001) 264–270.

11] L. Masse, D.I. Masse, K.J. Kennedy, S.P. Chou, Neutral fat hydrolysis andlong-chain fatty acid oxidation during anaerobic digestion of slaughter-house wastewater, Biotechnol. Bioeng. 79 (1) (2002) 43–52.

12] C.N. Weng, J.S. Jeris, Biochemical mechanisms in the methane fermenta-tion of glutamic and oleic acids, Water Res. 10 (1976) 9–18.

13] M.J. Broughton, J.H. Thiele, E.J. Birch, A. Cohen, Anaerobic batch diges-tion of sheep tallow, Water Res. 5 (1998) 1423–1428.

14] C. Gallert, S. Bauer, J. Winter, Effect of ammonia on the anaerobic degra-dation of protein by a mesophilic and thermophilic biowaste population,Appl. Microbiol. Biotechnol. 50 (4) (1998) 495–501.

15] Z. Wang, C.J. Banks, Evaluation of a two stage anaerobic digester forthe treatment of mixed abattoir wastes, Process Biochem. 38 (2003)1267–1273.

16] B. Kherrati, M. Faid, M. Elyachioui, A. Wahmane, Process for recyclingslaughterhouses wastes and by-products by fermentation, Bioresour. Tech-nol. 63 (1998) 75–79.

17] B. Calli, B. Mertoglu, B. Inanc, O. Yigun, Effects of high free ammo-nia concentrations on the performances of anaerobic bioreactors, ProcessBiochem. 40 (2005) 1285–1292.

18] K.-L. Rosenwinkel, H. Meyer, Anaerobic treatment of slaughterhouseresidues in municipal digesters, Water Sci. Technol. 40 (1) (1999) 101–111.

19] E. Salminen, J. Einola, J. Rintala, The methane production of poultryslaughtering residues and effects of pretreatments on the methane pro-duction of poultry feather, Environ. Technol. 24 (2003) 1079–1086.

20] C.J. Banks, Z. Wang, Development of a two phase anaerobic digester for thetreatment of mixed abattoir wastes, Water Sci. Technol. 40 (1999) 69–76.

21] B. OıKelly, Sewage sludge to landfill: some pertinent engineering proper-ties, J. Air Waste Manage. 55 (2005) 765–771.

22] X. Gomez, M.J. Cuetos, J. Cara, A. Moran, A.I. Garcıa, Anaerobic co-digestion of primary sludge and the fruit and vegetable fraction of themunicipal solid wastes. Conditions for mixing and evaluation of the organicloading rate, Renew. Energy 31 (2006) 2017–2024.

23] E.F. Castillo, D.E. Cristancho, V. Arellano, Study of the operational con-ditions for anaerobic digestion of urban solid wastes, Waste Manage. 26(2006) 546–556.

24] X. Gomez, A. Moran, M.J. Cuetos, M.E. Sanchez, The production ofhydrogen by dark fermentation of municipal solid wastes and slaugh-terhouse waste: a two-phase process, J. Power Sources 157 (2006) 727–732.

25] E. Salminen, J. Rintala, Anaerobic digestion of organic solid poultryslaughterhouse waste—a review, Bioresour. Technol. 83 (2002) 13–26.

26] M. Edstrom, A. Nordberg, L. Thyselius, Anaerobic treatment of ani-mal byproducts from slaughterhouses at laboratory and pilot scale, Appl.Biochem. Biotechnol. 109 (2003) 127–138.

27] D.R. Vartak, C.R. Engler, M.J. McFarland, S.C. Ricke, Attached-film mediaperformance in psychrophilic anaerobic treatment of dairy cattle wastew-ater, Bioresour. Technol. 62 (1997) 79–84.

28] M.C. Sterling Jr., R.E. Lacey, C.R. Engler, S.C. Ricke, Effects of ammonia

nitrogen on H2 and CH4 production during anaerobic digestion of dairycattle manure, Bioresour. Technol. 77 (2001) 9–18.

29] APHA, Standard Methods for the Examination of Water and Wastewater,20th ed., American Public Health Association, Washington, DC, USA,1998.

[

ering Journal 40 (2008) 99–106

30] MAPA, Metodos oficiales de analisis, Ministerio de Agricultura Pesca yAlimentacion, Direccion General de Polıtica Alimentaria, vol. III, MAPA,Madrid, 1994.

31] E. Salminen, J. Rintala, L.Ya. Lokshina, V.A. Vavilin, Anaerobic batchdegradation of solid poultry slaughterhouse waste, Water Sci. Technol. 41(3) (2000) 33–41.

32] A. Fernandez, A. Sanchez, X. Font, Anaerobic co-digestion of a simulatedorganic fraction of municipal solid wastes and fats of animal and vegetableorigin, Biochem. Eng. J. 26 (2005) 22–28.

33] R. Borja, C.J. Banks, Z. Wang, A. Mancha, Anaerobic digestion ofslaughterhouse wastewater using a combination sludge blanket and filterarrangement in a single reactor, Bioresour. Technol. 65 (1998) 125–133.

34] I.W.A. Cramer, Inhibition of methanogenesis from acetate in granularsludge by long chain fatty acids, Appl. Environ. Microbiol. 53 (2) (1987)403–409.

35] I. Angelidaki, B.K. Ahring, Effect of free long-chain fatty acids on ther-mophilic anaerobic digestion, Appl. Microbiol. Biotechnol. 37 (1992)808–812.

36] M. Perle, S. Kimchie, G. Shelef, Some biochemical aspects of the anaerobicdegradation of dairy wastewater, Water Res. 29 (6) (1995) 1549–1554.

37] J.A. Lalman, D.M. Bagley, Anaerobic degradation and inhibitory effectsof linoleic acid, Water Res. 34 (2000) 4220–4228.

38] Y.T. Wang, M.T. Suidan, J.T. Pfeffer, I. Najm, Effects of same alkyl phenolon methanogenic degradation of phenol, Appl. Environ. Microbiol. 54 (5)(1988) 1277–1279.

39] Q. Wang, M. Kuninobu, H.I. Ogawa, Y. Kato, Degradation of volatile fattyacids in highly efficient anaerobic digestion, Biomass Bioenergy 16 (1999)407–416.

40] E. Salminen, J. Einola, J. Rintala, Characterisation and anaerobic batchdegradation of materials accumulating in anaerobic digesters treating poul-try slaughterhouse waste, Environ. Technol. 22 (2001) 577–585.

41] R. Braun, P. Huber, J. Meyrath, Ammonia toxicity in liquid piggery manuredigestion, Biotechnol. Lett. 3 (1981) 159–164.

42] M. Henze, P. Harremoes, Anaerobic treatment of wastewater in fixed filmreactors—a literature review, Water Sci. Technol. 15 (1983) 1–101.

43] G. Tchobanoglous, F.L. Burton, Ingenierıa sanitaria. Tratamiento, evac-uacion y reutilizacion de aguas residuals, in: Diseno de instalaciones parael tratamiento y vertido del fango, Metcalf and Eddy Inc., McGraw Hill,Interamericana de Espana, SA, 1995, pp. 865–880.

44] H. Bouallagui, R. Ben-Cheikh, L. Marouani, M. Hamdi, Mesophilic biogasproduction from fruit and vegetable waste in a tubular digester, Bioresour.Technol. 86 (2003) 85–89.

45] M. Murtor, L. Bjornsson, B. Mattiasson, Impact of food industrial wasteon anaerobic co-digestion of sewage sludge and pig manure, J. Environ.Manage. 70 (2004) 101–107.

46] E.A. Salminen, J.A. Rintala, Anaerobic digestion of poultry slaughteringwastes, Environ. Technol. 20 (1999) 21–28.

47] M.A. Pereira, A.J. Cavaleiro, M. Mota, M.M. Alves, Accumulation of longchain fatty acids onto anaerobic sludge under steady state and shock load-ing conditions: effect on acetogenic and methanogenic activity, Water Sci.Technol. 48 (6) (2003) 33–40.

48] M.A. Pereira, D.Z. Sousa, M. Mota, M.M. Alves, Mineralization of LCFA

associated with anaerobic sludge: kinetics, enhancement of methanogenicactivity and effect of VFA, Biotechnol. Bioeng. 88 (4) (2004) 502–511.

49] I. Angelidaki, B.K. Ahring, Thermophilic anaerobic digestion of livestockwaste: the effect of ammonia, Appl. Microbiol. Biotechnol. 38 (1993)560–564.