mesophilic and thermophilic anaerobic co-digestion of rendering plant and slaughterhouse wastes

9
Mesophilic and thermophilic anaerobic co-digestion of rendering plant and slaughterhouse wastes Suvi Bayr , Marianne Rantanen, Prasad Kaparaju, Jukka Rintala 1 University of Jyväskylä, Department of Biological and Environmental Science, P.O. Box 35, FI-40014 University of Jyväskylä, Finland article info Article history: Received 7 July 2011 Received in revised form 21 September 2011 Accepted 24 September 2011 Available online 7 October 2011 Keywords: Anaerobic digestion Mesophilic Rendering plant waste Slaughterhouse waste Thermophilic abstract Co-digestion of rendering and slaughterhouse wastes was studied in laboratory scale semi-continuously fed continuously stirred tank reactors (CSTRs) at 35 and 55 °C. All in all, 10 different rendering plant and slaughterhouse waste fractions were characterised showing high contents of lipids and proteins, and methane potentials of 262–572 dm 3 CH 4 /kg volatile solids (VS) added . In mesophilic CSTR methane yields of ca 720 dm 3 CH 4 /kg VS fed were obtained with organic loading rates (OLR) of 1.0 and 1.5 kg VS/m 3 d, and hydraulic retention time (HRT) of 50 d. For thermophilic process, the lowest studied OLR of 1.5 kg VS/m 3 d, turned to be unstable after operation of 1.5 HRT, due to accumulating ammonia, volatile fatty acids (VFAs) and probably also long chain fatty acids (LCFAs). In conclusion, mesophilic process was found to be more feasible for co-digestion than thermophilic process, methane yields being higher and process more stable in mesophilic conditions. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Anaerobic digestion is one option to sustainably produce energy from organic matter including biodegradable wastes. In anaerobic digestion process, micro-organisms convert biodegradable waste to biogas, mainly consisting of methane and carbon dioxide, and digestate. Methane can be used to produce heat and/or electricity or vehicle fuel whilst, nutrient rich digestate can be used for exam- ple as a soil conditioner. In slaughterhouse, animals are slaugh- tered and processed into meat products for human consumption. Besides meat products, animal by-products not intended for hu- man consumption are produced. Treatment of these wastes is reg- ulated due to risk of disease spread, mainly Bovine Spongiform Encephalopathy (BSE). In European Union (EU), treatment of slaughterhouse wastes is regulated by the Animal By-product Reg- ulation (ABPR 1069/2009/EC – replacing the ABPR 1774/2002/EC, European Parliament and Council, 2009). According to ABP regula- tion, animal by-products and their process products and wastes are classified into three categories depending upon the risk they pose towards human, animals and environment (European Parliament and Council, 2009). This regulation also defines the corresponding treatment and utilisation possibilities for these materials. Most of the animal by-products from slaughterhouses are treated by ren- dering (European Commission, 2005). Rendering is processing of edible and non-edible animal by-products, in most cases with heat (Woodgate and van der Veen, 2004). Raw materials for rendering process includes carcases, parts of carcases, heads, feet, offal, ex- cess fat, excess meat, hides, feathers, bones and blood (European Commission, 2005; Woodgate and van der Veen, 2004). In render- ing process, raw materials are ground to a uniform size (<50 mm) and subjected to heat treatment (e.g. 133 °C for 20 min at 3 bars) for sterilization. Solid and liquid parts are separated, water is evap- orated and fat is separated from protein and bone (Woodgate and van der Veen, 2004). The finished fat (e.g. tallow, lard, yellow grease) and the solid protein (e.g. bone meal, poultry meal) are pressed into cake for processing into crude animal feed. Depending on the raw material characteristics, rendering is carried out as wet or dry processes and operated either as a single batch or multiple continuous process. Slaughterhouse and rendering wastes are considered as ideal substrates for biogas production, because they usually contain high concentration of organic matter and are rich in proteins and lipids. However, anaerobic digestion of these materials is extremely prone to failure due to production of inhibitory compounds such as ammonia, VFAs and LCFAs (Cuetos et al., 2008; Hejnfelt and Angelidaki, 2009). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.09.104 Abbreviations: CSTR, continuously stirred tank reactor; FM, fresh matter; GC, gas chromatograph; HRT, hydraulic retention time; LCFA, long chain fatty acid; NH 3 , free ammonia nitrogen; NH 4 , ammonia nitrogen; OFMSW, organic fraction of municipal solid waste; OLR, organic loading rate; SCOD, soluble chemical oxygen demand; TKN, total Kjehldahl nitrogen; TS, total solids; TVFA, total volatile fatty acids; uVFA, unionised volatile fatty acid; VFA, volatile fatty acid; VS, volatile solids. Corresponding author. Tel.: +358 45 6790087; fax: +358 14 2602321. E-mail address: suvi.bayr@jyu.fi (S. Bayr). 1 Present address: Tampere University of Technology, Department of Chemistry and Bioengineering, P.O. Box 541, FI-33101 Tampere, Finland. Bioresource Technology 104 (2012) 28–36 Contents lists available at SciVerse ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech

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Page 1: Mesophilic and thermophilic anaerobic co-digestion of rendering plant and slaughterhouse wastes

Bioresource Technology 104 (2012) 28–36

Contents lists available at SciVerse ScienceDirect

Bioresource Technology

journal homepage: www.elsevier .com/locate /bior tech

Mesophilic and thermophilic anaerobic co-digestion of rendering plantand slaughterhouse wastes

Suvi Bayr ⇑, Marianne Rantanen, Prasad Kaparaju, Jukka Rintala 1

University of Jyväskylä, Department of Biological and Environmental Science, P.O. Box 35, FI-40014 University of Jyväskylä, Finland

a r t i c l e i n f o

Article history:Received 7 July 2011Received in revised form 21 September 2011Accepted 24 September 2011Available online 7 October 2011

Keywords:Anaerobic digestionMesophilicRendering plant wasteSlaughterhouse wasteThermophilic

0960-8524/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.biortech.2011.09.104

Abbreviations: CSTR, continuously stirred tank reacchromatograph; HRT, hydraulic retention time; LCFAfree ammonia nitrogen; NH4, ammonia nitrogen;municipal solid waste; OLR, organic loading rate; SCdemand; TKN, total Kjehldahl nitrogen; TS, total solacids; uVFA, unionised volatile fatty acid; VFA, volatile⇑ Corresponding author. Tel.: +358 45 6790087; fax

E-mail address: [email protected] (S. Bayr).1 Present address: Tampere University of Technolo

and Bioengineering, P.O. Box 541, FI-33101 Tampere, F

a b s t r a c t

Co-digestion of rendering and slaughterhouse wastes was studied in laboratory scale semi-continuouslyfed continuously stirred tank reactors (CSTRs) at 35 and 55 �C. All in all, 10 different rendering plant andslaughterhouse waste fractions were characterised showing high contents of lipids and proteins, andmethane potentials of 262–572 dm3 CH4/kg volatile solids (VS)added. In mesophilic CSTR methane yieldsof ca 720 dm3 CH4/kg VSfed were obtained with organic loading rates (OLR) of 1.0 and 1.5 kg VS/m3 d,and hydraulic retention time (HRT) of 50 d. For thermophilic process, the lowest studied OLR of1.5 kg VS/m3 d, turned to be unstable after operation of 1.5 HRT, due to accumulating ammonia, volatilefatty acids (VFAs) and probably also long chain fatty acids (LCFAs). In conclusion, mesophilic process wasfound to be more feasible for co-digestion than thermophilic process, methane yields being higher andprocess more stable in mesophilic conditions.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Anaerobic digestion is one option to sustainably produce energyfrom organic matter including biodegradable wastes. In anaerobicdigestion process, micro-organisms convert biodegradable wasteto biogas, mainly consisting of methane and carbon dioxide, anddigestate. Methane can be used to produce heat and/or electricityor vehicle fuel whilst, nutrient rich digestate can be used for exam-ple as a soil conditioner. In slaughterhouse, animals are slaugh-tered and processed into meat products for human consumption.Besides meat products, animal by-products not intended for hu-man consumption are produced. Treatment of these wastes is reg-ulated due to risk of disease spread, mainly Bovine SpongiformEncephalopathy (BSE). In European Union (EU), treatment ofslaughterhouse wastes is regulated by the Animal By-product Reg-ulation (ABPR 1069/2009/EC – replacing the ABPR 1774/2002/EC,European Parliament and Council, 2009). According to ABP regula-tion, animal by-products and their process products and wastes are

ll rights reserved.

tor; FM, fresh matter; GC, gas, long chain fatty acid; NH3,

OFMSW, organic fraction ofOD, soluble chemical oxygenids; TVFA, total volatile fatty

fatty acid; VS, volatile solids.: +358 14 2602321.

gy, Department of Chemistryinland.

classified into three categories depending upon the risk they posetowards human, animals and environment (European Parliamentand Council, 2009). This regulation also defines the correspondingtreatment and utilisation possibilities for these materials. Most ofthe animal by-products from slaughterhouses are treated by ren-dering (European Commission, 2005). Rendering is processing ofedible and non-edible animal by-products, in most cases with heat(Woodgate and van der Veen, 2004). Raw materials for renderingprocess includes carcases, parts of carcases, heads, feet, offal, ex-cess fat, excess meat, hides, feathers, bones and blood (EuropeanCommission, 2005; Woodgate and van der Veen, 2004). In render-ing process, raw materials are ground to a uniform size (<50 mm)and subjected to heat treatment (e.g. 133 �C for 20 min at 3 bars)for sterilization. Solid and liquid parts are separated, water is evap-orated and fat is separated from protein and bone (Woodgate andvan der Veen, 2004). The finished fat (e.g. tallow, lard, yellowgrease) and the solid protein (e.g. bone meal, poultry meal) arepressed into cake for processing into crude animal feed. Dependingon the raw material characteristics, rendering is carried out as wetor dry processes and operated either as a single batch or multiplecontinuous process.

Slaughterhouse and rendering wastes are considered as idealsubstrates for biogas production, because they usually contain highconcentration of organic matter and are rich in proteins and lipids.However, anaerobic digestion of these materials is extremelyprone to failure due to production of inhibitory compounds suchas ammonia, VFAs and LCFAs (Cuetos et al., 2008; Hejnfelt andAngelidaki, 2009).

Page 2: Mesophilic and thermophilic anaerobic co-digestion of rendering plant and slaughterhouse wastes

S. Bayr et al. / Bioresource Technology 104 (2012) 28–36 29

Ammonia is produced by the biological degradation of thenitrogenous matter, mostly proteins (Kayhanian, 1999). In anaero-bic digestion process, proteins are hydrolysed to amino acids,which are degraded to VFAs (Mata-Alvarez, 2003). In the anaerobicdigestion process, ammonia exists in two forms viz. ammoniumions (NHþ4 ) and ammonia gas (NH3, free ammonia), the relativeamount of each depends on pH (Hobson and Wheatley, 1993;Kayhanian, 1999). Free ammonia has been suggested to be moreinhibitory than total ammonia nitrogen (Angelidaki and Ahring,1994), because it can readily diffuse across the cell membrane(Kadam and Boone, 1996). Besides pH, temperature also affectsthe dissociation constant of ammonia nitrogen and concentrationof free ammonia in the process; the higher is the temperaturethe higher is the concentration of free ammonia (Kayhanian,1999). In the literature a wide range of inhibitory levels, 0.045–1.1 g/l, have been reported for NH3 (e.g. Angelidaki and Ahring,1994; Hansen et al., 1998; Kayhanian, 1999). Similarly, for NH4–N, concentrations of 3.86 and 5.60 g/l were found to decreasemethane production by 50% in treating synthetic substrate (petfood) to simulate organic fraction of municipal solid waste(OFMSW) at 35 and 55 �C, respectively (Benabdallah El Hadjet al., 2009), whilst concentration of 6 g/l led to inhibited steadystate at 55 �C, but was less inhibitory at 35 �C in CSTRs treatingswine manure (Hansen et al., 1998).

Similarly, under anaerobic conditions, lipids are first hydrolysedto LCFAs, which are oxidised to acetate or propionate and hydrogenthrough the b-oxidation pathway (Cirne et al., 2007; Mata-Alvarez,2003). Inhibitory concentrations of individual LCFAs vary, as e.g.30 mg/l of oleic or linoleic acid inhibited acetoclastic methanogen-esis, whilst 100 mg/l of stearic acid was not inhibitory (Lalman andBagley, 2000, 2001). Furthermore, oleic acid was found less inhib-itory to acidogenesis than for methanogenesis (Beccari et al.,1996). LCFAs were speculated to be reason for process failures inCSTRs treating poultry slaughterhouse wastes at 35 �C (Cuetoset al., 2010) and pig slaughterhouse waste with manure at 35and 55 �C (Hejnfelt and Angelidaki, 2009). Moreover, temperaturewas reported to effect oleic acid inhibition as acetate-utilisingmethanogenesis was more susceptible to oleic acid toxicity at55 �C than at 35 �C (Hwu and Lettinga, 1997). Nevertheless LCFAinhibition has been shown to be temporary, but recovery timemight be long (Cirne et al., 2007). Studies on anaerobic digestionof rendering wastes are scarce whereas, several studies on theanaerobic digestion of slaughterhouse waste, especially, poultrywaste have been reported in the literature. For rendering wastes,methane potentials of 497 and 487 dm3 CH4/kg VS were reportedfor bone flour and fat, respectively, in batch assays at 55 �C(Hejnfelt and Angelidaki, 2009). In a similar study, methane poten-tial of 351–381 dm3 CH4/kg VS was reported for meat and bonemeal at 35 �C (Wu et al., 2009). For slaughterhouse wastes, meth-ane potentials of 225–619 dm3 CH4/kg VS were reported for pigwaste at 55 �C (Hejnfelt and Angelidaki, 2009) and 580 dm3 CH4/kg VS at 35 �C (Rodríguez-Abalde et al., 2011). Similarly, methanepotentials of 460 dm3 CH4/kg VS were reported for poultry wasteat 35 �C (Rodríguez-Abalde et al., 2011) and 210–910 dm3 CH4/kg VS both at 35 and 55 �C; highest potential was obtained for offal(Salminen et al., 2003). On the other hand, methane yields of 520–700 dm3 CH4/kg VS were reported during semi-continuous diges-tion of poultry slaughterhouse wastes in CSTR experiments at35 �C (Cuetos et al., 2008; Salminen and Rintala, 2002).

Co-digestion of slaughterhouse wastes with other materialscontaining low nitrogen and/or lipid content is one option to im-prove the digestion process stability and increase methane yields.Methane yields of 270–500 dm3 CH4/kg VS have been reportedduring the co-digestion of slaughterhouse wastes with other sub-strates such as manure, sewage sludge and OFMSW (Alvarez andLiden, 2008; Cuetos et al., 2008; Hejnfelt and Angelidaki, 2009;

Luste and Luostarinen, 2010). In many cases, large quantities ofslaughterhouse and rendering wastes are generated at the sameprocessing plant. Moreover, treatment and use of these organicwastes, rich in protein and lipids, through anaerobic digestion isconsidered as a sustainable solution for simultaneous recovery ofenergy and nutrients. As far as we know, there are no studies onsemi-continuous anaerobic digestion of rendering plant wastesalone or co-digestion of rendering plant wastes with slaughter-house wastes. The objective of the present study was to evaluatethe feasibility of anaerobic semi-continuous co-digestion of ren-dering plant wastes with slaughterhouse wastes in laboratory scaleCSTRs at 35 and 55 �C. In addition, methane potentials of 10 differ-ent rendering plant and slaughterhouse waste fractions were stud-ied in batch assays at 35 �C.

2. Methods

2.1. Substrates and inocula

Seven different types of rendering plant wastes and three dif-ferent slaughterhouse by-products were used as substrates in theexperiments. Characteristics of the substrates are presented inTable 1. The rendering wastes viz. melt (sterilized (133 �C,20 min, 3 bar) mass), biosludge (sludge from wastewater treat-ment), fat from fat separation well (fat separated with H2O2

from wastewater of production equipments and rooms), separa-tor sludge (water, protein and fat extracted in final purificationby centrifuge from sterilized and solids separated fat), decantersludge (solids, separated by centrifuge from fat separated bypressing from sterilized mass), fat (sterilized and purified fat)and boneflour (solids separated by pressing from sterilized mass)were collected from a rendering plant (Honkajoki Ltd., Finland).The above mentioned waste fractions were chosen in the studyas there is need to develop waste treatment method. Slaughter-house wastes viz. the contents of stomach and intestines of bo-vine (without rumen and reticulum) and swine were from ameat producing factory (Saarioinen Ltd., Jyväskylä). Discardedpoultry (turkey) was delivered by Honkajoki Ltd., Finland. Atthe laboratory, the slaughterhouse by-products were macerated(5 mm) by using a meat mincer (Talsa W 22). The well homog-enised materials were stored at �20 �C until further use. All thesubstrates, rendering plant as well as slaughterhouse wastes, areheterogeneous, which needs to be taken into account when con-sidering the results.

Digested sludge from a municipal wastewater treatment plant(Nenäinniemi, Jyväskylä, Central Finland) was used as inoculum inmesophilic experiments. For thermophilic experiments, digestedmaterials from a full-scale biogas plant (Stormossen, Vaasa, Finland)treating putrescible organic fraction of municipal waste was used asinoculum. Characteristics of the inocula are presented in Table 1.

2.2. Batch experiments

Biochemical methane potential experiment was carried out in1 l glass bottles with a working volume of 750 ml. To each bottle,rendering plant wastes (seven materials) or slaughterhouse wastes(three materials) and inoculum (250 ml/bottle) were added at asubstrate to inoculum (Nenäinniemi) VS ratio of 0.25. Tap waterwas added in order to obtain the working volume of 750 ml. NaH-CO3 (3 g/l) was added as buffer. Thereafter, bottles were flushedwith N2 for 3 min in order to create anaerobic conditions andsealed with silicon stoppers. Assays containing only inoculumand water were used as controls. The methane production fromcontrol assays was subtracted from the sample assays. Theprepared assays were incubated statically at 35 ± 1 �C. The

Page 3: Mesophilic and thermophilic anaerobic co-digestion of rendering plant and slaughterhouse wastes

Tabl

e1

Chem

ical

char

acte

rist

ics

and

met

hane

pote

ntia

ls(S

TP)

ofth

est

udie

dsl

augh

terh

ouse

and

rend

erin

gpl

ant

was

tes

and

inoc

ula.

Stan

dard

devi

atio

nin

pare

nthe

sis

whe

nap

plic

able

.

Mat

eria

lB

atch

TS (%)

VS

(%)

VS/

TS(%

)TK

N(g

/kg

FM)

NH

4(g

/kg

FM)

Prot

ein

(g/

kgFM

)Pr

otei

n(g

/kg

VS)

Lipi

d(g

/kg

FM)

Lipi

d(g

/kg

VS)

Met

han

epr

od.

(dm

3C

H4/k

gV

S ad

ded

)M

eth

ane

prod

.(d

m3

CH

4/k

gFM

add

ed)

Theo

reti

cal

met

han

epr

od.

(dm

3C

H4/k

gV

S)**

Part

offe

ed(%

ofFM

)*

Ren

deri

ngw

aste

sFa

tfr

omfa

tse

para

tion

124

.122

.292

4.2

0.80

2611

719

889

227

5(5

2)61

(12)

962

1.4

278

.376

.097

N.d

.N

.d.

N.d

.N

.d.

N.d

.N

.d.

N.d

.N

.d.

N.d

.Se

para

tor

slu

dge

12.

22.

091

0.29

0.01

210

016

800

572

(187

)12

(4)

861

4.0

222

.421

.194

N.d

.N

.d.

N.d

.N

.d.

N.d

.N

.d.

N.d

.N

.d.

N.d

.M

elt

198

.466

.668

690.

2842

964

422

033

051

5(5

4)34

3(3

7)65

41.

12

98.4

74.5

76N

.d.

N.d

.N

.d.

N.d

.N

.d.

N.d

.N

.d.

N.d

.N

.d.

Dec

ante

rsl

udg

e1

98.2

62.0

6361

0.26

384

619

221

356

476

(164

)29

5(1

02)

669

1.3

298

.974

.675

N.d

.N

.d.

N.d

.N

.d.

N.d

.N

.d.

N.d

.N

.d.

N.d

.B

iosl

udg

e1

1.0

0.9

901.

20.

748

844

111

116

(23)

0.1

(0.2

)53

22.

22

2.3

2.0

87N

.d.

N.d

.N

.d.

N.d

.N

.d.

N.d

.N

.d.

N.d

.N

.d.

Fat

199

.399

.210

01.

10.

027

793

594

340

6(3

9)40

3(3

9)95

9–

Bon

eflou

r1

98.7

56.0

5772

0.34

450

804

100

179

287

(123

)16

1(6

9)58

0–

Slau

ghte

rhou

sew

aste

sPo

ult

ry1

38.2

33.1

8726

.30.

7216

448

315

145

326

6(7

4)90

(25)

699

38.2

232

.629

.590

N.d

.N

.d.

N.d

.N

.d.

N.d

.N

.d.

262

(93)

76(2

7)–

Swin

e1

31.8

28.2

8915

.81.

199

351

148

525

428

(25)

120

(7)

706

25.2

Bov

ine

153

.252

.699

5.6

0.24

3557

461

767

572

(89)

296

(46)

806

26.7

Inoc

ula

Mes

oph

ilic

11.

490.

7752

1.3

0.78

N.d

.N

.d.

N.d

.N

.d.

––

––

Ther

mop

hil

ic1

2.67

1.24

46N

.d.

1.73

N.d

.N

.d.

N.d

.N

.d.

––

––

N.d

.=n

otde

term

ined

.*

Part

offe

ed,w

ater

dilu

tion

not

take

nin

toac

cou

nt.

**

Cal

cula

ted

base

don

prot

ein

and

lipi

dco

nte

nts

.

30 S. Bayr et al. / Bioresource Technology 104 (2012) 28–36

produced biogas was collected into aluminium gas bags. Bot-tles were mixed manually before each gas analysis. On day24, as the methane production slowed down, one of the repli-cate bottles digesting rendering plant wastes was moved from35 to 55 �C in order to study the effect of change in tempera-ture on the residual methane potential.

2.3. Reactor experiments

Anaerobic co-digestion of rendering plant and slaughter-house wastes was studied in two semi-continuously fed stain-less steel CSTRs with a total volume of 13 l and liquid volumeof 10 l. Reactors, R1 and R2, were operated at 35 and 55 �C,respectively. Temperature in the reactors was maintained bya heating coil wrapped around the insulated reactor and con-trolled thermostatically. Reactors were mixed mechanicallywith a timer (13 min on/16 min off). Reactors were fed manu-ally on five days per week on days 14–48 and daily on days 49–178. Reactors were fed when stirring was off, at a feed rate of200 ml/d and HRT of 50 d (expect for days 112–168 in R2). Ateach feeding, an equal amount of effluent was collected intothe digestate storage container (10 l capacity) via the gas trap.The produced biogas was collected in aluminium gas bags.

Feed was prepared by mixing the five rendering plant andthree slaughterhouse waste streams according to the actualamount of the by-products produced at the factories as indi-cated in Table 1 (wet weight basis). The prepared feed was fro-zen in plastic containers (500 ml) as weekly feed portions.Frozen feed was thawed and diluted with tap water to obtainthe desired HRT and OLR (Tables 2 and 3).

During the start-up, 9.7 l of inoculum and 50 g of separatorsludge was added to each reactor. Reactors were flushed withN2 for 5 min to create anaerobic conditions. Separator sludgewas fed twice before actual feeding was initiated on day 14.The initial OLR used in both reactors was 1.5 kg VS/m3 d. Onday 21, OLR of mesophilic reactor (R1) was decreased from1.5 to 0.5 kg VS/m3 d. Thereafter, OLR was increased stepwiselyto 1 kg VS/m3 d on day 69, to 1.5 kg VS/m3 d on day 107 and to2.0 kg VS/m3 d on day 154. On the other hand, OLR of thermo-philic reactor (R2) was increased to 2.25 kg VS/m3 d on day107. Due to process inhibition, feeding in R2 was withheld be-tween days 112 and 168. However, R2 was fed 50–200 g onindividual days (day 113,119–120,133–168) at an averageOLR of 0.4 kg VS/m3 d and HRT 109 d.

2.4. Analyses

Total solids (TS) and VS were analysed according to standardmethods (APHA, 1998). pH was measured with Mettler ToledoSevenEasy pH-metre immediately after each sampling. Solublechemical oxygen demand (SCOD) was analysed according toFinnish Standard Methods (SFS 5504, Finnish Standard Associa-tion, 1988). Ammonia nitrogen (NH4) and total Kjeldahl nitrogen(TKN) were determined according to Tecator application note(effluents, Perstorp Analytical/Tecator AB, 1995) or standardmethod (substrates, method 984.13, AOAC, 1990). VFAs weredetermined by using a gas chromatograph (Perkin–Elmer Auto-system XL GC, PE FFAP column 30 m � 0.32 mm � 25 lm). He-lium was used as a carrier gas and operation conditions were:oven 100–160 �C (25 �C/min), detector 225 �C and injector230 �C. The samples for SCOD and VFA analysis were centrifuged(Sanyo Harrier 18/80 Refrigerated) at 2500 rpm (15 min), forVFA also at 12,000 rpm (10 min) and filtered through glassmicrofilter (VWR Glass microfibers filter 691, particle retention1.6 lm). Lipid content was analysed with ether extract afterhydrolysis with 3 M HCl (Anon, 1971).

Page 4: Mesophilic and thermophilic anaerobic co-digestion of rendering plant and slaughterhouse wastes

Table 2Process parameters and characteristics of feeds and effluents during semi-continuous anaerobic co-digestion of rendering plant and slaughterhouse wastes in CSTR at 35 �C. Allvalues, except for pH, are mean values during the run (standard deviation in parenthesis).

Days 14–20 21–68 69–106 107–153 154–178

OLR (kg VS/ m3d) 1.5 0.5 1.0 1.5 2.0HRT (d) 50 50 50 50 50CH4 yield (dm3/kg VSfed) 35 (–) 965 (489) 725 (28) 717 (45) 639 (37)

Feed Effluent Feed Effluent Feed Effluent Feed Effluent Feed EffluentpH 6.0–6.1 6.8–7.4 5.8–6.3 6.6–7.4 5.7–6.1 7.1–7.5 5.4–5.9 7.5–7.9 5.5–5.7 7.6–7.8SCOD (g/l) 7.9 (2.4) 2.1 (–) 3.3 (0.3) 1.8 (1.4) 8.6 (0.04) 0.7 (0.1) 12.3 (1.2) 1.2 (0.2) 13.4 (4.3) 3.0 (0.8)TVFA (mg/l) 586 (–) 1143 (381) 131 (27) 417 (554) 371 (48) 34 (9) 624 (101) 95 (98) N.d. 1110 (406)TKN (mg/l) 2682 (8) 1189 (–) 1546 (354) 1370 (184) 2815 (216) 1897 (162) 4027 (71) 2720 (320) 5160 (88) 3275 (121)NH4 (mg/l) 144 (4) 896 (–) 53 (4) 916 (70) 138 (5) 1029 (100) 214 (26) 1617 (283) 293 (–) 2230 (159)NH3 (mg/l) N.d. 16 (13) N.d. 15 (8) N.d. 21 (10) N.d. 68 (22) N.d. 94 (10)TS (%) 8.2 (–) 1.4 (0.5) 2.3 (0.4) 1.3 (0.1) 4.5 (0.4) 1.1 (0.04) 7.5 (0.1) 1.3 (0.1) 9.9 (0.3) 1.4 (0.04)VS (%) 7.9 (–) 0.9 (0.4) 2.2 (0.4) 0.7 (0.04) 4.2 (0.4) 0.7 (0.04) 7.1 (0.1) 0.9 (0.1) 9.4 (0.2) 1.0 (0.02)VSremoval (%) 89 68 83 87 89

N.d. = not determined.

Table 3Process parameters and characteristics of feeds and effluents during semi-continuous anaerobic co-digestion of rendering plant and slaughterhouse wastes in CSTR at 55 �C. Allvalues, except for pH, are mean values during the run (standard deviation in parenthesis).

Days 14–106 107–111 112–168

OLR (kg VS/m3 d) 1.5 2.25 0.4HRT (d) 50 50 109CH4 yield (dm3/kg VSfed) 766 (173) 617 (–) 401 (128)

Feed Effluent Feed Effluent Feed EffluentpH 5.6–6.1 7.7–8.1 5.9 7.9 5.4–5.9 7.5–7.9SCOD (g/l) 10.1 (1.7) 3.9 (0.8) 17.8 (–) 6.9 (0.6) 5.0 (0.6) 12.9 (1.8)TVFA (mg/l) 453 (66) 465 (461) 983 (–) 2181 (196) 258 (13) 4135 (438)TKN (mg/l) 3177 (507) 2785 (350) 5118 (–) 3392 (–) 1548 (43) 3368 (298)NH4 (mg/l) 168 (22) 2049 (255) 341 (–) 2647 (89) 86 (8) 2756 (221)NH3 (mg/l) N.d. 455 (137) N.d. 598 (–) N.d. 488 (123)TS (%) 7.4 (0.8) 2.2 (0.5) 11.6 (–) 1.7 (–) 2.9 (0.03) 2.4 (0.2)VS (%) 7.0 (0.9) 1.3 (0.2) 10.8 (–) 1.1 (–) 2.8 (0.04) 1.7 (0.2)VSremoval (%) 81 90 39

N.d. = not determined.

S. Bayr et al. / Bioresource Technology 104 (2012) 28–36 31

Gas composition (CH4, CO2 and H2) was measured by using a GC(Perkin–Elmer Arnel Clarus 500, Alumina column 30 m � 0.53 mm)equipped with a thermal conductivity detector (TCD, reactor exper-iments) or flame-ionization detector (FID, batch experiments).Argon was used as a carrier gas and operation conditions were:oven 100 �C, detector and injection port 225 �C. The biogas volumewas measured by water displacement method. Biogas results wereconverted to standard temperature and pressure conditions (STP,T = 273 K, p = 1 bar).

2.5. Calculations

In CSTR-studies methane yields were calculated on weekly val-ues. Protein content was calculated by 6.25 � TKN. Theoreticalmethane yields based on protein and lipid contents of the sub-strates (STP, T = 273 K, p = 1 bar) were calculated by X �496 dm3 CH4/kg VS + Y � 1014 dm3 CH4/kg VS, were X = % ofproteins and Y = % of lipids (Angelidaki and Sanders, 2004). Theunionised fraction of the ammonia nitrogen was calculated bythe following equation (Watcharasukarn et al., 2009):

FNH3 ¼ 1þ 10ðpkw�pkb�pHÞ� ��1

ð1Þ

The unionised fraction of VFA (uVFA) was calculated by the fol-lowing equation (Watcharasukarn et al., 2009):

FVFA ¼10ðpka�pHÞ

1þ 10ðpka�pHÞ ð2Þ

3. Results and discussion

3.1. Material characterisation

The characteristics of the 10 studied materials varied greatly(Table 1). Rendering materials, except for biosludge, separatorsludge and fat from fat separation, had TS more than 98%, withVS accounting for >85% of TS in all materials, except for melt, de-canter sludge and boneflour. Fat from fat separation unit, separatorsludge and biosludge varied greatly between two batches appar-ently due to differences in rendering and wastewater treatmentprocesses. Based on chemical composition, melt, decanter sludge,fat and boneflour of the rendering wastes and bovine from slaugh-terhouse wastes, were expected to produce high methane yieldsdue to high protein and lipid contents taken together (P495 g/kgfresh matter (FM)). All materials, except for biosludge, could beconsidered as promising substrates for methane production. Inthe reactor feed (without dilution) calculated proportion of lipidswas 22.7% of FM and calculated proportion of proteins was 10.5%of FM.

3.2. Batch experiments

Methane yields for the studied materials are presented in Ta-ble 1. Methane yields for rendering materials varied between 275and 572 dm3 CH4/kg VSadded (except for biosludge 16 dm3 CH4/kg VSadded). Similar methane yields of 262–572 dm3 CH4/kg VSadded

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32 S. Bayr et al. / Bioresource Technology 104 (2012) 28–36

were also obtained for slaughterhouse wastes. On VS basis, highestmethane yield of 572 dm3 CH4/kg VSadded, was obtained for separa-tor sludge and bovine. Both these materials had high VS/TS ratioand high concentration of proteins and lipids taken together. Onthe other hand, fat, with high lipid content (935 g/kg FMadded)and VS/TS ratio 100%, produced the highest methane yield perFM, (403 dm3 CH4/kg FMadded). No difference was noticed when as-says incubated at 35 �C were moved to thermophilic conditions (onday 24), compared to bottles left to mesophilic conditions (data notshown).

As far as we know this is the first study to report methane yieldsfrom rendering plant wastes widely. The methane yields obtainedfor bone flour (287 dm3 CH4/kg VSadded) and fat (406 dm3 CH4/kg VSadded) in the present study were slightly lower than the yieldsof 497 and 487 dm3 CH4/kg VS, reported for similar materials byHejnfelt and Angelidaki (2009). For meat and bone meal, methaneyields of 351–381 dm3 CH4/kg VS were reported (Wu et al., 2009).The methane yields obtained for pig waste (428 dm3 CH4/kg VSadded) in the present study, were within the range of 225–619 dm3 CH4/kg VS reported for pig wastes at 55 �C (Hejnfelt andAngelidaki, 2009). On the other hand, the methane yield obtainedfor poultry slaughterhouse waste in the present study(266 dm3 CH4/kg VSadded) was similar or lower than the yieldsreported at 35 and 55 �C. For instance, Salminen et al. (2003)reported methane yields of 210–910 dm3 CH4/kg VS, with highestyield reported for poultry offal. For bovine slaughterhouse wastethere are no comparable results reported in the literature.

3.3. Reactor experiments

3.3.1. OLRs and methane productionCo-digestion of rendering plant and slaughterhouse wastes was

studied in semi-continuously fed CSTR-reactors for 178 days at35 �C and 168 days at 55 �C. The reactor performances are shownin Figs. 1–3 and summarised in Tables 2 and 3.

In both reactors, daily feeding was started on day 14 with anOLR of 1.5 kg VS/m3 d. Methane production started immediatelyin thermophilic reactor, even though some low yields were de-tected around day 35. On the other hand, no methane productionwas noticed in mesophilic reactor and therefore, OLR of the meso-philic reactor was decreased from 1.5 to 0.5 kg VS/m3 d on day 21after which methane production rapidly peaked indicating metha-nation of accumulated VFAs. Subsequently, the OLR in mesophilicreactor was then increased stepwisely according to increasedmethane production up to OLR 2.0 kg VS/m3 d on day 154. Simi-larly, OLR in thermophilic reactor was increased as well, to2.25 kg VS/m3 d on day 107. However, methane production of ther-mophilic reactor started to decrease and from day 112 onwards, forrest of the run, reactor was fed with 50–200 g/d on individual days(d 113,119–120,133–168), average OLR being 0.4 kg VS/m3 d andaverage HRT 109 d.

Overall, better process performance and higher methane yieldswere noticed in the mesophilic reactor than in the thermophilicreactor (Figs. 1–3, Tables 2 and 3). During the stable period, meth-ane content of the biogas was 65–75% in the mesophilic reactor(days 25–178) and 55–72% in the thermophilic reactor (days 7–168). For mesophilic reactor the highest average methane yieldof 965 dm3 CH4/kg VSfed, was obtained when the reactor was oper-ated with an OLR of 0.5 kg VS/m3 d (Table 2, Fig. 1). However, someof the methane was from accumulated substrate from the initialOLR of 1.5 kg VS/m3 d period. Average methane yields 725 and717 dm3 CH4/kg VSfed were obtained with OLRs of 1 and1.5 kg VS/m3 d, respectively. Thus, it seems that mesophilic reactorcan be operated with OLR 1 kg VS/m3 d and apparently also withOLR 1.5 kg VS/m3 d as the experimental periods with these OLRslasted together for �2 HRTs. Good performance of the mesophilic

process was also supported with high VS reductions, between83% and 89% during the run. The lower VS reduction noticed duringdays 21–68 (68%) was due to the conversion of VFA that had accu-mulated during the first phase (day 14–20). On the other hand, inthe thermophilic process the highest average methane yield of766 dm3 CH4/kg VSfed, was obtained when the reactor was oper-ated with an OLR of 1.5 kg VS/m3 d with VS reduction of 81%. How-ever, after 1.5 HRT, thermophilic process became unstable and VFAaccumulation was noticed indicating that OLR of 1.5 kg VS/m3 dwas too high. These results are in agreement with previous studies,where OLRs of 0.8–1.7 kg VS/m3 d were successfully applied fortreating slaughterhouse wastes alone (Cuetos et al., 2008;Salminen and Rintala, 2002) and 1.85–3.7 kg VS/m3 d for co-diges-tion of slaughterhouse waste with OFMSW (Cuetos et al., 2008). Inthe present study, both reactors were operated with 50 d HRT (ex-cept for reactor R2 during days 112–168), which was the same orlower than HRTs (50–100 d) reported during the digestion of poul-try slaughterhouse waste in CSTRs at 35 �C (Salminen and Rintala,2002). Nevertheless, Cuetos et al. (2008) demonstrated that anaer-obic digestion of poultry slaughterhouse waste was possible at HRTof 25 d, if the HRT was progressively decreased from 50 to 25 d.This improvement in process stability in the above study wasmainly attributed due to the adaptation of micro-organisms. Thusthe need for low OLRs and long HRTs at the beginning of the exper-iment are crucial for process success. Before OLRs can be increased,the microbes need to adapt well for feedstocks with high lipid andprotein content such as slaughterhouse wastes (Cuetos et al., 2008;Edström et al., 2003).

As far as we know, there are no comparable studies on anaero-bic digestion of rendering plant wastes alone or co-digestion ofrendering with slaughterhouse wastes, especially semi-continuousreactor studies. Therefore, the methane yields obtained in the pres-ent study are compared with the yields obtained during digestionof slaughterhouse wastes alone or during co-digestion of slaugh-terhouse wastes and other wastes such as sewage sludge orOFMSW. The methane yields obtained in the present study weresimilar to the yields of 520–700 dm3 CH4/kg VS reported duringthe anaerobic digestion of slaughterhouse wastes alone at 35 �C(Cuetos et al., 2008; Salminen and Rintala, 2002) and were higherthan the yields of 380–430 dm3 CH4/kg VS, reported during the co-digestion of animal by-products and sewage sludge (VS ratios of1:3 and 1:7) at 35 �C (Luste and Luostarinen, 2010).

3.3.2. VFAs and SCODVFA production profiles are presented in Fig. 3. Total volatile

fatty acids (TVFA) concentration in the thermophilic reactor in-creased gradually from 25 mg/l (day 17) to reach the highest con-centration of 4750 mg/l on day 143 and thereafter dropped to3300 mg/l at the end of the run (day 168). On the other hand, TVFAbuild up in the mesophilic reactor was noticed during the initialstart-up (days 1–39), slight increase after day 140, and faster in-crease after day 156 reaching 1655 mg/l in the end, suggesting thatOLR of 2 kg VS/m3 d was too high. However, the VFA levels in themesophilic reactor were lower than the concentrations of900 mg/l of propionic acid considered as inhibitory for methano-gens and 2400 mg/l of acetic or butyric acid considered non-inhib-itory for methanogens (Wang et al., 2009). Acetic acid was themain VFA, accounting for >80% of the TVFA, presented in the mes-ophilic reactor indicating that aceticlastic methanogenesis was therate-limiting step in this reactor. A similar observation was also re-ported during the mesophilic digestion of poultry slaughterhousewaste (Salminen and Rintala, 2002). Lowest concentration of SCOD(ca 0.6 g/l) was measured in mesophilic process when TVFA wasthe lowest (<30 mg/l) with OLR of 1.0 kg VS/m3 d indicating thatthe studied waste was well biodegradable.

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0

1

2

3

0

1000

2000

3000

OL

R (k

gVS/

m3d)

Met

hane

yie

ld (m

l C

H4/g

VS)

6

7

8

9pH

0

2

4

6

SCO

D (

g/l)

0

1

2

3

NH

3or

NH

4-N

(g/

l)

0

1000

2000

3000

0 50 100 150 200

TV

FA (

mg/

l)

0

2

4

6

TK

N (g

/l)

Fig. 1. Characteristics of the effluent during semi-continuous anaerobic co-digestion of rendering plant and slaughterhouse wastes in CSTR at 35 �C; (h) methane yield,(–) OLR, (j) pH, (N) SCOD, (e) NH4–N, (�) NH3, (�) TKN and (s) TVFA.

S. Bayr et al. / Bioresource Technology 104 (2012) 28–36 33

Concentrations of calculated uVFA values fluctuated more in themesophilic than in the thermophilic reactor, but remained mainlyat the level of 0.01–2 mg/l in the mesophilic reactor and 0.01–4 mg/l in the thermophilic reactor. Highest calculated uVFA con-centrations of 23.1 and 5.6 mg/l were noticed on day 22 and day168 in the mesophilic and the thermophilic reactor, respectively.The increase in calculated uVFA concentrations in both reactors

were apparently due to an increase in TVFA concentrations asthe concentration of uVFA depends on concentration of TVFA, pHand temperature (Kayhanian, 1999).

3.3.3. Ammonia and pHDepending upon the applied OLR, feed TKN concentration ran-

ged from 1.5 to 5.1 g/l with NH4–N accounting for 3–7% of TKN.

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0

1

2

3

0

400

800

1200

OL

R (k

gVS/

m3 d

)

Met

hane

yie

ld (m

l C

H4/

gVS)

6

7

8

9pH

0

6

12

18

SCO

D (

g/l)

0

1

2

3

NH

3or

NH

4-N

(g/

l)

0

2000

4000

6000

0 50 100 150 200

TV

FA (

mg/

l)

0

2

4

6

TK

N (g

/l)

Fig. 2. Characteristics of the effluent during semi-continuous anaerobic co-digestion of rendering plant and slaughterhouse wastes in CSTR at 55 �C; (h) methane yield,(–) OLR, (j) pH, (N) SCOD, (e) NH4–N, (�) NH3, (�) TKN and (s) TVFA.

34 S. Bayr et al. / Bioresource Technology 104 (2012) 28–36

NH4–N concentration in the effluent of mesophilic and thermo-philic reactor was 54–75% and 74–82% of TKN, respectively, indi-cating high ammonification of organic nitrogen in the feed.Salminen and Rintala (2002) also reported a similar ammonifica-tion of 52–67% ammonia of TKN in the effluent, compared to littleammonia present in the feed, during the anaerobic digestion of

poultry slaughterhouse waste at mesophilic conditions. These re-sults suggest that high amounts of ammonia would be formed dur-ing anaerobic digestion of the studied substrates that might inhibitmethanogenesis.

As expected, concentration of NH4–N was higher in the thermo-philic than in the mesophilic reactor. In thermophilic reactor,

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0

1000

2000

3000

4000

5000

0 50 100 150 200

VFA

(m

g/l)

Time (d)

0

500

1000

1500

2000

2500

3000

0 50 100 150 200

VFA

(m

g/l)

Fig. 3. TVFA, acetic acid, propionic acid and iso-valeric acid concentrations in semi-continuous CSTR experiments of rendering plant and slaughterhouse wastes at35 �C (upper) and 55 �C (lower); (j) TVFA, (e) acetic acid, (�) propionic acid, (s)iso-valeric acid. Concentrations of other acids were less than 100 mg/l at 35 �C andless than 280 mg/l at 55 �C.

S. Bayr et al. / Bioresource Technology 104 (2012) 28–36 35

NH4–N concentration increased throughout the experiment from1.6 to 3.0 g/l. On the other hand, NH4–N concentration in the mes-ophilic reactor was 0.8–1.1 g/l during days 1–101, but increased to2.4 g/l (on day 178), when OLR was increased to 1.5 kg VS/m3 d.However, the ammonia concentrations in the present study werebelow 3.86 and 5.60 g/l found to decrease methane productionby 50% in treating simulated OFMSW at 35 and 55 �C, respectively(Benabdallah El Hadj et al., 2009).

The calculated NH3 concentration, was 3–6 times higher inthermophilic than in mesophilic reactor and followed the sametrend as that of NH4–N. The calculated NH3 concentration in thethermophilic reactor was between 290 and 635 mg/l during thewhole experiment (except for days 1–14). The corresponding valuefor the mesophilic reactor was <115 mg/l (except for days 1–20).The calculated free ammonia values in the mesophilic process inthe present study were similar or lower than those reported inthe literature. For instance, Cuetos et al. (2008) reported NH3 con-centration of 115–270 mg/l during the anaerobic digestion ofslaughterhouse waste alone at 35 �C with an OLR of 0.9–1.7 kg VS/m3 d and 50–25 d HRT (pH 7.6–7.8) or 121–337 mg/lduring the co-digestion of slaughterhouse waste with OFMSW at35 �C with an OLR of 1.85–3.7 kg VS/m3 d and 50–25 d HRT (pH7.7–7.9). On the other hand, the calculated NH3 levels noticed dur-ing the thermophilic process in the present study were lower thanthe values of 980–990 mg/l reported during the thermophilic co-digestion of mixed pork waste with manure (OLR 12.5 g VS/day,21 d HRT, pH 8.05) and 1080–1330 mg/l reported during co-diges-tion of slaughterhouse waste and manure (OLR of 8.3 g VS/day,HRT of 21 d, pH 8.23) (Hejnfelt and Angelidaki, 2009). The highercalculated NH3 concentration in the thermophilic than in the mes-ophilic reactor was apparently due to higher operating tempera-ture, pH and NH4–N concentration in the former reactor.

Although pH in the thermophilic reactor in the present study re-mained more or less above 7.5, unstabilities in reactor operationwere seen in accumulating VFA and decreasing methane produc-tion. This is due to fact that the high ammonia concentration pro-vided necessary buffering capacity against pH drop (Angelidaki andAhring, 1994; Björnsson et al., 2000). On the other hand, pH in themesophilic reactor was more than 7 only from day 28 onwards.This low pH during the initial experimental period in the meso-philic reactor was apparently due to VFA accumulation (Figs. 1and 3). However, VFA accumulation during the later period ofrun did not result in a pH drop.

3.3.4. LCFAs and inhibitionLCFAs are one possibility for inhibition of the thermophilic

reactor in the present study, as also previously speculated in thetreatment of different slaughterhouse wastes in mesophilic andthermophilic CSTR processes (Cuetos et al., 2010; Hejnfelt andAngelidaki, 2009). From batch assays with individual LCFAs, it isknown that already low concentrations of some individual LCFAscan be inhibitory (Lalman and Bagley, 2000, 2001) and furthermoreit was shown that acetate-utilising methanogenesis was moresusceptible to oleic acid toxicity at 55 �C than at 35 �C (Hwu andLettinga, 1997). Thus it cannot be unambiguously concluded theseparate and/or combined role of LCFAs, ammonia or/and VFAson the failure in the present thermophilic CSTR.

In this study it was shown that long term co-digestion with onlywastes containing much lipids and proteins, viz. slaughterhouseand rendering plant wastes, is possible at least in mesophilic pro-cess. Methane production is high, but especially in thermophilicprocess apparently only relatively low OLRs can be applied and stillthe long term process stability cannot be ensured. In order to in-crease process stability, co-digestion with low lipid and proteinsubstrates have been studied (Cuetos et al., 2008; Luste andLuostarinen, 2010), However, further research is needed about spe-cific inhibition mechanisms and possibilities to increase processstability e.g. by removal of nitrogen before anaerobic process e.g.by ammonia stripping.

4. Conclusions

In this study 10 different rendering plant and slaughterhousewaste fractions were characterised showing high contents of lipidsand proteins and methane potentials of 262–572 dm3 CH4/kg VSadded. Co-digestion of rendering and slaughterhouse wasteswas shown to be feasible in mesophilic process with OLRs 1 and1.5 kg VS/m3 d and HRT of 50 d with methane yields of ca720 dm3 CH4/kg VS. For thermophilic process lowest studied OLR,1.5 kg VS/m3 d showed methane yield of 766 dm3 CH4/kg VS, butturned to be unstable after 1.5 HRT of operation apparently dueto accumulating ammonia, VFAs and probably also LCFAs.

Acknowledgements

This project was part of the project BIOVIRTA (processing biogasplant digestates into value-added products, 40281/08) co-financedby The Finnish Funding Agency for Technology and Innovation(Tekes) and several companies. Authors also greatly acknowledgeMervi Koistinen for her work in the laboratory.

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