Biochemical Engineering Journal 26 (2005) 22–28

Anaerobic co-digestion of a simulated organic fraction of municipalsolid wastes and fats of animal and vegetable origin

Anna Fernandez, Antoni Sanchez∗, Xavier FontEscola Universitaria Politecnica del Medi Ambient, Universitat Autonoma de Barcelona,

Rbla Pompeu Fabra 1, 08100 Mollet del Valles (Barcelona), Spain

Received 10 February 2005; accepted 18 February 2005

Abstract

The potential of mesophilic anaerobic digestion for the treatment of fats of different origin through co-digestion with the organic fractionof municipal solid wastes (OFMSW) has been evaluated. Co-digestion process was conducted in a pilot plant working in semi-continuousregime in the mesophilic range (37◦C) and the hydraulic retention time (HRT) was 17 days. During the start-up period the digester wasfed with increasing quantities of a simulated OFMSW (diluted dry pet food). When the designed organic loading was reached, co-digestionprocess was initiated. The fat used consisted of waste from the food industry (animal fat), its composition was very similar to that of thes p to 28%,a taining theo , total fatr FMSW. Inc a renewables©

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imulated OFMSW in relation to the long-chain fatty acid (LCFA) profile. Fat content in the feedstock was gradually increased und hence the organic loading, with stable operation of the digester. Animal fat was suddenly substituted by vegetable fat mainrganic loading. No accumulation of LCFA or volatile fatty acids (VFA) was detected in either case. After a short adaptation periodemoval throughout the experiment was over 88%, whereas biogas and methane yields were very similar to those of simulated Oonclusion, anaerobic co-digestion of OFMSW and fat wastes appears to be a suitable technology to treat such wastes, obtainingource of energy from biogas.2005 Elsevier B.V. All rights reserved.

eywords: Anaerobic processes; Biogas; Fat; Long-chain fatty acids; Organic fraction of municipal solid wastes; Waste treatment

. Introduction

During the last few decades, anaerobic digestion ofrganic matter has been presented as a suitable technologysed for treatment of organic wastes and production ofnergy from combustion of biogas[1–3]. Anaerobic diges-

ion technology has evolved quickly and, at present, cane competitive with aerobic systems, especially for treating

ndustrial wastewater and organic solid wastes with highrganic loading[4].

In anaerobic digestion, co-digestion is the term used toescribe the combined treatment of several wastes with com-lementary characteristics, being one of the main advantagesf the anaerobic technology. There is abundant literaturebout the utilization of co-digestion, such as co-digestion

∗ Corresponding author. Tel.: +34 93 5796784; fax: +34 93 5796785.E-mail address: asanchez@eupma.uab.es (A. Sanchez).

of organic fraction of municipal solid wastes (OFMSW) aagricultural residues[5,6], organic solid wastes and sewasludge[7] or more specific wastes[8].

Recent works on co-digestion have been focused osearch of synergisms or antagonisms among the co-digsubstrates[9]. For instance, the optimization of the carbto nitrogen ratio when co-digesting municipal wastessewage sludge is pointed as beneficial to methane yield[10].The improvement of the buffer capacity is also reportea positive effect in the co-digestion process[11]. On theother hand, some authors have shown negative resuco-digestion processes, which are attributed to the spcharacteristics of the digested wastes[12]. Additionally, theconfiguration of the anaerobic reactor (batch or continuone or two stages, mesophilic or thermophilic) has beeobjective of some works[13,14]. However, there is scarinformation about the response of an anaerobic reactoring a typical waste when several co-substrates of sim

369-703X/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.bej.2005.02.018

A. Fernandez et al. / Biochemical Engineering Journal 26 (2005) 22–28 23

biochemical composition but with different reported levelsof inhibition to the anaerobic microbial communities areco-digested. This is of special interest in the operation ofindustrial anaerobic digesters, where the feed co-substratescomposition may be variable.

Among the co-digested wastes, one of the most commonlyused are lipids. Lipids, characterized either as fats or oils andgreases, are one of the major organic matters found in foodwastes and some industrial wastewaters, such as those com-ing from slaughterhouses, dairy industries or fat refineries[15]. Lipids included in waste and wastewater consist mainlyof triacylglycerides and long-chain fatty acids (LCFAs). Tria-cylglicerides can be hydrolyzed to LCFAs and glycerol. Theprofile of LCFAs found in wastes and wastewaters usuallydepends on the origin (animal or vegetable) of the lipids; how-ever, LCFAs usually contain an even number of carbon atoms,typically within 14–24. LCFAs are successively degraded viathe �-oxidation pathway to acetate and hydrogen, which inturn are converted to methane[16,17].

If compared with other organic matter of differentbiochemical composition, lipids are attractive for biogasproduction. This is due to the fact that they are reducedorganic materials and have a high theoretical methane poten-tial [18]. However, anaerobic treatment of organic wasteswith high lipid content presents several problems. On theone hand, adsorption of lipids onto biomass can cause sludgefl enw sta-b enicbO en-t ionsoa3 di tena obicp tationo on-c rted[ iona pula-t stedl

lityo n ofm ter-m per-

Table 1Main properties of the simulated OFMSW (dry pet food) according to man-ufacturer’s data

Parameter Value

Moisture (%) 9.0Total volatile solids (%, dry basis) 91.0Total protein (%, dry basis) 21.0Total fat (%, dry basis) 10.0Total cellulose (%, dry basis) 3.3Ashes (%, dry basis) 7.0

ational conditions at the steady state; (ii) to investigate theco-digestion of simulated OFMSW and increasing amountsof fats of animal origin (mainly composed of palmitic, stearicand oleic acid); and (iii) to determine the effects of chang-ing to a fat of vegetable origin (mainly composed of lauric,myristic and palmitic acid).

2. Materials and methods

2.1. Materials

Three different substrates were used in the anaerobicdigestion: simulated synthetic OFMSW, which consisted ofdiluted dry pet food (Dog Menu, Affinity Pet Care, Barcelona,Spain), and two kinds of residual fats used as co-substrates.Pet food was chosen as a basic substrate because of its nutri-tional similarity with OFMSW (fibre, protein and fat content)[26,27]. Pet food was diluted with distilled water accordingto the organic loading applied to the digester. Residual fatswere: animal fat from the food industry (lard), the composi-tion of which was very similar to that of simulated OFMSWespecially in the LCFA profile, and commercial vegetable(coconut) oil. The main properties of the substrates used areshown inTables 1 and 2.

2

itha ges-t of al onsa g allt sixfl ani ipals n oft

TP tes

S :0) (<1%)

P 14.9A 17.0V 3.3

otation and washout[18,19]. On the other hand, it has beidely reported that high LCFA concentrations can deilise anaerobic digesters due to inhibition of methanogacteria by possible damage to cellular membrane[20,21].n that point, however, different values of inhibition conc

ration for different LCFA are reported. Thus, concentratf inhibition are in the range of 30–300 mg l−1 for oleiccid [4,22–24], 100–300 mg l−1 for stearic acid[23,24] or0 mg l−1 for linoleic acid[17]. The variability of the reporte

nhibition values for LCFAs on anaerobic digestion is ofttributed to the different characteristics of the anaeropulation (granular or suspended). Nevertheless, adapf microorganisms to high loads of LCFA to degrade centrations well above the inhibition limits are also repo22,25]. However, to our knowledge, there is no informatbout the transition and adaptation of the anaerobic po

ion to a sudden change in the composition of a co-digeipid (and thus in the LCFA profile to be degraded).

The objectives of this work are: (i) to study the suitabif anaerobic digestion to treat a simulated organic fractiounicipal solid wastes with a high percentage of lipids, deining the optimal and stable organic loading and the o

able 2ercentage of the main LCFAs found in the fats from different substra

ubstrate Lauric (C12:0) Mystiric (C14:0) Palmitic (C16

et food 0.2 2.2 32.9nimal fat NDa 3.0 30.0egetable fat 45.5 18.5 10.4a ND: not detected.

.2. Anaerobic digester configuration and operation

A semi-continuous, completely mixed liquid reactor wworking volume of 14 l was used for the anaerobic di

ion of wastes. The volume was maintained by meansevel control. The digester is cylindrical and its dimensire: 20 cm diameter and 58 cm height. Stirring rate durin

he experiments was 24 rpm with four disc turbines withat blades. The digester was inoculated with sludge fromndustrial-scale anaerobic digestion plant treating municolid wastes (Barcelona, Spain). The initial concentratiootal volatile solids (TVS) of the reactor was 1.56 g l−1. The

Stearic (C18:0) Oleic (C18:1) Linoleic (C18:2) Other LCFA

33.8 5.6 10.438.0 6.0 6.08.7 2.2 11.4

24 A. Fernandez et al. / Biochemical Engineering Journal 26 (2005) 22–28

Fig. 1. Scheme of the digester.

digester was inside a chamber where temperature was main-tained to 37◦C by a heating device. The substrate was fedfour times a day using a time-controlled pump. Feed influentwas prepared twice a week. A recirculation system was peri-odically working 6 h a day to ensure a good homogenizationof the digester liquid by pumping a flow of 150 ml min−1.The hydraulic retention time of the digester was maintainedto 17 days during all the experiments. Biogas produced in thedigester was collected and measured daily. A detailed schemeof the digester is presented inFig. 1.

During start-up, the influent organic loading was increasedfrom 0.55 to 2.57 kg TVS m−3 day−1, using diluted pet food,to find the optimal value. After seven HRT under stable con-ditions, the co-digestion process was started using animal fat.Animal fat was gradually increased up to 28% of the organicloading. At this point, animal fat was substituted by vegetablefat (coconut oil) maintaining the organic loading. Fats weremixed previously with the simulated OFMSW in the feedinfluent tank. For each experimental condition, the reactorwas continuously operated for at least two HRT to ensurea situation of steady state. Calcium bicarbonate was addedoccasionally to maintain the alkalinity level.

2.3. Biochemical methane potential assays

Biochemical methane potential (BMP) was assayed tod tionp orf uslys

and the biogas and methane production were analyzed inbatch experiments during approximately 70 h (when a plateauin the biogas production was reached). In experiments withfats, these were previously diluted with diethylether (DE,10 g l−1) due to the low solubility of the fats (Lalman andBagley, 2000). BMP was calculated according to the rate ofmethane generation per kg of TVS. A blank experiment withonly simulated OFMSW was carried out, jointly with a con-trol experiment with simulated OFMSW and DE. Inhibitioncaused by fats was calculated as the percentage of decreasein the methane generation in relation to that of the controlexperiment. BMP was statistically analysed by the Tukey’smethod at 5% level of probability.

2.4. Lipolytic activity

Lipolytic activity was determined using a commercial kit(Roche/Hitachi LIP no. 1821792). Briefly, samples (adjustedto pH 8.0 with a Tris–HCl buffer 400 mM) were mixed withTriton X-100 (1%) to extract the lipases[30]. After extraction,samples were centrifuged (30 min, 3500× g) and filtered(0.45�m) to remove biomass and solids. This sample is thenused for lipolytic activity determination according to manu-facturer’s instructions.

2.5. Analytical methods

2week

f total

etermine the potential toxicity of fats in the co-digesrocess. One hundred and fifty milliliters of mixed liqu

rom the digester was placed in 300 ml bottles, previoealed and purged with N2. Bottles were incubated at 37◦C

.5.1. Routine analysisThe feed and digester content were analyzed twice a

or routine parameters. Water content, total solids (TS),

A. Fernandez et al. / Biochemical Engineering Journal 26 (2005) 22–28 25

volatile solids, pH and alkalinity were determined accordingto the standard methods[28].

2.5.2. Fat contentThe quantification of the total lipid content was carried

out using a standard Soxhlet method usingn-heptane (99%purity, Panreac, Spain) as organic solvent[29].

2.5.3. Biogas compositionMethane and CO2 content in 200�l biogas samples col-

lected from the headspace of the reactor were analyzed by gaschromatography (Perkin-Elmer AutoSystem XL Gas Chro-matograph) with a thermal conductivity detector (TCD) andusing a column Hayesep 3m 1/8 in. 100/120. The carrier gaswas He in splitless mode (column flow: 19 ml min−1). Oventemperature was maintained at 40◦C during analysis. Injectorand detector temperatures were 150 and 250◦C, respectively.The system was calibrated with pure samples of methane andCO2 (99.9% purity, Carburos Metalicos, Spain).

Volatile fatty acid analysis: 50�l of sulphuric acid (98%)were added to 0.6 ml of sample. The acidified sample was thencentrifuged (30 min, 3500× g) and the resulting supernatantfiltrated through a Millipore Millex-FGS filter (0.2�m). Thissample was used for VFA determination by gas chromatog-raphy. A Perkin-Elmer AutoSystem XL Gas Chromatographwith a flame ionization detector (FID) and a HP Innowax3 aswA r1m ectort mw ix-t ric,i ) ofcf

n-r for3 cen-t ntfie hro-m ro-m wax3 aswA r1m ctort mw ureo ica ther sw

2.6. Data analysis

Statistical significance of values of different parametersobtained for consecutive organic loadings applied to thedigester was carried out by means ofF-test (variance analy-sis) and Student’st-test (mean analysis) both at 5% level ofprobability.

3. Results and discussion

3.1. Digestion of simulated OFMSW

Anaerobic digestion of simulated OFMSW was carriedout in two steps. Initially, the organic loading of the reactorwas progressively increased to find the maximum value understable conditions. In the second step, the steady state reachedwas maintained for seven hydraulic residence times to obtainthe main operational parameters for anaerobic digestion ofsimulated OFMSW. Both periods are shown inFig. 2.

Start-up period was characterized by a parallel increaseof the biogas production and TVS removal as the organic

Fig. 2. Organic loading, total VFA concentration, biogas production andTVS removal during the period of digestion of simulated OFMSW.

0 m× 0.25 mm× 0.25�m column was used. The carrier gas He and with a split ratio of 13 (column flow: 5 ml min−1).n initial oven temperature of 120◦C was maintained fomin; then, it was increased to 245◦C at 10◦C min−1, andaintained at that temperature for 2 min. Injector and det

emperatures were 250 and 300◦C, respectively. The systeas calibrated with different dilutions of a standard m

ure of VFAs (including acetic, propionic, butyric, isovalesobutyric, valeric and isocaproic acid from Sigma, Spainoncentrations in the range of 0–100 mg l−1. Detection limitor VFA analysis was 5 mg l−1.

LCFA analysis: 5 ml ofn-heptane (99% purity, Paeac, Spain) was added to 5 ml of sample and mixed0 min to extract LCFA. The suspension was then

rifuged (30 min, 3500× g) and the resulting supernataltrated through a Millipore Millex-FGS filter (0.2�m). Thisxtract was used for free LCFA determination by gas catography. A Perkin-Elmer AutoSystem XL Gas Chatograph with a flame ionization detector and a HP Inno0 m× 0.25 mm× 0.25�m column was used. The carrier gas He and with a split ratio of 13 (column flow: 5 ml min−1).n initial oven temperature of 120◦C was maintained fomin; then, it was increased to 250◦C at 8◦C min−1, andaintained at this temperature for 7 min. Injector and dete

emperatures were 250 and 275◦C, respectively The systeas calibrated with different dilutions of a standard mixtf LCFAs (including lauric, mystiric, palmitic, stearic, olend linoleic acid from Sigma, Spain) of concentrations inange of 0–100 mg l−1. Detection limit for LCFA analysias 5 mg l−1.

26 A. Fernandez et al. / Biochemical Engineering Journal 26 (2005) 22–28

Fig. 3. Concentrations of different VFAs during the period of digestion ofsimulated OFMSW.

loading increased, showing a good acclimation of sludge tosimulated OFMSW. However, from day 60, total VFA con-centration reached values above 1 g l−1. Although literaturevalues for inhibition caused by VFA are lower than thosefound in the start-up period[31], the decrease in the biogasproduction and TVS removal confirmed that the maximumorganic loading had been reached (Fig. 2). The relatively lowvalues of VFA inhibition in this process were probably causedby the type of VFA detected. InFig. 3, it can be observed thatalthough acetic acid was by far the dominant VFA produced,propionic acid and isovaleric acid were also detected. Theseacids are reported to be inhibitory for methanogenic bacteriaeven at trace concentrations[32].

To avoid problems of reactor instability and to permit astable co-digestion process, organic loading was fixed to alow value of 0.97 kg TVS m−3 day−1. The reactor was thenmaintained under these conditions during seven consecutiveHRTs showing a highly stable operation (Fig. 2), with valuesof total VFA concentration below 0.1 g l−1 and undetectedlevels of acetic acid, propionic acid and other VFA. Themain results corresponding to the steady state period are pre-sented inTable 3. Values of methane content in biogas andbiogas and methane yields were similar to those found formesophilic anaerobic digesters[33], whereas TVS removal

TO teadys

P ion

OTBTBMMTp

can be considered high when compared with values foundfor other organic wastes[33], which is probably due to thehigh biodegradability of simulated OFMSW. Nevertheless,it must be pointed that literature data show some dispersionin the values obtained for typical operational parameters ofanaerobic digesters, especially with organic solid wastes.

3.2. Inhibition of biochemical methane potential by fats

Inhibition experiments were carried out previously to theco-digestion process. Thus, anaerobic sludge from the reac-tor was incubated with different concentrations of animal fat(lard), the composition of which was very similar to that ofsimulated OFMSW (Table 2) being palmitic, stearic and oleicacid the dominant LCFAs. Results showed a percentage ofinhibition within the range of 10–20% calculated for fat addi-tions corresponding to increments of 10–25% of the organicloading at steady state. However, statistical analysis indicatedthat there were no significant differences among treatments.Nevertheless, it could be concluded that inhibition of BMPby animal fat should be low or inexistent and the co-digestionprocess could be initiated. Additionally, some authors havereported studies on acclimation of anaerobic sludge to severalinhibitors, including fats and LCFAs[4,22,25], which mightbe expected to occur in the anaerobic reactor.

3

tate.T hichL W( dsa tenti d bya dinga rep

erens nceo nots dur-i leica n-t hereh oor-g tw ithint ther ionb kedb tionofi ed( asd h the

able 3perational parameters for the digestion of simulated OFMSW at s

tate

arameter Value Standard deviat

rganic loading (kg TVS m−3 day−1) 0.97 –otal VFA concentration (g l−1) <0.1 –iogas production (l day−1) 8.0 1.5VS removal (%) 73 4iogas yield (m3 kg−1 TVS removed) 0.8 0.3ethane yield (m3 kg−1 TVS removed) 0.5 0.2ethane in biogas (%) 58 2

otal alkalinity (meq. CO32− l−1) 50 5H 8.0 0.2

.3. Co-digestion with animal fat

Anaerobic co-digestion was started from the steady she first co-substrate used was animal fat (lard), wCFA profile was very similar to that of simulated OFMSTable 2), with palmitic, stearic, oleic and linoleic aciccounting for the 91% of the total LCFA content. Fat con

n the influent of the reactor was progressively increasepercentage of 4, 7, 14, 21 and 28% of the organic loat steady state. InFig. 4, the main results for this period aresented.

Throughout the co-digestion process, levels of VFA wegligible (total content below 0.1 g l−1) indicating a hightability of the reactor, which was confirmed by the presef steady profiles of pH and alkalinity of the reactor (datahown). On the other hand, LCFA content was very lowng the co-digestion with animal fat, with only traces of ocid detected (below 12 mg l−1). These values of LCFA co

ent are quite lower than those reported in other studies, wigher contents cause inhibition to methanogenic micranisms[4,17,22–24]. Additionally, no accumulation of faas detected in the reactor, and total fat removal was w

he range of 88–94%, whereas TVS removal was withinange of 70–80%. It is likely that the similar compositetween animal fat and fat from simulated OFMSW provooth the animal fat fast hydrolysis and the quick consumpf hydrolysis products (glycerol and LCFA)[34]. To con-rm this, the lipolytic activity of the reactor was determin28% animal fat period). However, no lipolytic activity wetected throughout the co-digestion process. Althoug

A. Fernandez et al. / Biochemical Engineering Journal 26 (2005) 22–28 27

Fig. 4. Biogas production, biogas and methane yield for the different treat-ments in the co-digestion of simulated OFMSW and fats. Solid bars corre-spond to average values and vertical lines correspond to standard deviation.Consecutive treatments with different letters are statistically different.

reason for this is not clear, it may be hypothesized that lipoly-tic activity was beyond the detection level or that lipases wereused as substrate as soon as they hydrolyzed the fats. Moreover, some authors point out that the extraction of lipases fromsludge is not easy and that lipase activity remains immobi-lized on the organic particulate matter and/or intracellularmembrane or cell wall bound[30,35].

Biogas production, biogas and methane yields are pre-sented inFig. 4for each period of animal fat with increasingconcentration. Data inFig. 4 for each period was analysedfor statistical significance. Periods that are statistically dif-ferent are shown inFig. 4with different letters. A significantdecrease (p < 0.05) was observed for all the parameters whenanimal fat co-digestion was initiated, which was probably dueto a partial inhibition of the methanogenic activity as it was

also observed in the BMP assays. The inhibition observed inthe reactor was in the range of 25% for the studied parameters.However, when the fat loading was progressively increased,biogas and methane yields recovered the steady state levelsand biogas production increased according to an increase inthe total organic loading of the reactor. These results seemedto confirm that sludge was progressively acclimated to thenew co-substrate, as it has been reported in other works[22,25].

During the co-digestion with animal fats, methane con-tent in biogas slightly increased from 58 (steady state) to 61%(28% animal fat period). However, in the case of our study, thelow increase in the methane content did not rise significantlythe methane yield (Fig. 4). In other works, although methaneyield increases when lipids are used as co-substrates, the theo-retical methane yield calculated according to the�-oxidationof LCFA is not usually reached, which is often explained bysome degree of inhibition caused by LCFA[15,16].

3.4. Co-digestion with vegetable fat

Animal fat (28% period) was suddenly substituted by veg-etable fat (coconut oil) maintaining fat concentration and therest of operational conditions of the reactor. Results for thislast period of co-digestion are also presented inFig. 4. It isworthwhile to notice that LCFA profile for vegetable fat isc tedO hem cida pitet waso datan tinep wereo heret do igh( robics micalc bolicc d int

4

arst ofs s int ervedw a fato le.T arei cli-m triala trates

-

ompletely different from that of animal fat and simulaFMSW (Table 2) being short-chain-saturated LFCA tost predominant (lauric acid, mystiric acid and palmitic accounting for the 74% of the total LCFA content). Des

his fact, only a slight increase in the VFA concentrationbserved during the first days of vegetable fat feeding (ot shown), whereas LCFA concentration and other rouarameters (pH and alkalinity) remained steady. Therether operational parameters, such as methane yield, w

here was no statistical difference (p < 0.05) from that founn of animal fat, whereas total fat removal was very h97%). These results showed that an acclimatized anaeludge can degrade fats from different sources (and cheomposition). Besides, it seems that no important metahanges are implied in the hydrolysis of different fats anhe degradation of their LCFAs.

. Conclusions

A pilot size digester was operating for nearly two yereating wastes with high lipid content. From this periodtudy, the main conclusion is that no important differencehe performance of the anaerobic co-digestion were obshen a fat from animal origin was suddenly changed byf vegetable origin with a completely different LCFA profihis may indicate that no important metabolic changes

mplied in the degradation of different LCFAs with an acatized sludge. This is of special interest in the induspplication of co-digestion process, where the co-subs

28 A. Fernandez et al. / Biochemical Engineering Journal 26 (2005) 22–28

may be variable in composition and, thus, in expected inhi-bition.

Additionally, other relevant conclusions from this workare:

• During the steady state of the digester, treating a simulatedOFMSW, high percentages of TVS removal were obtained(73%), jointly with typical yields of biogas and methanegeneration (0.8 m3 biogas per kg of TVS degraded, 0.5 m3

methane per kg of TVS degraded, 58% of methane in bio-gas).

• After an adaptation period, the co-digestion with fatincreases the amount of biogas produced according to theapplied organic loading. The yields of biogas and methanegenerated per kg of TVS degraded are similar to thosefound with OFMSW, being the methane content slightlyhigher in the presence of fats.

• Both fats from animal or vegetal origin were almost com-pletely degraded in high percentages (94% for animal fatand 97% for vegetal fat), which confirmed that anaerobicdigestion is a suitable technology for treatment of thesewastes.

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