Bioresource Technology 100 (2009) 1903–1909

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Bioresource Technology

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Feasibility of anaerobic co-digestion as a treatment option of meat industry wastes

Inmaculada M. Buendía *, Francisco J. Fernández, José Villaseñor, Lourdes RodríguezChemical Engineering Department, ITQUIMA, University of Castilla-La Mancha, Avda. Camilo José Cela S/N 13071, Ciudad Real, Spain

a r t i c l e i n f o

Article history:Received 1 July 2008Received in revised form 9 October 2008Accepted 12 October 2008Available online 28 November 2008

Keywords:Ammonia inhibitionAnaerobic biodegradabilityBiodegradable fractionsCo-digestionMeat industry wastes

0960-8524/$ - see front matter � 2008 Elsevier Ltd. Adoi:10.1016/j.biortech.2008.10.013

* Corresponding author. Tel.: +34 902 204 100; faxE-mail address: inmaculadam.buendia@uclm.es (I.

a b s t r a c t

The anaerobic biodegradability of meat industry wastes was investigated in mesophilic batch reactorsand combined with a mathematical model for describing their biodegradable fractions. The characteris-tics and methane yield achieved when digesting waste sludge, suggested the use of this as co-substratefor enhancing the biodegradability of the other wastes. The co-digestion experiments showed that itwould be feasible to co-digest cow manure or ruminal waste with waste sludge, but biodegradabilityof pig/cow slurries was not improved, being strongly influenced by the ammonium concentration ofco-digestion mixture. By applying the mathematical model, it was observed that when increasing theamount of waste sludge in the co-digestion mixtures, the amount of inert and slowly biodegradable frac-tions decreased leading to an increase in readily biodegradable fractions, volatile solid removal efficien-cies and methane yields. These results suggest that using readily biodegradable wastes as co-substrate,the anaerobic biodegradability of complex organic wastes can be improved.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

A prolonged storage of organic waste (e.g. disposal in landfills)can cause natural and uncontrolled decomposition of the organicmatter, polluting soils, groundwater, surface waters and releasinggases such as methane and carbon dioxide (Møller et al., 2004;Vedrenne et al., 2008). Due to the increasing regulation on wastedisposal and as a result of the global warming effects resultingfrom the release of greenhouse gases, biological treatments of or-ganic wastes are increasing in importance as an option to reducethe water and soil pollution and greenhouse gas emissions.

The treatment of the organic wastes through anaerobic diges-tion processes has been widely recognized as a way to controlgreenhouse gas emissions and to use them for energy generation(Lettinga, 1995, 2001; Barton et al., 2008). In that respect, the EUcountries have agreed on supporting the production of biogas asa renewable energy source in combined heat and power plants inorder to decrease the greenhouse gas emissions according to theKyoto protocol (CEC, 2001). Additionally, the use of the anaerobicdigestion reduces the volume of waste, which after an aerobictreatment as composting, could be used as soil amendment.

Meat industries and farms are important sources of animalwastes including rumen, stomach and intestinal content fromslaughterhouses, slurry (low solid content) and manure (high solidcontent) from farms. For enhancing yields during the anaerobicdigestion when treating animal wastes, some studies show severalpossibilities, such as modifications in mechanical pre-treatment,

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: +34 926 295 242.M. Buendía).

temperature and mode of mixing (Karim et al., 2005; Kaparajuet al., 2008). Nevertheless, due to the high organic content, highbiological oxygen demand and low carbon/nitrogen ratio com-pared to domestic or vegetal waste, the enhancement of yields inthe anaerobic treatment of the meat industry wastes can beaccomplished by anaerobic co-digestion (Rosenwinkel and Meyer,1999; Carucci et al., 2005; Macias-Corral et al., 2008). It should benoticed that anaerobic co-digestion is not a treatment option forthose animal wastes presenting a risk for transmissible spongiformencephalopathy, which must be completely disposed of a waste byincineration, co-incineration or landfill (Edström et al., 2003).

However, the large ammonia concentrations in animal wastesare found to inhibit anaerobic treatments (Nielsen and Angelidaki,2008), a problem that is further accentuated for proteins-richwastes, such as slaughterhouse wastes, for which the ammoniaconcentration rises significantly during their fermentation (Salmi-nen and Rintala, 1999; Chen et al., 2008). Capela et al. (2008) foundthat the buffer capacity of wastes can be balanced and the effect oftoxic or inhibitory compounds on the process may be minimizedusing different substrates as co-digestates, improving the stabilityand process performance. In addition, anaerobic co-digestion al-lows for digestion of poorly biodegradable wastes, which cannotbe digested alone (e.g. fat or protein wastes), when mixing withother more degradable wastes (Alatriste-Mondragón et al., 2006).The interest in the co-treatment of this type of organic wastes to-gether with the sludge generated in the municipal wastewatertreatment plants is increasing, since existing treatment plants witha complete infrastructure for gas utilization and wastewater treat-ment can be used (Weiland, 2000). In addition, the co-digestion ofactivated sludge with other organic wastes is a common practice

1904 I.M. Buendía et al. / Bioresource Technology 100 (2009) 1903–1909

adopted in the wastewater treatment plants in order to improveperformances of the anaerobic digesters (Bolzonella et al., 2006).

The objective of this work was to study the feasibility of usingthe waste sludge generated in a biological wastewater treatmentplant, treating the wastewater of the meat industry, as co-sub-strate to enhance the anaerobic digestion, under mesophilic condi-tions, of cow manure, ruminal waste and pig/cow waste slurry interms of methane productivity and VS reduction. In addition, thiswork was aimed at estimating the different biologically degradablefractions (readily, slowly and inert fractions) in meat industrywastes when they were used as co-digestates of the waste sludgein order to provide information on the changes of biodegradabilityof the different wastes during co-digestion.

2. Methods

2.1. Inoculum and substrate sources

Mesophilically digested material from the anaerobic digester ofa conventional activated sludge wastewater treatment plant wasused as seed sludge. The anaerobic inoculum was kept at 35 �C dur-ing one week for removing any remaining biodegradable fraction.

The four organic wastes used as substrates in this study camefrom a meat industry located in Valdepeñas (Spain): waste sludge(WS), cow manure (CM), ruminal waste (RW) and pig and cowwaste slurries (PCS). WS was produced in a high load activatedsludge process used to treat the wastewater generated in theslaughterhouse. CM mixed with straw as bedding material (15%in weight), RW obtained from rumen paunch material generatedin the slaughterhouse and PCS, were all taken from animal stablesclose to the slaughterhouse.

These organic wastes were collected in samples of more than50 kg in order to obtain representative samples. Afterwards, thosesamples were homogenized using a double-helix mixer and trans-ferred into 5 l containers until being used as feeding substrate. Thecharacteristics of the different substrates are showed in Table 1.

2.2. Chemical analyses

The characterization of the wastes was performed by analysis oftotal solids (TS), volatile solids (VS), total-nitrogen (T-N),total-phosphorus (T-P), ammonium–nitrogen (NHþ4 –N) and pHaccording to Standard Methods (APHA et al., 1998). Total-carbonconcentration (T-C) was analysed using a TOC Shimadzu 5050Aequipment. Ca2+, K+, Fe2+ and Mg2+ were determined by flameatomic absorption spectrometry and heavy metals were measuredby ICP-Mass Spectrometry. Methane and carbon dioxide concen-trations in gas samples were determined with gas chromatographyusing a thermal conductivity detector.

Table 1Characteristics of the organic wastes used in the experiments.

Parameter Waste sludge Cow ma

Total Solid (%) (w/w) 17 31Volatile Solid (%) (w/w) 11 20pH 8.4 7.7Total-C (g kg�1 waste) 650 470Total-N (g kg-1 waste) 41 12NH4

+–N (g kg�1 waste) 1.3 1.1Total-P (g kg�1 waste) 7.0 11Ca2+ (g kg�1 waste) 37 41K+ (g kg�1 waste) 18 27Mg2+ (g kg�1 waste) 9.0 3.0Fe2+ (g kg�1 waste) 1.5 1.5Metals (g kg�1 waste) <LOD <LOD

LOD: Limit of detection (Cd: 1.5 ppb; Cr: 4 ppb; Cu: 2 ppb; Hg: 8.5 ppb; Ni: 5.5 ppb; Pb

2.3. Anaerobic experiments

Thirty-four anaerobic batch experiments (seventeen in dupli-cate) were carried out in 1 l-batch reactors with an active volumeof 400 ml operated under mesophilic conditions (35 �C). The differ-ent wastes were mixed in different co-digestion ratios from zero%to 100% of the total VS fed to the reactor, keeping the same food/microorganism ratio (F/M) at 1.2 g VS g VS�1. Blank experimentswithout any substrate added were included. The biogas releaseto the gas phase was measured with a manometric sensor incorpo-rated at the top of the reactors and two sampling valves were fittedto the reactor wall to take both liquid and gas samples during theincubation. Stirring was performed with a magnetic stirrer to avoidthe stratification of solids and to ensure a good degree of contactbetween substrate and microorganisms. The experimental designof the batch experiments is presented in Table 2.

2.3.1. Cumulative methane productionCumulative methane production, expressed as litres of CH4 per

kg of VS loaded to the reactor, was assessed by measurement of thebiogas production continuously registered in the manometric sen-sor placed in the anaerobic reactor and the methane compositiondata. The maximum methane production rate (Rmax) and lag phase(k) were determined by fitting the modified Gompertz model de-scribed by Zwietering et al. (1990) and Lay et al. (1998), to theexperimental cumulative methane production curves correctedfor biogas production in the blanks. Consequently, the equationused to obtain the maximum methane production rate had the fol-lowing form:

M ¼ P � exp � expRmax � e

Pðk� tÞ þ 1

� �� �ð1Þ

M, cumulative methane production (L CH4 kg VS�1loaded; P, meth-

ane potential (L CH4 kg VS�1loaded); t, time (days);. Rmax (L

CH4 kg VS�1loaded) and k (days).

‘GraphPad Prism 5.0’ software package was used for estimatingthe value of the model parameters (P, Rmax and k) Eq. (1) with theminimum residual sum of squared errors between the experimen-tal data and model curve.

2.3.2. Quantification of ammonia inhibitionTo describe the influence of ammonium/ammonia inhibition,

the concentration of free ammonia [NH3] was calculated takinginto account the ammonium concentration ½NHþ4 �, pH and temper-ature (T) into the anaerobic reactor (Gallert and Winter, 1997)

½NH3� ¼½NHþ4 � � 10PH

k b=kw þ 10PH ) kb=kw ¼ eð6433=TÞ ð2Þ

nure Ruminal waste Pig and cow slurries

13 2.511 1.48.2 8.0450 45015 410.6 8.51.0 9.037 232.0 232.0 7.06.0 1.3<LOD <LOD

: 14 ppb).

Table 2Experimental design of digestion and co-digestion experiments.

Mixing ratio (% of VS) Experiments

WS:CM WS:RW WS:PCS

0:100 x x x10:90 – – x25:75 x x x35:65 – – x50:50 x x x75:25 x x x100:0 x x x

I.M. Buendía et al. / Bioresource Technology 100 (2009) 1903–1909 1905

kb, ionization constant of ammonia; kw, water dissociation constantand T (K).

The following extended Monod model was selected for assess-ing the influence of ammonia or ammonium concentration onanaerobic digestion at a specific pH (Sung and Liu, 2003):

Rmax ¼ R0 1� II�

� �n SSþ ksð1� ðI=I�ÞÞm� �

ð3Þ

R0, maximum methane production rate without inhibition(L CH4 kg VS�1

loaded); I, inhibitor concentration (g m�3); I*, lethalinhibitor concentration (g m�3); S, substrate concentration(g VS m�3); kS, half-saturation constant (g VS m�3); n, dimension-less coefficient and m, dimensionless coefficient.

The different parameters involved in the extended Monod equa-tion (R’, I*, ks, n and m) were estimated using AQUASIM 2.0 softwarepackage by minimizing the sum of the squares of the weighteddeviations between the experimental Rmax, at different ammoniumconcentrations, and the model results using the secant algorithm(Ralston and Jennrich, 1978). The absolute-relative sensitivityfunction described by Reichert (1998) was also used to determinethe values of the most sensitive parameters and to assess the effectof the different parameters (R’, I*, ks, n and m) on the maximummethane production rate.

2.3.3. Estimation of biodegradable fractionsThe biodegradable fractions of the organic wastes was divided

into readily (SS) and slowly (X) biodegradable fractions in accor-dance with Spanjers and Vanrolleghem (1995) and De Lucaset al. (2007). Their consumption can be described using Monodkinetics without mutual interactions by a modification of theMethane Production model (MP model) as described by Rodríguezet al. (2007). Thereby, the different biodegradable fractions and thekinetic parameters of the different co-digestion mixtures weredetermined. Due to the low growth rate of the anaerobic microor-ganisms involved in the anaerobic digestion process, the mass bal-ances applied to the anaerobic batch processes were

dSdt¼ dSs

dtþ dX

dtþ dSI

dtð4Þ

dsbiodegradable

dt¼ �rmax;Ss �

Ss

KSs þ Ss� rmax;X �

Xkx þ X

ð5Þ

dCH4

dt¼ YCH4 �

dsbiodegradable

dtð6Þ

SS, concentration of readily degradable fraction (g VS m�3); X, con-centration of slowly degradable fraction (g VS m�3); SI, concentra-tion of inert fraction (g VS m�3); rmax,Ss, maximum apparentdegradation rate for readily degradable fraction (g VS m�3 d�1);kSs, half-saturation coefficient for readily degradable fraction(g VS m�3); rmax,X, maximum apparent degradation rate for slowlydegradable fraction (g VS m�3 d�1); kX, half-saturation coefficientfor readily degradable fraction (g VS m�3) and YCH4 ,methane yield(L CH4 g VS�1 m3).

In order to determine the effect of the different parameters(rmax,i, ki, SS and X) on the reduction of biodegradable VS and meth-ane production, the absolute-relative sensitivity analysis describedby Reichert (1998) was performed using ‘AQUASIM 2.0’ softwarepackage. This analysis showed that the sensitivity of the calculatedmodel variables to the parameter ki was much lower in comparisonto that showed to the parameters rmax,i, SS and X, leading to a largeruncertainty of the estimate ki. Therefore, the ki values were takenfrom literature (Angelidaki et al., 1999; Siegrist et al., 2002).

To assess the biodegradable fractions (SS and X) and the kineticparameters (rmax,Ss and rmax,X), the set of Eqs. (5) and (6) was solvedsimultaneously using the Gauss–Newton algorithm. An initial setof values was assigned to those parameters and, after several iter-ations, the values of those parameters that yielded the minimum ofthe sum of squared errors (SSE) were chosen as the best estimate.The SSE expression used to estimate the parameters was

vðpÞ ¼

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiPni¼1ðymeas;i � yðpÞÞ

2

s

yþ

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiPni¼1ðxmeas;i � xiðpÞÞ2

s

xð7Þ

where y (biodegradable VS reduction) and x (methane production)are the model variables, ymeas,i and xmeas,i are the ith measurement,yi(p) and xi(p) are the calculated value of the model variable corre-sponding to the i th measurement and evaluated at the time andlocation of this measurement. p ¼ ðp1; :::; pmÞ are the modelparameters, y and x are the mean between the maximum and min-imum value of the measurements and n is the number of datapoints.

In order to ensure a correct estimation of the model parameters,their initial values were chosen according to Orhon et al. (1997).

3. Result and discussion

3.1. Methane productivity: digestion experiments

The cumulative methane production of the four meat industrywastes is presented in Fig. 1.

The curves corresponding to the digestion of the raw wastes arepresented as 100:0 or 0:100. Taken overall, these results suggestthat, based purely in the biogas production, it would be interestingto improve methane yields of CM, RW and PCS by using anaerobicco-digestion. Due to its good anaerobic biodegradability, WS wasthe most promising waste that could be used as co-substrate.

CM and RW were slowly degraded only reaching 84 L CH4 kgVS�1

loaded and 45 L CH4 kg VS�1loaded, respectively, in comparison to

WS (500 L CH4 kg VS�1loaded) after 50 days. These low methane yields

could be due to the slowly degradable lignocellulosic compoundsthat are present in CM and RW, and for which hydrolysis is difficultand slow (Schmidt and Thomsen, 1998; Møller et al., 2004). Theruminal waste (RW) may contain a fraction of blood, and the lowmethane yield could also be due to the accumulation of high levelsof ammonia resulting from the degradation of nitrogen rich proteincomponents of blood (Banks and Wang, 1999). Pig/cow waste slur-ries (PCS) could not be biodegraded and it produced even lessmethane than the blank experiments. This may be explained byammonia inhibition taking into account the NH4

+ level in PCS,8.5 g L�1 (Table 1) (Hansen et al., 1998; Chen et al., 2008).

3.2. Methane productivity: co-digestion experiments

The average values of the cumulative methane production inco-digestion experiments (Table 2) are presented in Fig. 1. In orderto interpret influence of adding the readily biodegradable substrate(WS), the Gompertz model was used to describe the methane pro-duction of the different experiments.

0

100

200

300

400

500

600WS:CM

L C

H4 k

g V

S-1 load

ed

Time (days)

0

100

200

300

400

500

600WS:RW

L C

H4 k

g V

S-1 load

ed

Time (days)

0 10 20 30 40 50 60

0

100

200

300

400

500

600WS:PCS

100:0 90:10 75:2565:35 50:50 25:75 0:100

L C

H4 k

g V

S-1 load

ed

Time (days)

8070

0 10 20 30 40 50

0 10 20 30 40 50

a

c

b

Fig. 1. Mean cumulative methane production from the anaerobic experiments ofthe different wastes. Co-digestion of waste sludge (WS) with: (a) cow manure(WS:CM); (b) ruminal waste (WS:RW); (c) pig/cow waste slurry (WS:PCS). The co-digestion ratios are expressed as weight percentage of total VS in the mixture. Notethe different time scale in WS:PCS experiments.

1906 I.M. Buendía et al. / Bioresource Technology 100 (2009) 1903–1909

Fig. 2a shows an example of the fitting of the Gompertz equa-tion to the experimental cumulative methane production of theco-digestion experiment carried out with WS and PCS (75:25).The methane production rates deriving both from the experimen-tal data and the model description are shown in Fig. 2b. As canbe seen in Fig. 2a, the accuracy of the curve fitting described bythe fitting residuals was lower than 2%.

Table 3 shows the model parameters for the Gompertz modelEq. (1) (methane potential, P; maximum methane production rate,Rmax and lag phase, k) determined for the digestion and co-diges-tion experiments.

For each co-digestion mixture, it can be seen that the methanepotential increased with the amount of volatile solids from WS (Ta-ble 3). Since the WS was the waste with higher anaerobic biode-

gradability when digesting alone, the increase in the amount ofWS in the co-digestion mixture resulted in higher methane poten-tial. In the same way, it can also be observed that the lag phase (k)and the maximum methane production rate (Rmax) also depend onthe percentage of WS in the co-digestion mixtures. For example, theco-digestion experiment carried out with 25% of WS and 75% of CMpresented a k of 4.3 d and an Rmax of 11.7 L CH4 kg VS�1 d�1, whilethe mixture of 75% of WS and 25% of CM presented a value of kand Rmax of 1.2 d and 29.2 L CH4 kg VS�1 d�1, respectively. In everymixture the same trend was observed, that is, with increasing addi-tion of WS, k decreased and Rmax increased (Table 3). Thus, these re-sults can contribute to the design of treatment of complex wasteslike meat industry wastes at different co-digestion ratios.

According to Fig. 1 and Table 3, the use of WS as co-substrateimproved the process performance with respect to the methaneyield reached in the digestion of the raw wastes (CM, RW andPCS). For instance, a two-fold increase in methane production(from 84 L CH4 kg VS�1

loaded to 177 L CH4 kg VS�1) was observedwhen co-digesting CM with WS in a ratio of 25:75 (WS:CM) andan almost four-fold increase (from 49 L CH4 kg VS�1 to194 L CH4 kg VS�1) was achieved by co-digesting RW with WS atthe same co-digestion ratio (25:75; WS:RW). However, whenfocussing the attention on the digestion of the raw WS, it could alsobe said that the methane yield decreased as the amount of waste(CM, RW and PCS) increased in the binary mixtures. That is,500 L of methane per kg of VS loaded were obtained when WSwas treated alone, in comparison to the 346 L CH4 kg VS�1

loaded thatwere obtained when 75% of the total VS came from WS and 25%from RW. These trends were the same for every co-digestion mix-ture (Table 3).

In order to explain whether the overall methane yield did in factincrease by co-digestions, it was necessary to make a thoroughanalysis of methane productivity. To establish whether digestionor co-digestion of the wastes would be the most feasible option,the theoretical methane yield of the different co-digestion experi-ments was assessed based on the methane potential of the rawwastes when digesting alone and, afterwards, compared with theexperimental methane potential reached in the different co-diges-tion experiments.

For the co-digestion experiments of WS with CM and WS withRW, the methane yield seemed to be the same from both experi-mental and theoretical productions. This means that it would befeasible to co-digest CM and RW with WS. However, for WS:PCSco-digestion experiments, the experimental methane potentialsshowed lower values than the theoretical ones. These results indi-cate that co-digesting PCS with WS yielded lower methane produc-tion than digesting the raw wastes separately. Since this may bedue to ammonia inhibition, the influence of ammonia on WS:PCSexperiments was evaluated in depth.

3.3. Effects of ammonia concentration

The effects of ammonia concentration on the anaerobic biode-gradability of the mixtures of WS and PCS were described usingthe extended Monod model Eq. (3) at a specific pH. The initialpH for every WS:PCS co-digestion experiment had a variationof ± 0.1, therefore it was assumed that the Eq. (3) could be usedfor assessing the effect of ammonia in the WS:PCS co-digestionmixtures.

The values of estimated parameters from the extended Monodmodel and their sensitivities are shown in Table 4. The small sen-sitivity of the methane production rate to the parameters kS and m,2.6 � 10�3 and 2.1 � 10�3 L CH4 kgVS�1 d�1, respectively, led to anuncertainty of the estimation of those parameters. Because of that,their values were taken from literature (Rodríguez et al., 2007;Sung and Liu, 2003).

0

50

100

150

200

250

300

-4-2024

CH4 prod. data

Gompertz mod.

L C

H4 k

gVS-1

load

ed

Time (days)

Res

idua

ls (

%)

0.0

1.5

3.0

4.5

6.0

7.5

9.0

L C

H4 k

gVS-1

load

ed d

-1

Time (days)

R data R model

15 45 60 75 90300

15 45 60 75 90300

15 45 60 75 90300

a b

Fig. 2. Parameter estimation from Gompertz model: (a) Cumulative methane production and curve fitting residuals; (b) Methane production rate for the co-digestionexperiments between WS and PCS when they were mixed in a co-digestion ratio of 75:25 (expressed as weight percentage of the total VS added to the reactor). Dots representthe experimental data, while straight lines indicate the model curve.

Table 3Summary of the estimated parameters from Gompertz equation for digestion and co-digestion experiments at different co-digestion ratios expressed as weight percentage oftotal VS in the mixture.

Mixture (%/%) P (L CH4 kgVS�1) Rmax (L CH4 kgVS�1 d�1) k (days) aR2

WS:waste 100:0 500 ± 2.96 32.9 ± 0.48 4.2 ± 0. 16 0.998

WS:CM 0:100 84 ± 0.43 4.8 ± 0.14 5.7 ± 0.27 0.99725:75 177 ± 0.80 11.7 ± 0.29 4.3 ± 0.20 0.99850:50 301 ± 2.29 10.7 ± 0.23 3.1 ± 0.14 0.99875:25 389 ± 1.20 29.2 ± 0.56 1.2 ± 0.30 0.987

WS:RW 0:100 49 ± 0.97 1.7 ± 0.08 6.5 ± 0.61 0.98425:75 174 ± 0.94 7.2 ± 0.26 2.3 ± 0.47 0.99750:50 305 ± 0.37 14.2 ± 0.12 1.5 ± 0.10 0.99975:25 346 ± 1.17 20.7 ± 0.37 0.1 ± 0.15 0.989

WS:PCS 0:100 – – – –25:75 36 ± 3.46 0.4 ± 0.89 13.1 ± 0.72 0.94950:50 206 ± 1.60 3.3 ± 0.04 12.7 ± 0.31 0.99865:35 219 ± 0.85 6.1 ± 0.09 9.2 ± 0.27 0.99775:25 258 ± 0.59 8.0 ± 0.09 6.1 ± 0.18 0.99890:10 352 ± 0.60 16.2 ± 0.18 5.8 ± 0.13 0.999

–: Not possible to estimate.a Coefficient of determination: R2.

Table 4Values of the estimated parameters for the extended Monod equation and the meanabsolute-relative sensitivities for the co-digestion experiments carried out with WSand PCS from 100:0 to 0:100.

Parameter Value A-R sensitivity (L CH4 kg VS�1 d�1)

R’ (L CH4 kg VS-1 d�1) 41.7 ± 1.33 6.20I* (g NH4

+–N L�1) 8.4 ± 1.98 1.38n 5.3 ± 0.62 1.27aks (g SV L�1) 0.2 2.6�10�3

am 2.0 2.1�10�3

a Not estimated.

I.M. Buendía et al. / Bioresource Technology 100 (2009) 1903–1909 1907

As a result of the fitting procedure of the extended Monod equa-tion, the lethal ammonium concentration, I*, causing a 100% inhibi-tion on methane production, would occur at 8.4 g L�1 (0.6 g L�1

NH3–N at pH7.83) (Table 4). According to Sung and Liu (2003),the ammonia inhibition was characterized by an uncompetitiveinhibition when treating a mixture of WS and PCS since the valuesof m (2.0) and n (5.3), derived from the extended Monod equation,were greater than zero.

The methane production rate of the different co-digestion mix-tures was strongly influenced by the ammonium concentration.Thus, a 50% inhibition of maximum methane production rate wasobserved at a co-digestion ratio of 90:10 (WS:PCS), correspondingto 1.13 g L�1 of NH4

+–N (0.07 g L�1 NH3–N) in the liquid phase,increasing to 80% when the ammonium level was 2.77 g L�1

(0.23 g L�1 NH3–N) corresponding to a co-digestion ratio of 65:35(WS:PCS). These values are in accordance with those found in liter-ature, which reported that ammonium inhibition started to be no-ticed in a range from 1.5 to 2.5 g L�1 when treating cattle manureunder mesophilic conditions with methanogenic cultures not pre-viously acclimated to high ammonia concentrations (Koster andLettinga, 1984; Hashimoto, 1986). In terms of free-ammonia, Braunet al. (1981) stated that a level of 0.15 g L�1 could cause growthinhibition in unadapted cultures when treating liquid pig manure.Gallert and Winter (1997) also reported that at a pH of 7.6, 0.22–0.28 g L�1 of NH3 could cause 50% inhibition of mesophilic glucosefermentation and methane production. The difference found be-tween the ammonia level in the surveys above mentioned and thisstudy for 50% of inhibition (0.068 g L�1; pH7.75), could be attrib-uted to the different nature of the wastes subjected to digestion

0

25

50

75

100 0

25

50

75

100

WS:CM

X (%

)S I (%

)

SS (%)

0

25 75

100

WS:RW

X ((%

)

0 25 75 10050

a

b

1908 I.M. Buendía et al. / Bioresource Technology 100 (2009) 1903–1909

and the different inoculum used in each study (Alatriste-Mond-ragón et al., 2006).

3.4. Estimation of biodegradable fractions

In order to estimate how the co-digestion of the differentwastes affects to the different biodegradability fractions, the Eqs.(2)–(4) were fitted to the experimental data of both the cumulativemethane production and the reduction of biodegradable VS. As anexample, Fig. 3 shows the cumulative methane production and VSremoval curves of both the experimental data and the modified MPmodel for the 75:25 (WS:PCS) co-digestion experiment.

Table 5 summarizes the estimated kinetic coefficients resultingfrom the mathematical fitting and VS removal efficiencies for thedifferent digestion and co-digestion experiments.

The estimated readily biodegradable fraction (SS), slowly biode-gradable fraction (X) and inert fraction (SI) for the different co-digestion experiments can be seen in Fig. 4.

The distribution of the different fractions from the co-digestionof WS with CM is shown in Fig. 4a. It can be observed that whenincreasing the amount of WS in the mixture, the amount of SI

and X fractions decreased, leading to an increase in the readily bio-degradable fraction (SS). In the same way, the mixtures with higher

0 10 20 30 40 50 600

20

40

60

80

100

0

30

60

90

120

150

180

210

240

270

VS bi

odeg

rada

ble(%

)

Time (days)

MP data MP model VS data VS model

L C

H4 k

g V

S-1

load

ed

Fig. 3. Fitting of the modified methane potential model for the determination of thedifferent biodegradable fractions, cumulative methane production and biodegrad-able VS from the co-digestion experiment of WS:PCS (75:25).

Table 5Values of the different biodegradable fractions expressed as weight percentage of thetotal VS added to the reactor, maximum apparent degradation rate and VS removalefficiency for the different organic wastes and mixtures.

Mixture (%:%) (% VS) (g VS m�3 d�1) (% VS)

SS X SI rmax,Ss rmax,X Removal

WS 74 9 17 420 ± 5.8 42 ± 0.3 82CM 34 20 46 323 ± 7.3 82 ± 0.2 54RW 1 48 51 312 ± 10.3 25 ± 0.9 49PCS – – – – – 0

WS:CM 25:75 39 19 42 175 ± 2.9 65 ± 0.1 5750:50 47 14 39 162 ± 5.9 53 ± 0.6 6175:25 67 10 23 338 ± 12.3 49 ± 0.6 77

WS:RW 25:75 53 22 25 228 ± 5.9 87 ± 1.1 7550:50 58 18 24 262 ± 17.3 63 ± 0.7 7675:25 66 15 19 385 ± 22.3 62 ± 0.6 81

WS:PCS 25:75 1 11 88 100 ± 6.1 32 ± 0.4 1150:50 3 36 61 149 ± 4.7 58 ± 2.2 3965:35 6 52 42 172 ± 5.0 65 ± 1.7 5875:25 38 30 32 208 ± 2.5 56 ± 0.4 6890:10 50 21 29 213 ± 0.4 74 ± 2.6 71

–: Not possible to estimate.

50

75

100 0

25

50

%)S I

SS (%)

0

25

50

75

100 0

25

50

75

100

WS:PCS

X (%

)

100:0 90:10 75:2565:35 50:50 25:75 0:100

S I (%

)

SS (%)

0 25 75 10050

0 25 75 10050

c

Fig. 4. Distribution of SS (readily biodegradable fraction), X (slowly biodegradablefraction) and SI (inert fraction) for the different mixtures at different co-digestionratios. Co-digestion of waste sludge (WS) with: (a) cow manure (CM); (b) ruminalwaste (RW) and (c) pig/cow waste slurry (PCS). The co-digestion ratios areexpressed as weight percentage of total VS in the mixture and the fractions areexpressed as weight percentage of the total VS added to the reactor.

proportion of WS presented an increase in both the percentage ofVS removal efficiency and the maximum apparent degradation ratefor the readily degradable fraction (Table 5). Moreover, the maxi-mum apparent degradation rate observed for the SS fraction was al-most a ten-fold higher than that observed for X. The mixtureconsisting of 75% of CM achieved a VS removal of 57%, while mix-tures with 25% of CM reached a value of 77% of solids reduction.

I.M. Buendía et al. / Bioresource Technology 100 (2009) 1903–1909 1909

The same trend was observed for the co-digestion experimentscarried out with a mixture of WS and RW, i.e. increasing the per-centage of WS in the mixture, decreased the fractions of X and SI

(Table 5). However, in the digestion experiment of raw RW, almostno SS fraction was observed ( � 1%), and just by co-digesting RWwith WS in a co-digestion ratio of 25:75 (WS:RW), the amount ofSS increased up to 50% (Fig. 4b), indicating that the biodegradablefractions of the wastes are mainly readily biodegradable. TheWS:RW mixtures presented a VS removal that varied between 75and 81%, a range that was similar to the VS removal efficiency ofWS when was digested alone (82%). In addition, the values of thekinetic coefficients found for the WS:RW mixtures were similarto those estimated for the raw WS (Table 5).

Regarding WS and PCS co-digestion experiments, two differentzones can be distinguished in the distribution of the different esti-mated fractions (Fig. 4c). The first zone, from 100:0 to 65:35(WS:PCS), shows that the increase in the amount of PCS led to anincrease of X with decreasing SS content in the mixture, but alsoa fraction of slowly degradable fraction (X) was converted to inertfraction (SI). As the proportion of SI increased from 17% (100:0) to42% (65:35), the VS removal efficiency decreased from 82% (100:0)to 58% (65:35). The second zone, from 65:35 to 0:100 (WS:PCS),shows that the biodegradable fractions were converted to inertfraction, drastically lowering the reduction of VS from 39%(50:50) to 0% (0:100). Consequently, the kinetic coefficients werenegatively affected when increasing the amount of PCS in the co-digestion mixtures.

These results may be linked to the different ammonia levelspresent for the co-digestion experiments carried out with WSand PCS. It can be observed that when increasing the amount ofPCS in the mixture, the inert fraction (SI) increased gradually(Fig. 4). This does not mean that the WS:PCS mixtures have no bio-degradable fractions, but rather that the microorganisms popula-tion was partially inhibited and therefore not able to degrade thewhole organic waste when the mixture had an ammonium concen-tration higher than 2.77 g L�1 (65:35; WS:PCS).

3.5. Conclusion

Overall, this study contributes with a methodological approachfor feasibility studies of co-digestion of complex waste mixtures.The use of waste sludge generated in the wastewater treatmentplant of a meat industry as co-substrate, is considered to be aninteresting option for treating complex organic wastes, in this casegenerated in the same industry, because it is both economical andenvironmentally sound. Thereby, transport and chemical costs canbe reduced and the simultaneous treatment of different complexorganic wastes would be more efficient, because those wastescan be treated in the same facility, improving the energy balanceat the plant.

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