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Mesophilic and thermophilic co-fermentation of cattle excretaand olive mill wastes in pilot anaerobic digesters

M. Goberna a,*, M.A. Schoen b, D. Sperl a, B. Wett b, H. Insam a

a University of Innsbruck, Institute of Microbiology, Technikerstrasse 25d, 6020 Innsbruck, Austriab University of Innsbruck, Institute of Infrastructure, Technikerstrasse 13, 6020 Innsbruck, Austria

a r t i c l e i n f o

Article history:

Received 28 April 2009

Received in revised form

6 November 2009

Accepted 11 November 2009

Available online 3 December 2009

Keywords:

Animal waste

Codigestion

Biogas

Methane

Two-phase olive mill wastes

* Corresponding author. Tel.: þ43 512 507 59E-mail address: marta.goberna@uibk.ac.a

0961-9534/$ – see front matter ª 2009 Elsevidoi:10.1016/j.biombioe.2009.11.005

a b s t r a c t

Cattle excreta and two-phase olive mill wastes (TPOMW) were codigested at a 3:1 ratio in

two 75 L continuous stirred tank reactors at 37 �C and 55 �C to analyse their biogas

production. The contribution of each residue to the total gas production at 37 �C was

evaluated in reactors digesting either 3:1 excreta:water or 3:1 water:TPOMW. The meso-

philic co-fermentation of cattle excreta with TPOMW at an organic loading rate (OLR) of

5.5 g COD L�1 d�1 rendered 1096 mL biogas L�1 sludge d�1. This was 337% higher than that

of excreta alone. The methane yield resulting from the codigestion was 179 L CH4 kg�1 VS

loaded, of which 42% was attributed to the quarter of the reactor corresponding to TPOMW.

Under thermophilic conditions, the codigestion yielded 17.3% more methane than meso-

philically. In the reactor digesting TPOMW alone (OLR ¼ 3.8 g COD L�1 d�1) the ratio VFA/

alkalinity exceeded 0.8 after 21 d, leading to its acidification and inhibition of methano-

genesis. Farm-scale digestion of animal excreta and TPOMW should be promoted in

Mediterranean countries as an environmentally sound option for waste recycling and

renewable energy production.

ª 2009 Elsevier Ltd. All rights reserved.

1. Introduction sustainable land-use system is unevenly distributed, i.e. most

Biogas generated from the anaerobic digestion of animal

excreta is increasingly used for the production of renewable

energy. In Europe there are ca. 4240 farm-scale biogas plants

[1], which mainly treat cow, pig or poultry manure. These are

widespread and ready-to-use residues which yield sufficient

biogas to account for the energy consumption of the farm and

its digester, as well as a net energy surplus that is fed into the

grid as green electricity. Furthermore, the stabilised end-

product can be applied as an organic amendment to the

agricultural soils. Land-spreading anaerobically digested

sludge instead of untreated manure reduces malodours, helps

controlling pathogens, provides soils with a more balanced

nutrient mix and higher nutrient bioavailability [2]. This

95; fax: þ43 512 507 2928.t (M. Goberna).er Ltd. All rights reserved

plants are located in a few regions in central Europe, and thus

the EU will encourage farmers to set-up biogas facilities all

over the territory to reduce the needs for energy imports and

improve the CO2 balance [3].

However, cattle excreta are already-digested substances,

rich in lignocellulose with a relatively low C/N ratio, and high

water content [4]. Therefore, these have a low methane

production potential [4,5], and this threatens the economic

viability of farm-scale biogas plants [1]. The anaerobic

biodegradation of cattle excreta renders 20 m3 biogas t�1 (wet

weight), whereas 650 m3 t�1 can be obtained from bakery

residues and up to 1200 m3 t�1 from vegetable oils [6]. Co-

fermenting animal excreta with other residues is an option to

increase their biogas production potential. Olive mill effluents

.

Fig. 1 – Schematic depiction of the pilot scale anaerobic

reactors. Source: [26].

b i o m a s s a n d b i o e n e r g y 3 4 ( 2 0 1 0 ) 3 4 0 – 3 4 6 341

(OME) are an interesting candidate to be used in Mediterra-

nean countries, where they are intensively produced [7,8].

OME can balance the C/N ratio of the N-rich excreta and

supplement them with fats, with their enormous biogas

production potential. On the other hand, the digestion of OME

alone is limited by their low pH, alkalinity and ammonia N

content [9], and by the presence of recalcitrant phenolic

compounds that can inhibit microbial growth [7]. High OME

digestion efficiencies can be achieved, even at high organic

loading rates, by pretreating the wastes [10,11] or supple-

menting them with nutrients and buffering solutions [12,13].

However, it is economically and environmentally more sound

to compensate the acidity and nutrient deficiency of OME by

their combined digestion with other biodegradable wastes

with complementary properties [13].

The anaerobic digestion of olive mill effluents has proven

feasible in combination with cattle manure, household waste,

sewage sludge [9], poultry manure [14], wine-grape and

slaughterhouse wastewater [15], and laying hen litter [16].

Most of these studies have been performed in 100–250 ml

serum vials or 1–2.5 L tank reactors using olive mill waste-

waters. Not much has been published, however, on the use of

two-phase olive mill wastes (TPOMW), i.e. the paste produced

during oil extraction with a two-phase centrifugation system,

as a substrate for codigestion experiments. The volume of

TPOMW, containing the stone and pulp of the fruits and high

moisture, has increased together with the expansion of the

two-phase technology after its adoption in the early 90s [7,8].

Appropriate TPOMW management and valorisation are still to

be achieved and require further research. In this survey, we

investigated the performance and biogas production of four

75 L pilot anaerobic reactors digesting cattle excreta and

TPOMW, both separately and in co-fermentation.

Table 1 – Physical and chemical properties of the feedingsolutions.

Parametera Excreta 3:1 excreta:TPOMW

3:1 water:TPOMW

TS (% w/v) 4.7 11.0 7.5

VS (% w/w of TS) 71.1 75.1 21.8

Total COD (g L�1) 40.2 118.5 81.1

Soluble COD (g L�1) 14.7 43.6 28.4

NH4–N (mg L�1) 1204 831 BDLb

pH 7.40 6.68 4.93

EC (mS cm�1) 17.2 14.4 4.1

Alkalinity (mmol Hþequ L�1) 166 124 10

a TS, total solids; VS, volatile solids; COD, chemical oxygen

demand; NH4–N, ammonia nitrogen; EC, electrical conductivity.

b BDL: Below detection limit.

2. Materials and methods

2.1. Operation of the reactors and feeding solutions

The experiment was performed in four anaerobic continuous

stirred tank reactors (CSTR), which are 1.4 m high, 0.36 m in

diameter and have a working volume of 75 L (Fig. 1). All reactors

were initially charged with 75 L cattle excreta (Table 1) and

heated up to 37 �C. This start-up strategy had previously proven

successful [17,18]. Reactors were fed daily with 3.5 L d�1 of

cattle excreta and an equivalent amount of effluent sludge was

removed, corresponding to a hydraulic retention time (HRT) of

21.4 d. These conditions were selected in order to simulate

those under which the full-scale BIO4GAS� plant in Rotholz

(Tirol, Austria) is operated [19].

Once steady state conditions, i.e. stable biogas produc-

tion, were achieved in all reactors, cattle excreta were

substituted by specific feed solutions at the same flow rate

(3.5 L d�1). The specific operation conditions for all reactors

and the characteristics of the feed solutions are summarised

in Tables 1 and 2. Reactors EX_OL37 and EX_OL55 were fed

with a mixture of excreta:TPOMW at a ratio of 3:1 operated

at 37 �C and 55 �C, respectively. The other two reactors were

loaded with either cattle excreta:water (EX37) or water:

TPOMW (OL37) at a ratio of 3:1. Both were operated at 37 �C.

This experimental set-up was designed to quantify the

individual contribution of either cattle excreta or TPOMW

and the possible benefits of co-fermenting these materials

under mesophilic conditions. The influence of temperature

was evaluated through the comparison of EX_OL37 with

EX_OL55, which was run at 55 �C.

Excreta (mixed excrements and urine) were collected on 5

December 2007 from the storage tank located underneath the

cattle stable in the agricultural school in Rotholz (Tirol,

Austria). This was sieved (<5 mm) prior to its use to remove

straw and avoid blocking of reactor pipes. TPOMW was

obtained from the agricultural cooperative COATO in Totana

(Murcia, Spain) on 7 December 2007 and transported to the

laboratory in sealed tanks. TPOMW was not further pre-

treated, so the material is representative of that resulting from

Table 2 – Operation of reactors.

Reactor Feedingsolutiona

Temperature(�C)

FFRb

(L d�1)OLRc

(g CODL�1 d�1)

EX_OL37 3:1 excreta:TPOMW 37 3.5 5.5

EX_OL55 3:1 excreta:TPOMW 55 3.5 5.5

EX37 3:1 excreta:water 37 3.5 1.4

OL37 3:1 water:TPOMW 37 3.5 3.8

a TPOMW: Two-phase Olive Mill Wastes.

b FFR: Feed Flow Rate.

c OLR: Organic Loading Rate.

b i o m a s s a n d b i o e n e r g y 3 4 ( 2 0 1 0 ) 3 4 0 – 3 4 6342

the mill processing effluent line. Both materials were stored at

4 �C until reactor start up on 19 December 2007.

2.2. Sampling of gas and sludge

Reactors were operated with the specific feed solutions for

30 d. The volume of biogas produced was quantified using

a gas meter located on top of each reactor (Fig. 1). Gas samples

were collected daily in gas-tight bags and gas composition

(CH4, CO2 and H2) measured using a Biogas Monitor BM 2000

(Geotechnical Instruments, Leamington, UK). Three sludge

samples (ca. 500 ml) were collected every 3.5 d from a tap

located in the bottom of each reactor (Fig. 1).

2.3. Analytic methods

Total solids (TS), volatile solids (VS), pH and electrical

conductivity (EC) were determined following EU Horizontal

standards [20]. Total C and N contents were measured in an

automated CNHS analyser (TruSpec�, LECO, Michigan, USA).

Total and soluble chemical oxygen demand (COD) were ana-

lysed with the LCK 014 test kit and ammonia nitrogen (NH4–N)

with the LCK 303 test kit (both from Hach Lange, Dusseldorf,

Germany). Particulate COD was calculated as the difference

between total and soluble COD. Determination of volatile fatty

acids (VFAs) was performed within 24 h of sampling. Briefly,

15 ml of sludge were centrifuged (14,000 rpm, 30 min) and the

0.2 mm filtered supernatant analysed for VFAs by High Pres-

sure Liquid Chromatography (HPLC Shimadzu LC-20A promi-

nence) using 20 ml injection volumes. VFAs were separated

through an ion exclusion column (300 � 7.8 mm, Aminex�

HPX-87H) and photometrically detected at 210 nm. An iso-

cratic flow (0.7 ml min�1) with 5 mM H2SO4 was used as the

running medium at 60 �C for 90 min.

2.4. Multivariate analysis

Principal component analysis (PCA) based on a correlation

matrix was performed to inspect the relationship between

biogas production, effluent sludge properties and the factors

‘‘reactor’’ and ‘‘sampling date’’, using SPSS 15.0 for Windows.

3. Results

3.1. Biogas quantity and quality

All reactors were run at 37 �C and fed daily with cattle excreta

at an equivalent feeding rate until a stable and similar biogas

production was reached, averaging 18.1 � 0.4 L d�1

(mean � SD) with a methane content of 68.7 � 0.4% v/v. This

steady state was maintained for three days (days 1–3 in Fig. 2)

prior to the start of the experiment. In day 3, the temperature

of reactor EX_OL55 was set to 55 �C. In day 4 (solid line in

Fig. 2), the feeding with specific solutions was initiated (Tables

1 and 2).

Immediately after starting the specific load, reactor

EX_OL37 showed an increase in biogas production (Fig. 2). This

stabilised at day 8, i.e. four days after starting the load with

specific feeding solutions, until the end of the experiment

averaging 82.0 � 4.8 L d�1. Methane content in the biogas was

constant, with 62.8 � 1.2%. In EX_OL55, there was an increase

in biogas production with lower methane content from days

3–4, which was a direct effect from the temperature rise to

55 �C (Fig. 2). This was followed by a decrease in biogas

production and its CH4 content from days 4–7, which

progressively recovered from day 8 up to day 14 when it sta-

bilised at 94.5 � 10.5 L biogas d�1 with 62.0 � 1.5% methane

content. During the stable period, non-stirring conditions due

to technical problems occurred, which resulted in a slight

decrease in biogas production (dashed lines in Fig. 2). Reactor

EX37 remained stable throughout the experiment, its biogas

production averaging 17.7 � 1.7 L d�1 with 64.8 � 1.2% CH4

(Fig. 2). Finally, reactor OL37 rapidly reacted to the initial

change in feed, reaching a maximum biogas production at day

8 (Fig. 2). Biogas production remained relatively high from

days 7–14 (59.5 � 3.1 L d�1) and then decreased rapidly.

Table 3 summarises the gas production parameters during

the steady state after the period corresponding to one HRT

(21.4 d). Data are average of three consecutive sampling dates.

Reactor EX_OL37 produced 337% more biogas and 73.3%

higher methane yield than EX37. Reactor EX_OL55 showed the

highest biogas production and methane yield. These were,

respectively, 17.6% and 17.3% higher than those of EX_OL37.

During this steady state, reactors EX_OL37 and EX_OL55 were

the most efficient in VS removal.

3.2. Effluent sludge properties

At the start of the experiment, while feeding was performed

with excreta alone, the amount of total solids (TS) in the

effluent sludge of all reactors was approximately 4.0� 0.1%, of

which 66.5 � 0.8% were volatile solids (VS). Total and soluble

chemical oxygen demand (COD) averaged 27.0 � 0.6 g L�1 and

7 � 0.7 g L�1, respectively. TS, VS, total and soluble COD

increased together with the change in feed in the effluent

sludge of reactors EX_OL37, EX_OL55 and OL37 (except for TS

in OL37), whereas all parameters slightly decreased in EX37

(Table 3).

The main volatile fatty acids (VFAs) detected in all reactors

were acetate and propionate (Fig. 3). VFAs showed relatively

constant values in reactors EX_OL37 and EX37, their levels

Fig. 2 – Biogas production (L dL1) and methane content in the biogas (% v/v) in the reactors (EX_OL37, 3:1 excreta:TPOMW at

37 8C; EX_OL55, 3:1 excreta:TPOMW at 55 8C; EX37, 3:1 excreta:water at 37 8C; OL37, 3:1 water:TPOMW at 37 8C). Solid line

indicates start of feeding with specific mixtures. Dashed lines indicate period under non-stirring conditions.

b i o m a s s a n d b i o e n e r g y 3 4 ( 2 0 1 0 ) 3 4 0 – 3 4 6 343

being always below 0.8 and 0.2 g VFA L�1, respectively. Both

reactors showed stable pH values above 7.6 throughout the

experiment (Fig. 3). However, EX_OL55 and OL 37 reached

peaks of 4.8 and 3.8 g VFA L�1. The increase in effluent VFAs in

EX_OL55 was mainly due to the accumulation of acetate, and

to a lesser extent propionate, after temperature was changed

from 37 �C to 55 �C on day 3. A subsequent decrease in pH was

detected from an initial 7.8 down to 7.4. This reverted on day

13 after acetate levels started to stabilise. In reactor OL37, both

acetate and propionate progressively accumulated, which

was accompanied by a decrease in pH down to 6.5 (Fig. 3).

Total ammonia nitrogen (NH4–N) averaged 1.3 � 0.03 g L�1

in all reactors before the shift to the specific feed solutions on

day 4 (Fig. 4). In EX_OL37 and EX_OL55, NH4–N decreased on

day 11 (i.e. five days after changing the feed) until the end of

Table 3 – Steady state parameters after the periodcorresponding to one HRT (21.4 d). Standard deviationsare given in brackets for n [ 3.

Parametera EX_OL37 EX_OL55 EX37 OL37

Gas production

Biogas production

(mL L�1 sludge d�1)

1096 (81) 1289 (158) 251 (39) –

Methane yield (L CH4 kg�1

VS added)

179 (18) 210 (30) 103 (16) –

VS removed (%) 53.4 (1.7) 54.0 (1.8) 45.8 (6.3) –

Effluent characteristics

TS (% w/v) 5.2 (0.1) 5.2 (0.2) 2.9 (0.2) 3.4 (0.1)

VS (% w/w of TS) 73.6 (0.6) 73.4 (0.6) 62.3 (1.9) 72.6 (1.5)

Particulate COD (g L�1) 27.9 (0.2) 26.0 (0.6) 16.5 (0.5) 22.4 (1.1)

Soluble COD (g L�1) 10.9 (0.7) 16.9 (0.9) 5.5 (0.2) 13.1 (0.7)

Total C (g L�1) 41.5 (2.4) 44.1 (3.6) 41.7 (2.2) 46.4 (2.5)

Total N (g L�1) 3.5 (0.1) 3.5 (0.2) 3.2 (0.3) 3.4 (0.2)

EC (mS cm�1) 16.1 (0.1) 16.7 (0.3) 15.3 (0.2) 10.8 (1.4)

a TS, total solids; VS, volatile solids; COD, chemical oxygen

demand; EC, electrical conductivity.

the experiment to values averaging 0.9 � 0.03 g L�1 and

1.1 � 0.05 g L�1, respectively. In EX37, NH4–N oscillated

between 1.1 and 1.3 g L�1, whereas in OL37 it decreased

progressively to final values of 0.5 g L�1. Alkalinity was highly

correlated with NH4–N (Pearson’s correlation

coefficient ¼ 0.80, p < 0.001) and followed similar temporal

patterns (Fig. 4).

3.3. Principal component analysis

The two principal components (PCs) extracted from the ordi-

nation of the data explained 76.6% of the total variance. Fig. 5A

shows the distribution of the samples within the biplot that

both PCs define. At the start of the experiment (day 4), all

reactors were located near to each other in the negative pole of

PC1, which was highly correlated with ammonia N (Fig. 5B). As

the digestion with the specific feed solutions proceeded, the

reactors followed divergent trajectories in the diagram. Both

reactors codigesting excreta and TPOMW (EX_OL37 and

EX_OL55) followed similar trajectories irrespective of their

operating temperature. These moved towards the area of high

biogas production, and high levels of TS and COD, responding to

the change of feed from excreta alone (OLR¼ 1.9 g COD L�1 d�1)

to excreta:TPOMW (OLR ¼ 5.5 g COD L�1 d�1). EX37 showed the

shortest trajectory, thus the most similar performance

throughout the experiment, towards the sector of low TS and

COD, due to the decrease in OLR from 1.9 (excreta) to

1.4 g COD L�1 d�1 (diluted excreta). Finally, OL37 advanced in the

direction of the area correlated with low biogas production,

alkalinity, pH and ammonia N, but high total C, total N and

propionate.

4. Discussion

Four pilot reactors were used to evaluate the potential biogas

production from the codigestion of cattle excreta and two-

Fig. 3 – Acetate, propionate (g LL1) and pH in the reactors (EX_OL37, 3:1 excreta:TPOMW at 37 8C; EX_OL55, 3:1

excreta:TPOMW at 55 8C; EX37, 3:1 excreta:water at 37 8C; OL37, 3:1 water:TPOMW at 37 8C).

b i o m a s s a n d b i o e n e r g y 3 4 ( 2 0 1 0 ) 3 4 0 – 3 4 6344

phase olive mill wastes (TPOMW). Prior to the start of the

experiment with specific feeding solutions and temperatures,

the reactors were run with cattle excreta at 37 �C until they

operated similarly in terms of gas production, methane

content and sludge properties (day 4 in Fig. 5A). This demon-

strated that all four reactors function similarly if operated

under equal conditions. The diverse combinations of wastes

led to distinct reactor performance (Fig. 5).

The reactors codigesting excreta and TPOMW at 37 �C

(EX_OL37) and 55 �C (EX_OL55), and the reactor digesting

excreta alone (EX37) operated stably and achieved a constant

biogas production and methane yield. After the period corre-

sponding to one HRT (21.4 d), 1096 mL L�1 d�1 biogas were

produced from the sludge in EX_OL37 while EX37 yielded only

251 mL L�1 d�1. Therefore, 337% more biogas was produced

when a quarter of the effective volume of the digester was

Fig. 4 – Ammonia N (mg LL1) and alkalinity (mmol HD equiv LL

EX_OL55, 3:1 excreta:TPOMW at 55 8C; EX37, 3:1 excreta:water a

filled with TPOMW instead of water. These values are well

above those reported previously in experiments using olive

mill wastewaters. Gelegenis et al. [14] obtained

520 mL biogas L�1 reactor d�1 from the digestion of poultry

excreta and olive mill wastewasters (pH 1.6) at a ratio 3:1 in

a 25 L reactor. This value was 25% higher than the biogas

produced by a similar reactor fed only with poultry excreta. Up

to 155% additional biogas was obtained in a batch experiment

in 100 ml vials when laying hen litter was digested with olive

mill effluent compared to laying hen litter alone, at

10 g COD L�1 load with 10% TS [16].

The methane yield of the mesophilic reactor codigesting

cattle excreta and TPOMW at a ratio 3:1 was 179 L CH4 kg�1 VS

loaded. A total 57.7% of this methane can be attributed to the

three quarters of the reactor that were filled up with excreta,

considering the results from the excreta digesting reactor

1) in the reactors (EX_OL37, 3:1 excreta:TPOMW at 37 8C;

t 37 8C; OL37, 3:1 water:TPOMW at 37 8C).

Fig. 5 – Biplot of the two main axes after principal

component (PC) analysis, showing the relationship

between the reactors (EX_OL37, 3:1 excreta:TPOMW at

37 8C; EX_OL55, 3:1 excreta:TPOMW at 55 8C; EX37, 3:1

excreta:water at 37 8C; OL37, 3:1 water:TPOMW at 37 8C),

the sampling dates (days 4, 7, 11, 14, 18, 21, 25 and 28) and

the variables: biogas production (Biogas), methane

production (CH4), total solids (TS), volatile solids (VS), total

chemical oxygen demand (CODtot), soluble COD (CODsol),

acetate (Acet) and propionate (Prop) concentration, total C

(Ct), total N (Nt), C/N ratio (C/N), ammonia N (NH4–N), pH,

alkalinity (Alk) and electrical conductivity (EC).

b i o m a s s a n d b i o e n e r g y 3 4 ( 2 0 1 0 ) 3 4 0 – 3 4 6 345

(103.1 L CH4 kg�1 VS). Thus, 42.3% of the total methane yield in

EX_OL37 (75.6 L CH4 kg�1 VS) was produced from the use of

TPOMW as a codigestate.

The thermophilic codigestion of excreta and TPOMW yiel-

ded 1289 mL biogas L�1 sludge d�1, which is within the range

of 1250–1550 mL L�1 d�1 reported by Angelidaki and Ahring [9]

using 2 L CSTRs codigesting cattle excreta and oil mill efflu-

ents at ratios 1:1 and 1:3 at 55 �C with HRT ¼ 13 days and

OLR ¼ 7.8 g COD L�1 d�1. Our reactor operated at 55 �C

produced 17.6% more biogas than that run at 37 �C. It is likely

that an increased biogas production at the end of the experi-

ment in the thermophilic reactor was due to VFA accumula-

tion during the non-stirring period. However, it should be

noticed that the first of the three consecutive dates used for

the calculations of the steady state parameters fell within the

non-stirring period, so it is unlikely that biogas production

was overestimated. Our data fit those obtained in a batch

experiment with 160 ml serum vials, in which the codigestion

of wastewasters from olive mill, slaughterhouse and wine-

grape industries resulted in 14–35% more biogas at 55 �C than

at 37 �C [15].

The mesophilic and thermophilic digesters were similarly

efficient in removing the pollutant load of the sludge (ca. 54%

of VS removed). These values are lower than those found in

the literature in codigestion experiments using other olive

mill effluents [9,14]. The calculated values do not fully agree

with those that should be expected from methane production.

A possible reason for this disagreement could be some sedi-

mentation of sludge in the tank affecting the effluent port at

the bottom of the reactor. Previous experiments in the same

digesters showed that stirring was enough to ensure an even

vertical distribution of the solids (unpublished data). Still,

some sedimentation could have occurred forced by the pieces

of olive stones in TPOMW. In the reactor digesting excreta

alone (EX37), VS removal efficiency averaged 45.8% after the

period corresponding to one HRT. These low values are

coherent with previous reports [5,21].

The reactor digesting TPOMW alone (OL37) at an OLR of

3.8 g COD L�1 d�1 showed a decrease in its buffering capacity,

together with the washout of the cattle excreta used to start it

up, until it collapsed. The reactor underwent a progressive

acidification during the experiment due to the increase in VFA

content together with the parallel decrease in its capacity to

neutralize acids. The ratio VFA/alkalinity, expressed in

equivalents of acetic acid/equivalents of calcium carbonate

was below 0.1 at the start of the experiment indicating process

stability [22]. However, the VFA/alkalinity ratio progressively

went up, exceeding 0.8 in the period after one HRT. This

threshold (VFA/alk � 0.8) has been pointed before as an indi-

cator of imminent process failure [23]. The washout of nutri-

ents was discarded as a reason of reactor breakdown.

Ammonia N decreased sharply due to its negligible levels in

the influent TPOMW. However, due to the degradation of N

containing biopolymers, NH4–N was above 0.5 g L�1 until the

end of the experiment. Deficiencies in other nutrients (K, P,

Ca, Mg, S, Fe) were not detected either. These were present at

similar levels in reactor digesting TPOMW alone and in all

other reactors [24]. It is more likely that the process imbalance

could be due to the supply with TPOMW of heavy metals and/

or phenolic substances that inhibit microbial growth [7,8].

Copper, a strong inhibitor of methanogenesis [25], was more

available in the acidified reactor digesting TPOMW (192 ppm

dry weight) than in all other reactors [24].

Borja et al. [12] demonstrated that TPOMW can be

successfully digested using a complex nutrient-trace solution

in the start-up phase and increasing the OLR stepwise, thus

allowing microbial acclimatisation. These authors obtained

0.84 L CH4 L�1 d�1 and a COD removal efficiency over 90% from

the digestion of 40% diluted TPOMW (74.9% VS) with an OLR of

3.24 g COD L�1 d�1 in a 1 L reactor. It should be tested whether

gradually increasing the TPOMW load of reactors digesting

animal excreta allows for a more efficient VS removal and an

even enhanced biogas production than that reported here.

5. Conclusions

High and stable methane production (179 L CH4 kg�1 VS

loaded) was obtained from the mesophilic codigestion of 3:1

b i o m a s s a n d b i o e n e r g y 3 4 ( 2 0 1 0 ) 3 4 0 – 3 4 6346

cattle excreta:two-phase olive mill wastes with an OLR of

5.5 g COD L�1 d�1 and a HRT of 21.4 d. The quarter of the

reactor filled up with TPOMW, with its high biodegradability,

contributed to 75.6 L CH4 kg�1 VS of the total methane yield.

The excreta buffered the low pH and alkalinity of the TPOMW,

allowing its anaerobic biodegradation without the need of any

pre-treatment or chemical additive. Thermophilically, the

codigestion of both wastes rendered 17.3% more methane

than mesophilically. Reactor efficiency in terms of VS removal

was higher (ca. 54%) thanks to the codigestion of the residues.

The mixing ration should be optimized according to the

precise composition of the input materials.

Acknowledgements

We thank the Agricultural School in Rotholz (Tirol, Austria)

and the cooperative COATO in Totana (Murcia, Spain) for

providing us with the cattle excreta and TPOMW, respectively.

We also thank M.A. Sanchez Monedero, A. Roig and C. Mon-

dini for arranging the transport of TPOMW to our laboratory.

We thank C. Mondini for providing nutrient and heavy metal

contents of the digestion end products and E.A. Eladawy for

providing Fig. 1. M. Goberna was supported by the Marie Curie

Actions (MEIF-CT-2006-041034). The support of the Tiroler

Zukunftsstiftung for the K-Regio Center BioTreaT is

appreciated.

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