Bioresource Technology 167 (2014) 495–502

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Application of Anaerobic Digestion Model No. 1 to describe

the syntrophic acetate oxidation of poultry litter in
thermophilic anaerobic digestion
Víctor Rivera-Salvador a, Irineo L. López-Cruz b, Teodoro Espinosa-Solares a,c,⇑, Juan S. Aranda-Barradas d,
David H. Huber c,e, Deepak Sharma c,e, J. Ulises Toledo c
a
Departamento de Ingeniería Agroindustrial, Universidad Autónoma Chapingo, Chapingo, Estado de México 56230, Mexico
b
Posgrado en Ingeniería Agrícola y Uso Integral del Agua, Universidad Autónoma Chapingo, Chapingo, Estado de México 56230, Mexico
c
Gus R. Douglass Institute, West Virginia State University, Institute, WV 25112-1000, USA
d
Unidad Profesional Interdisciplinaria de Biotecnología, Instituto Politécnico Nacional, Mexico City, DF 07340, Mexico
e
Department of Biology, West Virginia State University, Institute, WV 25112-1000, USA

h i g h l i g h t s g r a p h i c a l a b s t r a c t

 Hydrogenotrophic methanogens 1200

Acetate
dominate poultry litter anaerobic Propionate
1000 Butyrate
digestion.
Volatile fatty acids [mg·L ]

Valerate
-1

DEA Simulated Acetate

 Syntrophic acetate oxidation was DEA Simulated Propionate
800
DEA Simulated Butyrate
modeled for poultry litter TAD. DEA Simulated Valerate

 Differential evolution algorithms 600

applied to evaluated ADM1

400
parameters.
 Evaluation of ADM1 syntrophic
200
acetate oxidation kinetic parameters.
0
0 20 40 60 80
Time [ d ]

a r t i c l e i n f o a b s t r a c t

Article history: A molecular analysis found that poultry litter anaerobic digestion was dominated by hydrogenotrophic
Received 9 April 2014 methanogens which suggests that bacterial acetate oxidation is the primary pathway in the thermophilic
Received in revised form 2 June 2014 digestion of poultry litter. IWA Anaerobic Digestion Model No. 1 (ADM1) was modified to include the bac-
Accepted 4 June 2014
terial acetate oxidation process in the thermophilic anaerobic digestion (TAD). Two methods for ADM1
Available online 12 June 2014
parameter estimation were applied: manual calibration with non-linear least squares (MC-NLLS) and
an automatic calibration using differential evolution algorithms (DEA). In terms of kinetic parameters
Keywords:
for acetate oxidizing bacteria, estimation by MC-NLLS and DEA were, respectively, km 1.12 and
Differential evolution algorithms
Non-linear least squares
3.25 ± 0.56 kgCOD kg1 1
COD d , KS 0.20 and 0.29 ± 0.018 kgCOD m
3
and Yac-st 0.14 and 0.10 ± 0.016 kgCOD kg1
COD.

Kinetic parameters Experimental and predicted volatile fatty acids and biogas composition were in good agreement.
Parameter estimation Values of BIAS, MSE or INDEX demonstrate that both methods (MC-NLLS and DEA) increased ADM1
Anaerobic digestion model accuracy.
Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction
⇑ Corresponding author at: Departamento de Ingeniería Agroindustrial, Univers-
idad Autónoma Chapingo, km 38.5 Carretera México-Texcoco, Chapingo, Estado de
Anaerobic digestion has the capacity for treatment of organic
México 56230, Mexico. Tel.: +52 (595) 952 1730. wastes and energy recovery via biogas as well as reduced CO2
E-mail address: t.espinosa.s@taurus.chapingo.mx (T. Espinosa-Solares). emissions and use of residues as fertilizer which make it a

http://dx.doi.org/10.1016/j.biortech.2014.06.008
0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
496 V. Rivera-Salvador et al. / Bioresource Technology 167 (2014) 495–502
sustainable technology (Shen et al., 2013; Zamanzadeh et al.,
2013). Methane can be produced either by aceticlastic methano-
gens, which use acetate, or by hydrogenotrophic methanogens
which utilize H2 and CO2. It has been reported that two thirds of
methane produced by activated sludge derives from acetate
whereas just one third is obtained from hydrogenotrophic metha-
nogenesis (Boone et al., 1989).
Methanogenic acetate degradation is carried out by an aceticlas-
tic reaction or an anaerobic syntrophic acetate-oxidizing reaction
(Hattori, 2008; Westerholm et al., 2011). In conventional anaerobic
digestion, acetate may be degraded by aceticlastic methanogens
which limit the accumulation of acetate. In aceticlastic methano-
genesis, acetate is cleaved to methyl and carboxyl groups; the
methyl group is directly converted to methane by several biochem-
ical reactions while the carboxyl group is oxidized to CO2 (Hattori,
2008). However, an alternative pathway for acetate degradation
involves a syntrophic relationship between acetate-oxidizing
bacteria and hydrogenotrophic methanogens (Westerholm et al.,
2011). This is known as syntrophic acetate oxidation.
Syntrophy is an essential intermediate step in the anaerobic
conversion of organic matter to methane. In this mutualistic rela-
tionship, metabolically distinct microorganisms are tightly linked
by the need to maintain the exchanged metabolites at very low
concentrations (McInerney et al., 2009). Syntrophic metabolism
requires reverse electron transfer, close physical contact and
metabolic synchronization of the syntrophic partners (McInerney
et al., 2009). Anaerobic digestion is also an expression of the
syntrophic relationships among different microbes (Sasaki et al.,
2011; Shen et al., 2013).
Syntrophic acetate oxidation is a two step methanogenesis from
acetate by a coculture where acetate is oxidized to CO2 and H2 by
one organism and H2 is subsequently used by a second organism to
reduce CO2 to CH4 (Westerholm et al., 2011). During the methano-
genic mineralization process, syntrophic acetate oxidation is
thermodynamically unfavorable (DG°0 = +104.6 kJ mol1) (De Vrieze
et al., 2012), and proceeds only if hydrogen partial pressures are
kept low. By coupling hydrogen-consuming methanogens to the
process, hydrogen partial pressures can be maintained between
10 and 40 Pa (72 ± 5 Pa in some cases) (Hao et al., 2011; Schink,
1997), and Gibbs free energies fluctuate near 20 kJ mol1 (Hao
et al., 2011). Thus, interspecies hydrogen transfer between second-
ary fermenting bacteria and hydrogenotrophic methanogens is
important for the oxidation of substrates such as fatty acids, etha-
nol, propionate, butyrate and acetate (Hattori, 2008; Zamanzadeh
et al., 2013).
Therefore, hydrogenotrophic methanogens play a crucial role in
constantly removing H2 and producing methane which makes the
oxidation of substrate by proton reduction energetically feasible.
Hence, the syntrophic association between substrate oxidizers
and hydrogenotrophic methanogens is necessary for sustaining
the overall process of anaerobic degradation (Luo et al., 2002).
The acetate oxidation process is more common at thermophilic
temperatures, although this process can also be developed at
mesophilic conditions (Westerholm et al., 2010). However, if stress
factors arise, ammonia accumulation for instance, aceticlastic
methanogenesis can be inhibited (Schink, 1997; Wilson et al.,
2012). There is a considerable difference in the acetate degradation
rate for aceticlastic methanogenesis and syntrophic acetate
oxidation which suggests that syntrophic acetate oxidation might
be difficult to observe if both mechanisms are active at the same
time (Schink, 1997).
There is also evidence that syntrophic acetate-oxidizing bacte-
ria are important at high ammonia levels in thermophilic anaerobic
digesters and high acid concentrations (Hao et al., 2011; Wilson
et al., 2012). Acetate-oxidizing bacteria have been found to be
important for the start-up of methanogenesis from high organic
loadings in thermophilic anaerobic sequenced batch reactors
(ASBR) at 55 °C (Hao et al., 2011). However, information about syn-
trophic acetate oxidation, the organisms involved, and their role in
the methanogenic environment is currently limited, and more
research is required to elucidate the kinetic and ecological charac-
teristics of these bacteria (Hao et al., 2011; Westerholm et al.,
2011). Recently, the microbial community structure of a pilot-scale
thermophilic CSTR digester stabilized on poultry litter was
characterized (Smith et al., 2014). Based on the predominance of
hydrogenotrophic methanogens, as well as digester chemistry,
bacterial acetate oxidation was proposed as the primary pathway
for control of acetate levels in this digester.
Anaerobic Digestion Model No. 1 (ADM1) is a mechanistic
model that explains complex substrates through their principal
components (Batstone et al., 2002). It includes several steps that
describe the biochemical and physicochemical processes involved
in the anaerobic biodegradation of organic compounds. However,
syntrophic acetate oxidation is not currently a part of the model
but some suggestions have been made by Batstone et al. (2002)
to take into consideration. Mathematical modeling has become a
popular support tool for design, operation and control of activated
sludge systems (Lübken et al., 2007). It can be used to predict
process behavior in different situations and to assist operational
management in order to develop strategies that will improve
stability (Silva et al., 2009). These predictions can not only improve
operational decision making in agricultural biogas plants but also
assist the planning of research experiments (Zhou et al., 2011).
Because little progress has been made in the mathematical
modeling of bacterial acetate oxidation (Shimada et al., 2011),
the objective of this work was to incorporate bacterial acetate
oxidation into a well-known mathematical model. The present
paper simulates the TAD of poultry litter by incorporating the
bacterial acetate oxidation pathway in ADM1 as the main acetate
degradation process, rather than aceticlastic methanogenesis.
Two methods for parameter estimation were used: differential
evolution algorithms (DEA) and a coupled method of manual
calibration and non-linear least squares (MC-NLLS).
2. Methods
2.1. Lab-scale digester
The digester that was used in this experiment was described as
the control reactor in Sharma et al. (2013). This reactor was a 10 L
glass vessel with a round-cylindrical bottom and separate ports for
sampling, feeding and recirculation. The reactor was fed with a
2.2% TS chicken litter manure suspension during 90 days. The
hydraulic retention time (HRT) was 15 days and the chemical oxy-
gen demand (COD) was 24.77 ± 0.8 kgCOD m3. The feedstock was
supplied at a semi-continuous rate of 0.66 L d1. The poultry litter
(manure, feathers, and wood chips) was collected from Moorefield,
WV and New Market, VA.
The mixing consisted of a digestate recirculation system
without mechanical stirring. The digester was maintained at
thermophilic conditions (56 °C) by running hot water through an
external jacket. Temperature was monitored by a thermocouple.
pH was automatically measured by a pH probe (Cole Parmer)
connected in the re-circulation line. Biogas pressure was
measured with a pressure transducer (Omegadyne Inc., model no.
PX209-015G5V) when excess biogas was released into a cylinder
containing water.
2.2. Analytical methods
For the substrate, total solids (TS) were determined using the
standard methods of APHA (1998) and chemical oxygen demand
 V. Rivera-Salvador et al. / Bioresource Technology 167 (2014) 495–502
(COD) was measured according to the HACH water analysis hand-
book (HACH, 2004). In case of the digestate, samples for volatile
fatty acids (VFAs) profiles were collected twice per week. However,
four consecutive days of sampling were done at the end of each
30 days (two HRTs). VFA profiles were obtained for acetic, propi-
onic, butyric, iso-butyric, valeric and iso-valeric acids using gas
chromatography (Varian 3300 Gas Chromatograph, FID detector;
glass column packed with 80/120 Carbopack B-DA/4% Carbowax
20 M). Biogas composition was measured every day with a gas
chromatograph (Agilent 7890 GC with HP-PLOT Q column).
2.3. Microbial diversity methods
The detection and analysis of archaeal diversity was done using
standard methods. Archaeal diversity was sampled with PCR using
primers AR109f and AR915r which target 16S rRNA genes (Raskin
et al., 1994). To minimize bias caused by PCR artifacts, recondition-
ing PCR was used (Thompson et al., 2002). PCR reactions were done
in 25 lL reactions with Accuprime Taq polymerase (Invitrogen)
and 1.25 ng of sample DNA. PCR was carried out with two sets of
cycles: 20 cycle reaction followed by a 5 cycle reaction. PCR condi-
tions for the first round of cycles were: denaturation at 94 °C for
2 min, then 20 cycles of 94 °C for 1 min, 52 °C for 1 min, and
72 °C for 1.5 min, followed by a final extension at 72 °C for
3 min. The second ‘‘reconditioning’’ PCR reaction was done with
the same reaction conditions except 5 cycles were used. Amplicons
were cloned using TA cloning (Life Technologies), and plasmid
extractions were done with the Qiagen Miniprep kit. Clones were
sequenced with an Applied Biosystems 3130xl Genetic Analyzer.
Sequence analysis was done with the Ribosomal Database Project
(RDP) Classifier tool.
3. Model development
3.1. Model description and implementation
ADM1 is a mechanistic model with 35 dynamic state variables,
4 algebraic equations, 19 biochemical processes, 6 acid-base pro-
cesses and 3 liquid-gas transfer processes (Rosen and Jeppsson,
2004). With the addition of the bacterial acetate oxidation process,
the model was changed to 36 dynamic state variables and 21
biochemical processes. ADM1 assumes Monod-type kinetics.
Therefore, acetate oxidation was also assumed to have Monod-type
kinetics (Shimada et al., 2011) as shown in Eq. (1).
Sac
q11a ¼ km;acst   X st  I13 ð1Þ
K s;acst þ Sac
km,ac-st is the Monod maximum specific uptake rate (kgCODac
kgCODx1 d1) and KS,ac-st is the half saturation value for acetate-
oxidizing bacteria (Xst). The inhibition term (I13) considers low pH
inhibition (IpH,aa), inhibition for limiting nitrogen, (IIN,lim) and
hydrogen inhibition (Ih2,ac) (Eq. (2)).
I13 ¼ IpH;aa  IIN;lim  Ih2;ac ð2Þ
Table 1
Biochemical rate coefficients and kinetic rate equations for the syntrophic acetate oxidation process.
Process: j Component: i
7 8 10
Sac Sh2 SIC
(kgCOD m3) (kgCOD m3) (kmol m3)
P
20 Decay of  i¼1—9;11—25 C j  mi;20
Xac-st
P
11a Acetate oxidation 1 (1  Yac-st)  i¼1—9;11—25 C j  mi;11a
497
Hydrogen inhibition was considered as a non-competitive inhibi-
tion similar to propionate, butyrate and valerate as shown in
Eq. (3). According to previous work for hydrogen inhibition,
hydrogen partial pressure above 40 Pa has a KI,h2,ac value of
1  108 kgCOD m3, which is the concentration of dissolved
hydrogen in liquid medium (Hattori, 2008; Schink, 1997).
1
Ih2;ac ¼ ð3Þ
1 þ Sh2 =K I;h2;ac
The decay of acetate-oxidizing bacteria (Eq. (4)) was implemented
as a first order kinetic which was also used for the other microor-
ganisms in the model (Shimada et al., 2011). kdec,Xst has the value
of 0.04 d1 as in ADM1 for kdec of the model.
q20 ¼ kdec;Xst  X st ð4Þ
The modification for ADM1 is summarized in Table 1 as a Petersen
matrix. A new differential equation was proposed for syntrophic
acetate-oxidizing bacteria. Equations for complex matter (Xc), total
acetate (Sac) and dissolved hydrogen (Sh2) were modified due to
addition of acetate oxidation.
To avoid the modeling of aceticlastic methanogenesis, the vari-
able Xac was fixed to zero (Xac = 0 kgCOD m3) to represent the
absence of acetate-consuming methanogens in the system. This
avoids the growth of Xac, but keeps the process of aceticlastic
methanogenesis in the model. In previous studies, the inhibition
of aceticlastic methanogens was necessary to promote acetate oxi-
dation as a dominant methanogenic pathway (Wilson et al., 2012).
The digester was considered to be a continuous system with an
input flow of 6.66  104 m3 d1. The simulations were carried
out using Matlab 6.5.0 simulation platform.
3.2. Parameter estimation
Parameter values initially recommended by Batstone et al.
(2002) and Rosen and Jeppsson (2004) were used. The parameters
for acetate-oxidizing bacteria (km,ac-st, KS,ac-st and Yac-st) were
adapted from Beaty and McInerney (1989) and Dwyer et al.
(1988). The estimated kinetic parameters were as follows: Yfa,
Yc4, Ypro, Yh2, kdis, khyd,ch, khyd,pr, khyd,li, km,su, KS,su, Ysu, km,aa, KS,aa, Yaa,
km,fa, KS,fa, km,c4, KS,c4, km,pro, KS,pro, km,h2, KS,h2, km,ac-st, KS,ac-st and
Yac-st.
Two methods for the estimation of parameters were applied as
follows: manual calibration coupled to non-linear least squares
(MC-NLLS) and differential evolution algorithms (DEA).
Manual calibration coupled to non-linear least squares: Initially, a
manual calibration of the parameters was done to get a similar
trend between experimental results and model outputs. After this,
non-linear least squares were applied to refine the value of the
parameters. The nonlinear least squares method with the algo-
rithm of Levenberg–Marquardt was used. The ‘‘lsqnonlin’’ Matlab
function was used to minimize the objective function (Eq. (5)).
X
N
f ðpÞ ¼ ^i  yi Þ2
ðy ð5Þ
i¼1
Process rate
(kgCOD m3 d1)
11 13 25
SIN Xc Xac-st
(kmol m3) (kgCOD m3) (kgCOD m3)
(Nbac  Nxc) 1 1 q20 ¼ kdec;Xst  X st
(Yac-st)Nbac Yac-st Sac
q11a ¼ km;acst  K s;acst þSac  X st  I13
498 V. Rivera-Salvador et al. / Bioresource Technology 167 (2014) 495–502

where f (p) is a function that depends on the vector of parameters; Archaeal community structure was analyzed with 16S rRNA
^i is a vector of model output i as result of the vector of parameters
y clone libraries from a digester sample collected on day 30.
p and yi is a vector of observed data i. The reason for a coupled Thirty-four 16S rRNA sequences were randomly sequenced. Phylo-
method (MC-NLLS) is the problem of the over parametrization. This genetic analysis showed that 84.8% of the sequences were highly
means that the estimation of a large quantity of parameters (>10) similar (98% identity) to Methanothermobacter wolfeii (AB104858)
by least squares leads to inaccurate estimated values and inaccurate (Watanabe et al., 2004), 6.1% of the sequences were related to
predictions (Makowski et al., 2006). Methanoculleus thermophilus (AB065297) (Rivard and Smith,
Differential evolution algorithms: DEA are evolutionary algo- 1982) and 9% of the sequences were related to an uncultured
rithms based on a population of potential solutions represented archaeon (FJ205778) (Kröber et al., 2009) (Table 3). Both of these
as floating-point vectors. Such algorithms are simple and efficient genera are hydrogenotrophic methanogens. No known aceticlastic
compared with other global optimization methods. DEA reduces methanogens, such as Methanosarcina, were detected. The absence
the number of function evaluations required to converge to the of aceticlastic genera in this sample indicates that bacterial acetate
global optimum (López-Cruz et al., 2008). The DEA parameters oxidation is probably the dominant pathway (Shimada et al., 2011;
were: 0.2 for crossing over probability, differential variation factor Westerholm et al., 2011) although some researchers have sug-
of 0.9, population size of 60, 1  106 for accuracy, 25 variables for gested that the coupled growth of syntrophic acetate-oxidizing
optimization, 1000 generations, and the use of the standard differ- bacteria and Methanosarcina sp. may enhance the stability of reac-
ential evolution algorithm named DE/rand/1/bin. tors at high ammonia concentrations (De Vrieze et al., 2012).
Therefore, this analysis confirms that acetate oxidation in this
digester occurs primarily through the hydrogenotrophic pathway.
4. Results and discussion
4.3. Parameter estimation results
4.1. Substrate characterization
Parameters estimated by the two optimization methods are
Poultry litter was characterized in terms of its principal compo- shown in Table 4. Most of the parameters showed similar values
nents: carbohydrates, proteins, fats, volatile fatty acids and ammo- in both methods and model outputs showed similar behaviors.
nia. The results of this analysis are shown in Table 2. It is important However, important differences were found for km,su, km,aa, km,pro
to highlight the large concentration of hemicellulose (18.15%) and and km,h2. These differences may indicate that those parameters
protein (15.43%). Alkalinity was taken from previous results for could not affect model outputs but a sensitivity analysis is neces-
anaerobic digestion of poultry litter in a pilot plant digester sary to corroborate this hypothesis.
(Espinosa-Solares et al., 2006) as 6.21 kgCaCO3 m3 in a 5.46% TS Carbohydrates (Xch) are hydrolyzed faster than proteins and lip-
suspension. ids under these anaerobic conditions, which was associated to the
The substrate also contained a small concentration of lactic khyd values. Similar results have been previously considered in an
acid. The original ADM1 contains no modeling of the lactate degra- anaerobic digester fed with cattle manure and renewable energy
dation process, since concentrations as intermediates in most crops (Lübken et al., 2007).
digesters are low. Batstone et al. (2002) has pointed out that lactate When kinetic parameters for acetate-oxidizing bacteria are
is degraded quickly, and it has the same stoichiometry as glucose. needed, two main difficulties arise: isolation and the study of
Additionally the biological reaction stoichiometry is not affected by kinetics of single acetate-oxidizing bacterial populations (Hao
its omission from ADM1. et al., 2011; Hattori, 2008). The application of a parameter
estimation method, such as DEA or MC-NLLS, can be an adequate
alternative procedure to calculate those kinetic parameters.
4.2. Microbial diversity analysis However, DEA allows the estimation of more parameters than con-
ventional procedures, such as least squares (Shimada et al., 2011),
Acetate-oxidizing bacteria and hydrogenotrophic methanogens and reduces the problem of overparametrization. This method may
were previously found to be present in the 40 m3 pilot plant diges- increase the accuracy of the parameters values and the quality of
ter that was the source of inoculum used for this experiment the model prediction. Nevertheless, kinetic parameters for ace-
(Smith et al., 2014). To confirm that the microbial community tate-oxidizing bacteria (Xst) were calculated by DEA and coupled
had retained these methanogen populations, archaeal diversity MC-NLLS as estimation procedures to compare both methods.
was sampled in the lab-scale thermophilic digester. Using the DEA method, the value for km,ac-st was
3.25 ± 0.57 kgCOD kg1
COD d
1
and KS,ac-st was 0.29 ± 0.02 kgCOD m3,
which are similar to values derived by Dwyer et al. (1988) from
Table 2
syntrophic anaerobic bacteria in coculture with hydrogen-
Chemical composition of chicken litter manure.
oxidizing methanogens. On the other hand, the values obtained
Component % Dry basis here were similar to the ones reported by Shimada et al. (2011)
Hemicellulose 18.15 for mesophilic conditions in two-phase anaerobic digestion of
Lignin 3.61 waste activated sludge. For MC-NLLS; the value of km,ac-st
Crude protein 15.43 (2.8 kgCOD kg1 1
COD d in this paper) was slightly less than the one
Crude fats 1.56
Starch 1.51
(3.9 kgCOD kg1
COD d1
) reported by Shimada et al. (2011).
Water soluble carbohydrates 2.34 The yield parameter for syntrophic bacteria (Yac-st) was
Ash 7.95 estimated as 0.104 ± 0.016 kgCOD kg1 COD by DEA and 0.14 kgCOD
Ammonia-N 0.2386 kg1
COD by MC-NLLS, which are similar to the value reported by
Neutral detergent fiber 41.99
Shimada et al. (2011).
Acid detergent fiber 23.84
Volatile fatty acids 1.34 For some researchers, the transition of the dominant pathway
Acetic acid 0.6656 from syntrophic acetate oxidation to aceticlastic methanogenesis
Propionic acid 0.0315 was considered a minor difference. This may explain why syn-
Iso-butyric acid 0.0237 trophic oxidation has frequently been neglected or seldom evalu-
Butyric acid 0.1007
ated (Hao et al., 2011). Another research group (Sasaki et al.,
 V. Rivera-Salvador et al. / Bioresource Technology 167 (2014) 495–502 499

Table 3
Phylogenetic affiliations and relative abundance of archaeal populations in the anaerobic digester on day 30 based on 16S rRNA gene clone libraries.

Phylogenetic affiliation 16S rRNA% Similarity Methanogenesis Relative abundance at day 30

Methanothermobacter wolfeii 99.3% Hydrogenotrophic 84.8%
Methanoculleus thermophilus 99.8% Hydrogenotrophic 6%
Uncultured archaeon (FJ205778) 99.1% Unknown 9%

Table 4
Estimated parameters of the modified ADM1 models.

Parameter Uncertainty intervala MC-NLLS estimated value DEA estimated value (meand ± SD) Units
kdis 0.24–1.0 0.6032 0.55 ± 0.37 d1
khyd,ch 0.19–1.94 1.0675 1.05 ± 0.56 d1
khyd,pr 0.0096–1.0 0.5051 0.88 ± 0.23 d1
khyd,li 0.0096–0.4 0.2136 0.17 ± 0.17 d1
km,su 27–107 37.943 85.9 ± 27.0 kgCOD kg1
COD d
1

KS,su 0.05–0.2 0.1228 0.15 ± 0.04 kgCOD m3

Ysu 0.01–0.15 0.1066 0.13 ± 0.02 KgCOD kg1
COD
km,aa 27–107 27.465 70.4 ± 29.9 kgCOD kg1
COD d
1

3
KS,aa 0.05–0.2 0.1262 0.1 ± 0.05 kgCOD m
Yaa 0.086–0.15 0.0917 0.098 ± 0.02 kgCOD kg1
COD
km,fa 11–37 24.057 26.99 ± 9.7 kgCOD kg1
COD d
1

3
KS,fa 0.058–2.0 0.4022 0.076 ± 0.04 kgCOD m
Yfa 0.004–0.05 0.0262 0.023 ± 0.01 kgCOD kg1
COD
km,c4 5.3–13.7 6.3884 5.39 ± 0.17 kgCOD kg1
COD d
1

KS,c4 0.012–0.36 0.3230 0.36 ± 0.0001 kgCOD m3

Yc4 0.05–0.079 0.0789 0.06 ± 0.005 kgCOD kg1
COD
km,pro 5.5–33.0 10.012 23.4 ± 6.7 kgCOD kg1
COD d
1

3
KS,pro 0.02–0.392 0.3687 0.29 ± 0.12 kgCOD m
Ypro 0.05–0.089 0.0592 0.058 ± 0.009 kgCOD kg1
COD
km,h2 1.68–178.0 12.974 76.36 ± 44.34 kgCOD kg1
COD d
1

KS,h2 1  106–6  104 5.0  104 4  104 ± 1.95  104 kgCOD m3
Yh2 0.014–0.183 0.1715 0.095 ± 0.05 kgCOD kg1
COD
km,ac-stb 0.037–25.0 1.1204 3.25 ± 0.57 kgCOD kg1
COD d
1

KS,ac-stb 0.0005–0.3 0.2035 0.29 ± 0.02 kgCOD m 3

Yac-stc 0.0001–1.0 0.1350 0.104 ± 0.016 kgCOD kg1

COD

a
From Batstone et al. (2002).
b
From Dwyer et al. (1988).
c
From Beaty and McInerney (1989).
d
Mean of ten replicates.

2011), working with TAD and using labeled carbon, reported that
non-aceticlastic oxidation accounted for 74–88% of the total degra-
dation of acetate; consequently the aceticlastic cleavage of acetate
accounted for 12–26%.
In the present study, the kinetic values for syntrophic bacteria
(mainly km,ac-st and Yac-st) are different from those used in ADM1
for aceticlastic methanogens (km,ac and Yac). km,ac is much
larger (16 kgCOD kg1COD d
1
) than km,ac-st (1.12 kgCOD kg1COD d
1
and
3.25 ± 0.57 kgCOD kg1COD d 1
), and Y ac-st (0.14 kg COD kg 1
COD and
0.1 kgCOD kg1 1
COD) is more than double Yac (0.05 kgCOD kgCOD) which
was reported in ADM1. Therefore, the omission of a syntrophic
acetate oxidation pathway in this model may have different results
compared to an aceticlastic methanogenesis pathway.
4.4. Model outputs
Similar trends were found in the model in both cases, using DEA
and MC-NLLS (Fig. 1). However, manual calibration (MC), called
trial and error, has a subjective element which means that MC
depends on the judgment of the modeler, and replication of the
results may be difficult.
For the two methods, several measures of agreement between
calculated and measured data were determined for the variables
‘‘volatile fatty acids’’, ‘‘methane partial pressure’’ and ‘‘pH’’. For
each one, the next measurements were performed (Makowski
et al., 2006): bias, mean square error (MSE), relative root mean
square error (RRMSE), model efficiency (EF), correlation coefficient
(r) and the agreement index (Index). The results of these values are
in Table 5.
Both methods of parameter estimation showed a better
adjustment than the original ADM1 parameters, mainly in the
VFA outputs, as can be appreciated by an index close to 1.00
(0.95 for MC-NLLS and 0.85 for DEA). The values of bias indicate
that in all cases, the model under-predicts (Bias > 0) for VFA and
pH, but over-predicts (Bias > 0) for methane percentage.
Actually, it is accepted that a good model would have small bias
and a correlation coefficient (r) near to 1.00 (Makowski et al.,
2006). Both adjustment by DEA and MC-NLLS showed a small bias
and r near to one to describe VFA behavior. These values are better
in comparison with the ones obtained using the original
parameters of the ADM1. Therefore, the application of a parameter
estimation method (DEA or MC.NLLS) increased the quality of the
ADM1.
An agreement index of 1.00 indicates a perfect model, other-
wise if the model predictions are identical in all cases and equal
to the average of the observed values then the index is zero
(Makowski et al., 2006). In this case, both MC-NLLS and DEA
increased the quality of the original ADM1 for the VFA measured
data. Interestingly, the calculated index indicates good model
estimation for pH and methane partial pressure. However, these
values must be taken carefully, because the average of the
observed data and the model predictions had similar values that
500 V. Rivera-Salvador et al. / Bioresource Technology 167 (2014) 495–502

1200 1200
Acetate
(a) Propionate (b)
Acetate
Propionate
Butyrate Butyrate
1000 1000
Valerate
Volatile fatty acids [mg·L ]

Volatile fatty acids [mg·L ]

Valerate
-1

-1
MC-NLLS Simulated Acetate
DEA Simulated Acetate
MC-NLLS Simulated Propionate
800 DEA Simulated Propionate
MC-NLLS Simulated Butyrate 800
DEA Simulated Butyrate
MC-NLLS Simulated Valerate
DEA Simulated Valerate

600 600

400 400

200 200

0 0
0 20 40 60 80 0 20 40 60 80
Time [ d ] Time [ d ]
100 100

(c) Experimental Methane Data

(d)
MC-NLLS-Simulated Methane Experimental Methane Data
80 80 DEA-Simulated Methane
Biogas composition [% ]

Biogas composition [% ]
60 60

40 40

20 20

0 0
0 20 40 60 80 0 20 40 60 80
Time [d] Time [ d ]

Fig. 1. Comparison of modeled and experimental results for (a) VFA using MC-NLLS, (b) VFA using DEA, (c) biogas composition using MC-NLLS, (d) biogas composition
using DEA.

Table 5
Measures of agreement between measured and calculated values.

Variable of analysis Measure of agreement ADM1 (parameters from Batstone et al. ADM1 (estimation by MC-NLLS) ADM1 (estimation by DEA)
(2002) and Rosen and Jeppsson (2004))
Volatile fatty acids Bias (kgCOD m3) 0.4623 0.0366 0.1368
MSE ((kgCOD m3)2) 0.3049 0.0216 0.0471
RRMSE 1.0837 0.2883 0.4260
EF 2.0592 0.7834 0.5272
r 0.3262 0.9136 0.8551
Index 0.4797 0.9494 0.8502
Methane partial pressure Bias (bar) 0.0394 0.0085 0.0111
MSE (bar2) 0.0019 0.0082 0.0009
RRMSE 0.0861 0.1769 0.0588
EF 1.4699 9.4280 0.1526
r 0.5366 0.0328 0.0925
Index 1.0000 1.0000 1.0000
pH Bias 0.1449 0.4168 0.2266
MSE 0.0247 0.2054 0.0535
RRMSE 0.0202 0.0582 0.0297
EF 15.7067 138.1973 -35.2749
r 0.3841 0.0194 0.0097
Index 0.9999 0.9995 0.9999

MSE: mean square error; RRMSE: relative root mean square error, EF: model efficiency; r: correlation coefficient; Index: index agreement.

caused an index near to 1.00, which could affirm that the model is represented in the model, a peak-like perturbation occurred during
quasi-perfect. Nevertheless, the values of efficiency (EF) indicated the first days (Fig. 1c and d). The reasons for this behavior are
that the model could be a worse predictor than the average of unknown but are probably due to ‘‘auto-fitting’’ of the model.
the observed values (EF < 0) for pH and methane partial pressure According to Girault et al. (2012), during a non rate-limiting acido-
outputs (Table 5). genic stage, the peak at the beginning of an anaerobic digestion
In the case of methane partial pressure and pH, the model was process was the result of a rapid conversion into methane of
not as good as for VFA. Although biogas composition was well hydrogen (Girault et al., 2012).
 V. Rivera-Salvador et al. / Bioresource Technology 167 (2014) 495–502
0.6
Degrading microorganisms [kg COD·m-3 ]
Sugar degraders (Xsu)
0.5 Aminoacid degraders (Xaa)
Fatty acid degraders (Xfa)
Butyrate and valerate degraders (Xc4)
0.4
Propionate degraders (Xpro)
0.3
0.2
0.1
0.0
0 20 40 60
Time [d]
Fig. 2. Microbial biomass simulation during the chicken litter manure treatment.
In terms of the outputs, simulation of volatile fatty acids
(propionate, butyrate, valerate and acetate) showed a closer repre-
sentation to experimental data, mainly in the first 4 HRT́s (60 days)
for propionate, butyrate and valerate. On the other hand, propio-
nate was overestimated at the end of the process, mainly after
day 60 (Fig. 1a and b). In addition, acetate simulation did not rep-
resent the dynamic behavior of experimental data at the beginning
of the process.
Pseudo-stationary states were reached for all simulated volatile
fatty acids after 2.66 HRT (40 days). Furthermore, for simulated
microbial biomass, all species reached similar results. Sugar
degraders (Xsu) were the last group to reach a pseudo-stationary
state, following a significant biomass decrease (Fig. 2). The popula-
tions of microorganisms that were used in the model for methane
production were the hydrogenotrophic methanogens (Xh2) and the
acetate-oxidizing bacteria (Xst). This assumption was taken based
on the experimental results of this work which are in agreement
with the TAD reported by Sasaki et al. (2011). Aceticlastic metha-
nogens (Xac) remained zero during the entire simulation time, so
methane production from aceticlastic methanogenesis was not
carried out in the simulation. However, the accumulation of other
VFAs (propionate, butyrate) may have been due to the lack of
sufficient syntrophic bacteria, mainly at the beginning of the
simulation (Shi et al., 2013).
With respect to acidogenic and acetogenic bacteria, the amino
acid degraders (Xaa) and valerate and butyrate degraders (Xc4)
had the highest abundances. On the other hand, long chain fatty
acid degraders (Xfa) obtained the lowest abundance compared to
all the active microbial biomass. This was apparently due to the
low concentration of lipids in the substrate. The greater impor-
tance of the acetogenic step compared to the acidogenic step is
highlighted by the high abundance of acetogens. Lastly, the accu-
mulation of organic acids demonstrated their inhibitory effect on
methane production which is undesirable in anaerobic digestion.
5. Conclusion
TAD of poultry litter was dominated by hydrogenotrophic meth-
anogens, suggesting bacterial acetate oxidation as the primary
pathway. For syntrophic bacteria, the values estimated by MC-NLLS
were km,ac-st 1.12 kgCOD kg1COD d
1
, KS 0.20 kgCOD m3 and Yac-st
0.14 kgDQO kg1
DQO ; while using DEA they were km 3.25 ± 0.57 kgCOD
kg1
COD d
1
, KS 0.29 ± 0.02 kgCOD m3 and Yac-st 0.104 ± 0.016 kgDQO
kg1
DQO. Values of BIAS, MSE or INDEX demonstrate that both
methods (MC-NLLS and DEA) increased ADM1 accuracy. Thus, a
successful model was developed incorporating acetate-oxidizing
501
0.8
Methanogenic or acetate oxidizing
·m -3 ]
0.6
COD
microorganims [kg
0.4
Aceticlastic methanogens (Xac )
Syntrophic acetate oxidizers (Xst)
0.2 Hydrogenotrophic methanogens (Xh2)
0.0
80 0 20 40 60 80
Time [d]
syntrophic bacteria linked to hydrogenotrophic methanogens for
TAD.
Acknowledgements
The authors wish to acknowledge the financial support from
West Virginia State University (USA), USDA Grant 2010-02276
(USA), Gus R. Douglass Institute’s Agricultural and Environmental
Research Station (USA) and Consejo Nacional de Ciencia y
Tecnología (México). Furthermore express their gratitude to Dr.
Christian Rosen and to Dr. Ulf Jeppsson for providing the initial
Matlab implementation of ADM1.
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