Ind. Eng. Chem. Res.

2008, 47, 5337–5345 5337

Volatile Fatty Acid Anaerobic Degradation: Kinetic Modeling with an Inoculum

under Controlled Conditions
Karina Boltes,* Pedro Leton, and Eloy Garcia-Calvo
Departamento de Ingenierı́a Quı́mica, UniVersidad de Alcalá, Edificio de Ciencias, Campus UniVersitario,
28871 Alcalá de Henares, Spain

Due to the multiple reactions that are involved, the mathematical description of anaerobic degradation of
organic matter is normally complicated. Several efforts have been made for the development of the ADM1
model, that cover the major processes involved in complex organic substrate conversion. This model application
requires a large number of constants and coefficients, which were proposed, reviewed the information available
at the time of their publication. The more recent published papers about ADM1 model application report a
necessary revision of the kinetic parameters used for volatile fatty acid (VFA) degradation. This work presents
a kinetic study of VFA anaerobic degradation performed in batch and continuous stirred tank reactor. Acetic,
propionic, and butyric acids (mixed in a ratio 2:1:1 COD basis) and acetic acid only were used as substrate.
The biomass for kinetics assays was previously produced in a codigestion process using pig manure mixed
with sewage sludge obtained from anaerobic municipal digester. The inoculum build-up and maintenance
were conducted in a laboratory stirred tank digester, under controlled conditions to avoid any variability of
the resulting parameters obtained. Moreover, the black box approximation was applied in order to reduce the
number of parameters for a complete description. A set of lineal relations was obtained to estimate methane,
carbon dioxide, and mixed biomass production rates, from VFA degradation rates only. Finally, a good
simulation of experimental data was obtained for VFAs, biomass, methane, and carbon dioxide both in
continuous and batch operation modes.

1. Introduction different substrates; the growth and endogenous decay of each

trophic group of bacteria as well as the corresponding yield
The anaerobic degradation of organic matter to methane is a
coefficient for each microorganism on the substrate are needed.
highly complex process. On the microbial level, the process
All of these parameters were obtained from an extensive
requires the combined and coordinated action of a variety of
bibliography revision of the experimental data available at that
distinct bacteria. The reaction scheme proposed by Gujer and
time. The use of the ADM1 model to describe an anaerobic
Zhender1 includes the following processes: hydrolysis of
degradation process with accuracy is normally complicated, and
degradable particulate organic matter, fermentation of amino
only a few studies exist in implementing the ADM1 model for
acids and sugars, oxidation of long chain fatty acids, oxidation
experimental data simulation.9–11 The most recent published
of volatile fatty acids (VFAs), and methanogenesis. The more
works in which the ADM1 model was applied report a necessary
relevant characteristic of this biological system is that the
revision of the kinetic parameters used for volatile fatty acid
metabolism of each group of bacteria is dependent on the others,
(VFA) degradation. It would appear that the inhibition functions
and consequently, the degradation involves multiple reactions
associated with low pH values tend to overestimate the impact
in series and/or in parallel catalyzed by different groups of
of pH on biokinetic rates for acid-consuming bacteria.9 In
microorganisms. The volatile fatty acid (VFA) conversion is
addition, the kinetic parameters proposed in the ADM1 model
assumed to be the most important step of the global process
for propionate and acetate utilization, identified as the most
for degradation of organic matter to methane.2 Since 70% of
sensitive, were optimized manually for better data simulation;10
the methane produced comes from acetic acid degradation, long
therefore, it is evident that these kinetic constants must be
chain fatty acids need to be converted to acetic acid before they
revised. In other cases, the decay rate initially set for mesophilic
can be converted to methane. The microbial groups that
UASB reactors was increased by a factor of 5, resulting in more
degraded fatty acids, acetate and propionate, are slowly growing
realistic data like those reported by Batstone et al.11
bacteria and are therefore important for digester stability.3 In
The objective of this study is to contribute with a set of kinetic
this way, the inhibition processes that are involved in acetoge-
constants for the anaerobic digestion modeling and simulation.
nesis and methanogenesis are very recognized.
We present a kinetic study of VFA degradation to methane using
There are several studies published about mathematical
an inoculum developed and maintained in a laboratory reactor
modeling of organic matter anaerobic degradation.4–7 Even a
under controlled conditions. A mixture of acetic acid (HAc),
standardized reference model, namely, ADM1, has been estab-
propionic acid (HPr), and butyric acid (HBut) was used in a
lished by the IWA Task Group to provide a unified basis for
ratio of 2:1:1 (chemical oxygen demand (COD) basis) as the
anaerobic digestion modeling.8 The ADM1 model is the more
carbon source. In addition, only acetic acid was used in another
accepted and valued one because of its possible applicability
set of assays as the carbon source for methane production.
for different types of bioreactors and operational conditions. In
The inoculum build-up was performed in a previous stage
general, the application of this model requires a great number
using a stirred tank reactor. Pig manure and sludge were
of kinetics parameters to describe the conversion rates of
collected from a municipal sludge anaerobic digester. Manure
* To whom correspondence should be addressed. E-mail: contains a high content of proteins and urea, which upon
karina.boltes@uah.es. Phone: +34-918856422. Fax: +34-918855088. degradation release ammonia, a potent inhibitor of aceticlastic
10.1021/ie071583p CCC: $40.75  2008 American Chemical Society
Published on Web 07/02/2008
5338 Ind. Eng. Chem. Res., Vol. 47, No. 15, 2008
methanogens.12 Moreover, excessive VFA accumulation can
inhibit methanogenesis, as high hydrogen levels can inhibit
propionate and butyrate degrading acetogens.13 The original
manure and sludge mix was prepared to adjust the solids, acids,
and soluble chemical oxygen demand (COD) levels in order to
avoid high inhibition processes. For several months, this
bioreactor was followed by means of a methanogenic activity
test, VFA concentration, suspended solids, and biogas production.
The black box approximation was applied as a tool to reduce
the number of parameters for a complete system description.
From the black box application, it was found that it is necessary
to define only four rate conversion equations for the estimation
of the others. Principally, the most important is the methane
formation rate.
The kinetic constants for butyrate, propionate, and acetate
degradation under mesophilic conditions were obtained in
continuous and batch assays. Moreover, the decay constant of
the mixed biomass was then calculated. Finally, the experimental
data of VFA and methane were simulated for the different
operation modes, using the kinetic constant presented and the
lineal relation obtained from the black box description.
2. Black Box Aproximation
The most important aspect of microbial systems is that they
are open systems because microorganisms exchange mass and
energy with their environment. In microbial processes, we are
faced with an enormous complexity which could lead to
countless model parameters. Therefore, there is a need for
approximation to obtain a mathematical model with minimal
complexity. The main approximation applied in engineering is
the macroscopic approximation. Here, the system can be
considered to contain many so-called elementary volumes, which
in the case of a bioreactor could be 1 L of culture volume. This
elementary volume can be described by their average value,
which is a system property or the so-called macroscopic
properties. The properties denominated as intensive ones (pres-
sure, temperature) cannot be added up, but the extensive
properties (like mass and energy) are related to the system
quantities and can be added or can make balances.
For the full system definition in a constant volume system,
the establishment of the kinetics of all chemical compounds as
well as the transfer over the system boundary are necessary.
However, in the case of a microbial system, in which there are
almost 800 chemical compounds (intra- and extracellular)
involved, a mathematical model containing a large number of
parameters (about 1600) can result. Considering the extremely
large complexity described, the need of minimization of model
parameters can be satisfied by the following:14
-The assumption of the pseudostationary state of intracellular
compounds. Because there is no mass transfer over the cell
membrane, their concentration is generally very low and almost
constant due to the fact that microorganisms tend to maintain a
constant internal environment. This is the so-called black box
approach.
-The definition of the relevant compounds only. This means
that one can limit the chemical compounds to those which cross
the cellular membrane, at least: N-source, C-source, water, and
biomass.
-Application of conservation principles: conservation of mass,
elements, electric charge, and energy (1st law of thermodynam-
ics).
-Definition of a reaction scheme. If knowledge of metabolic
pathways exists, it is possible to specify some reactions like
anabolism, polymerization, oxidation of electron donor, and ATP
Table 1. Black Box Description of VFAs Anaerobic Degradation
net
elemental
composition
conversion
relevant compound rate
biomass CH1.4O0.4N0.2 rAx
acetic acid (electron donor) CH2O rAAc
propionic acid (electron donor) CH3O2/3 rAPr
butyric acid (electron donor) CH4O1/2 rABu
methane (product) CH4 rAm
ammonium (nitrogen source) NH4+ rAn
water H2O rAw
protons H+ rAp
bicarbonate (electron acceptor) HCO3- rAc
Scheme 1. General Form of System Matrices
production by electron transfer. In this paper, the system was
modeling using the black box approximation, defining a set of
relevant compounds, and applying conservation principles for
each one. The use of metabolic information is difficult because
biomass is a mixed culture, formed by different strains of
methanogens.
The most important simplification that was introduced is the
consideration of the mixed anaerobic biomass as another relevant
component of the reaction system with a fixed elemental
composition. Due to this, the cells can be considered as a black
box, exchanging only heat and compounds such as substrate
and products with the environments and dissipating Gibbs
energy, in the so-called black box thermodynamic approxima-
tion. Here, the pseudostationary state of intracellular compounds
was adopted and only the exchangeable ones are considered.15
These exchangeable compounds are the relevant denominated
ones, and the first step is the identification of them.
2.1. System Definition. In the anaerobic VFA mixture
degradation, we consider that there are three carbon sources in
a fixed proportion; methane is the only final product of
catabolism, because carbon dioxide is considered as the final
electron acceptor. The nitrogen source was NH4+ added in the
form of NH4Cl. The elemental composition of anaerobic
biomass can be represented by C5H7O2N.16 A detailed black
box definition of the anaerobic VFA consumption system is
summarized in Table 1.
2.2. Derivation of the Lineal Relations. A set of lineal
relations between the net conversion rates of relevant compounds
was obtained from elements, electrical charge, and Gibbs energy
balances.17,18 These equations allow estimation of dependent rates,
i.e., methane or biomass formation, as a function of those selected
as independent (VFA conversion and dissipated Gibbs energy).
Calculation of the lineal conservation relations is simplified
remarkably by use of lineal algebra from the definition of a
system of matrices and vectors. All of the information for the
black box description is contained in the elemental composition
matrix E. In addition, matrix M contains electrical charge and
Gibbs energy balances. Hence, matrix E is a submatrix of M,
as shown in Scheme 1.
The advantage of matrix M is evident because it contains
the Gibbs energy dissipation rate (Ds01) that can be estimated
by a simple correlation proposed by Heijnen and van Dijken.18

The lineal relations were obtained from matrix M and the net
conversion rate column vector r, applying the following conserva-
tion equation. The vector r includes the net conversion rates of all
relevant compounds and the Gibbs energy dissipation rate too.
M×r)0 (1)
The right definition of system matrices and a correct choice of
measured rate vector are essential for the successful use of lineal
relations. For this reason, we use a systematic procedure proposed
by Noorman et al.15 that involves the application of three tests:
the independency, consistency, and observability tests.
For the Gibbs energy balances, the values given by Roels17 for
the free energy of formation of relevant compounds were used.
See Table 1 for elemental composition and rate definition.
r ) [rAAc, rAPr, rABu, rAx, rAn, rAc, rAp, rAw, rAm, Ds01]
rAAc rAPr rABu rAx rAn rAc rAp rAw
C 1 1 1 1 0 1 0 0 1
H 2 2 2 1.4 4 1 1 2
2 1
O 1 /3 /2 0.4 0 3 0 1
M)
N 0 0 0 0.2 1 0 0 0
electrical 0 0 0 0 1 -1 1 0
charge
λ -186 -120.33 -94.5 -67 -80 -588 -40 -238 -51 1
The system results in four degrees of freedom, so four
independent rates are necessary to estimate the others. If
individual VFA conversions and Gibbs energy dissipation (Ds01)
were chosen as independent, the next lineal relations (in
C-mol · L-1 h-1) result:
rAx ) -1.845rAAc - 1.725rAPr - 1.290rABu - (1/18.7)Ds01
rAn ) 0.369rAAc + 0.345rAPr + 0.25rABu + (1/93.5)Ds01
rAc ) 0.422rAAc + 0.446rAPr + 0.27rABu + (1/37.4)Ds01
rAp ) 0.053rAAc + 0.101rAPr + 0.012rABu + (1/62.3)Ds01
rAw ) -1.529rAAc - 1.314rAPr - 0.794rABu - (1/17)Ds01
rAm ) 0.422rAAc + 0.279rAPr + 0.020rABu + (1/37.4)Ds01
Ds01 is the dissipated Gibbs energy and can be estimated (per
C-mol of biomass produced) using the next correlation.18
Ds01/rAx ) 200 + 18(6 - C)1.8 + exp[{(3.8 - γs)2}0.16(3.6 +
0.4C)] (3)
where C is the number of carbon atoms and γs is the degree of
reduction of carbon source. For the application of eq 3, we used
average values for C and γs based on the proportion of each
volatile acid present in the mixture and their number of carbon
atoms. For an acid distribution of HAc/HPr/HBut (2:1:1 COD
basis), the result is C ) 2.67, γs ) 4.376, and Ds01/rAx ) 406.96
kJ/C-mol.
Using the calculated value of Ds01/rAx, a major simplification
was obtained for the lineal relations (eq 2), as can be seen next:
rAx ) -0.081rAAc - 0.076rAPr - 0.057rABu
rAn ) 0.369rAAc + 0.345rAPr + 0.25rABu + 4.352rAx
rAc ) 0.422rAAc + 0.446rAPr + 0.27rABu + 10.881rAx
rAp ) 0.053rAAc + 0.101rAPr + 0.012rABu + 6.532rAx
rAw ) -1.529rAAc - 1.314rAPr - 0.794rABu - 23.939rAx
rAm ) 0.422rAAc + 0.279rAPr + 0.020rABu + 10.881rAx
With these lineal relations, it is possible to estimate biomass,
methane, and carbon dioxide production rates, and some not
Ind. Eng. Chem. Res., Vol. 47, No. 15, 2008 5339
usually measured as nitrogen source consumption, from VFA
net conversion rates only.
The required kinetic parameters to define VFA conversion
rates (acetic, propionic, and butyric acids) were obtained in a
kinetic study in continuous and batch modes.
3. Experimental Procedure
3.1. Inoculum Build-up. Pig manure collected in a pig farm
next to the city of Alcala de Henares and sludge from an
anaerobic digester of a wastewater municipal plant placed in
the same city was used to produce the anaerobic mixed biomass
that was later inoculated in reactors for kinetic assays. A high
concentration of long chain fatty acids (LCFAs) and ammonia
can be generated in hydrolysis of lipids and protein fermentation
steps, respectively. It is recognized that both of them produce
rAm Ds01 inhibition of acetogens and methanogens.12,13 For this reason,
0 the pig manure was pretreated prior to the codigestion. First,
4 0 the manure was diluted with distilled water in 1:4 volume
0 0 proportions and agitated vigorously; then, it was filtrated with
0 0 1 mm pore size metallic mesh in order to remove the major
0 0
solid contents for hydrolysis enhancement.19,20 To reduce the
long chain fatty acids and ammonia concentration, the reaction
volume of 5 L was prepared as follows: 20% v/v of the pig
manure filtrate fraction, 10% v/v of the anaerobic sludge, and
water.
The anaerobic codigestion was conducted, as previously
indicated, in a 5 L stirred tank reactor placed in a room with
temperature controlled at 37 °C. pH was adjusted daily (6.8-7.2)
by adding 0.5 M HCl or NaOH. For four months, the reactor
was followed by measuring the total and volatile solids, COD,
VFA concentrations, and gas production. Once the digester
showed a decrease in methane generation along with constant
values of methanogenic activity, a small fraction of reactor
content was separated and fresh media with 5 g/L of glucose
and nutrients were added. This procedure was repeated for
several months, measuring methanogenic activity, VFA, COD,
and solid contents in each new step of fresh synthetic media
(2) addition. The culture medium used was an Evans minimal
medium modification21 that includes chlorine salts of Fe2+,
Co2+, and Ni2+.
Methanogenic Activity. The anaerobic biomass evolution
was followed by a methanogenic activity test using a VFA
mixture (HAc, HPr, and HBut) as substrate, according to the
method proposed by Soto et al.22 The medium contains 2 g of
COD/L, 6 g of VSS/L, Na2S · 9H2O (0.1 mg/L), and NaHCO3
(1000 mg/L).
Tests were conducted by triplicate using 100 mL sealed
Erlenmeyer flasks in an orbital shaker at 200 rpm. A blank test
with no carbon source addition completes the series. Temper-
ature was maintained at 37 °C, and pH was adjusted to 7
initially. The methanogenic activity was evaluated by measuring
the methane generation rate in the free volume of the T-flask.
Methane accumulated was expressed as grams of COD gener-
ated per grams of VSS (biomass) and per day. The gas was
sampling manually, and the methane content was determined
by gas chromatography coupled by a flame ionization detector
according to the method described in part 3.3. Once the activity
test has shown acceptable and constant values, the biomass
maintained was transferred to the reactor for kinetic assays.
3.2. Substrate and Reactor for Kinetic Studies. The kinetic
studies were conducted in batch and continuous operation modes
(4)
at a controlled temperature of 37 °C. For continuous feeding, a
2 L stirred tank reactor equipped with a sedimentation vessel
for biomass recirculation was used. The reactor, operated at a
5340 Ind. Eng. Chem. Res., Vol. 47, No. 15, 2008
Scheme 2. Experimental Apparatusa
a
A, feed vessel; B, pump; C, pH controller; E, feed; F, refrigeration
system; G, gas exit; H, acid vessel; K, reactor; L, liquid exit; M, stirrer
motor; OH, basic vessel; P, gas measurement device; R, biomass recircula-
tion; S, mixed liquor exit; T, gas sampling port.
constant hydraulic retention time (HRT) of 9.12 days, was fed
with increasing organic loading rate from 0.087 to 0.634 g of
COD/g of VSS · d.
The anaerobic bioreactor was connected to a gas measurement
device that permits sampling, according to Scheme 2.
Batch assays was performed in a 2 L stirred tank reactor,
with a gas measurement device similar to that used in continuous
operation. As substrate, a mixture of HAc/HPr/HBut at an initial
concentration varying from 1 to 4.2 g of total COD/L was used.
The initial biomass concentration was always adjusted to around
5 g of VSS/L.
In all of the kinetic assays performed at reactor scale, the pH
was automatically controlled in the range previously indicated.
3.3. Analytical Methods. The analysis of VFAs was carried
out by gas chromatography (GC) equipped with a flame
ionization detector (FID). A 1 µL portion of a filtered sample
was injected onto a 30 m NUKOL capillary column (ID 0.25
mm, film 0.25 µm) with nitrogen as the carrier gas. The
temperature program includes a hold at 60 °C for 2 min followed
by an initial ramp rate of 8 °C · min-1 up to 80 °C, a next ramp
at a rate of 21 °C · min-1 up to 160 °C, and a final warming up
to 180 °C at a rate of 5 °C · min-1. The final temperature was
maintained by 4 min for purge. Both injector and detector
worked at 200 °C.
In the same way, the gas composition was analyzed by GC
with a thermal conductivity detector (TCD). A packed column
PORAPAK-N 80/100 (1/8 in. × 4.5 m) was used with helium
as the carrier gas. The column temperature was programmed
as follows: initial temperature 40 °C (2 min) and a ramp up to
150 °C at a rate of 20 °C · min-1. The TCD temperature was
maintained at 120 °C (filament 180 °C). A sample volume of
0.4 mL was manually injected.
The mixed liquor volatile suspended solid (VSS) was used
as biomass concentrations, which was measured according to
section 2540 E in Standard Methods. In addition, total solids
and total volatile solids were obtained as the solid remaining
after warming at 105 or 550 °C.
The soluble chemical oxygen demand (COD) was also
measured according to section 5220 D in Standard Methods.
4. Results and Discussion
4.1. Inoculum Build-up. The manure and sludge codigestion
was initiated with 10 g /L of soluble COD, 24 g/L of total solid,
and 17 g/L of volatile solid concentration in the culture broth.
pH was adjusted to near neutral by addition of sodium
bicarbonate in the reactor media. Figure 1 shows the evolution
of these variables over a period of 2 months as well as the gas
production.
The results indicate a reduction of the total and volatile solid
content with methane production at almost neutral pH. Acetate was
the major acid component of the reactor content in this first stage,
followed by valeric and propionic acids, both at low levels.
Results obtained from the methanogenic activity tests show
an increment of activity over the time during the first 2 months,
followed by maintenance of the methanogenic activity value
near 0.30 g of CH4-COD/g of VSS · d from the third month of
digestion (see Figure 1). The methanogenic activity measured
over the VFA mixture indicated that the biomass is an active
consortium that can be used for kinetic assays.
The microscopic examination of the biomass was conducted
by electronic microscopy. A sample of the liquid content of
the reactor was filtered and fixed for dehydration. Figure 2 shows
the morphologic forms found in the bioreactor prior to the use
of this biomass as inoculum. Here, it is evident that there are
two types of cocci and two types of rods.
The biomass produced was maintained as previously indicated
during a period of six months; during this time, the inoculum
was used both for batch test assays and for the start-up of the
continuous feeding reactor.
4.2. Kinetic Study. For a bioreactor with biomass recycling
in continuous operation, a difference exists for the retention time
of the liquid and solid biomass. In these systems, the specific
substrate consumption rate can be expressed by
SF - SE
q) (5)
X·HRT
where SF and SE are the substrate concentration in the feed and
effluent of the system as total soluble COD, X is the mixed
biomass concentration in the reactor at steady state, and HRT
is the hydraulic retention time.
The sludge age (θ), which is defined as the ratio of the
biomass content of the reactor to the net growth, can be
calculated by the next expression
X·V
θ) (6)
XE·QE
Here, V is the reaction volume, QE is the exit flow rate, and X
and XE are the biomass concentration in the reactor and liquid
exit (L in Scheme 2), respectively. Equation 6 was used for
sludge age estimation due to fact that the residence time of the
sludge in the settle is very short compared with the reactor
residence time. In addition, for steady state conditions, the net
growth of biomass was assumed to be equal to the biomass
exit from the system, because there is no biomass inlet in feed
flow and purge was not done.
On the other hand, the substrate consumption and the biomass
production are related through the biomass yield on substrate
(YXS).
1 1 Kd
q) + (7)
YXS θ YXS
For the experimental system with biomass recycling, the
previous equation was used to obtain the mixed biomass decay

Figure 1. Evolution of parameters during the inoculum production step.
Figure 2. Photographs taken of the biomass in the 5 L stirred tank (inoculum reactor).
constant (Kd) and the yield YXS, considering that, in the steady
state, the sludge age is equal to the inverse of the biomass
specific growth rate (µ).
Figure 3 shows the experimental data of methane production,
effluent COD, and biomass concentration during continuous
feeding at a hydraulic retention time (HRT) of 9.12 days and
at a feed substrate concentration varying from 2.2 to 18 g of
COD/L. In each run and after the increment of the organic
loading, the digester was operated until the stationary state was
reached.
Analysis of Figure 3 shows that an increment of organic
loading at 0.634 g of COD/g of VSS · d produces a reduction of
methane generation rate and an accumulation of effluent COD,
principally due to acetic and propionic acids (data not shown).
Although the HAc, HPr, and HBut contents in the feeding were
Ind. Eng. Chem. Res., Vol. 47, No. 15, 2008 5341
in a ratio of 2:1:1, respectively, the ratio of these acids in the
effluents was different at high organic loading.
High acetic acid levels produce results in inhibition processes
usually detected in anaerobic reactors, like product inhibition
for acetogenesis from propionic acid and substrate inhibition
for methanogenesis from acetic acid. We did not detect process
inhibition in continuous operation until reactor failure, so from
this data series, the Monod kinetics parameters, maximum
specific rate of consumption, and saturation constant were
obtained for individual VFA consumption and total COD
elimination. In addition, mixed biomass yield on substrate
expressed as the sum of VFA, YX/S, was calculated. The obtained
kinetic parameters for each volatile acid are summarized in
Table 2.
5342 Ind. Eng. Chem. Res., Vol. 47, No. 15, 2008
Figure 3. Effluent COD (SE), biomass concentration (X), and methane
production (CH4) with specific organic loading, q, (mg of COD/mg of
VSS · d).
Moreover, the kinetic parameters for mixed biomass were
obtained by fitting the steady state values for specific substrate
consumption rate (q) and sludge age (θ) according to eq 7. The
resulting biomass yield over the mixed substrate, expressed as the
sum of VFA concentration in COD basis, was 0.116 g of VSS/g
of COD, with a decay constant, Kd, of 0.0054 d-1.
Due to the fact that there was not inhibition detected in
continuous feeding, the more relevant inhibition processes
mentioned above were studied in batch assays. A modification
of the generalized form of Monod kinetics proposed by Han
and Levenspield23 to account for all kinds of product inhibition
was used to analyze the propionic and butyric acid degradation.
This model assumes that there exists a critical inhibitor
concentration above which the processes stop, and that the
constants of the Monod equation are functions of this limiting
inhibitor concentration. The next expression is proposed for both
HPr and HBut consumption:
HAc n S
(
qi ) qmi 1 -
HAc* ) S+K HAc m
(8)
Mi( 1-
HAc* ) example, for HAc degradation, in the majority of published
Here, the specific rate of substrate consumption (qi) depends
on the inhibitor concentration (HAc). HAc* represents the
critical inhibitor concentration above which reaction stops, qmi
is the maximum specific rate of substrate consumption, and KMi
is the Monod saturation constant. In addition, n and m are other
kinetic parameters to calculate. This equation can account for
several common patterns of inhibition processes.
From analysis of the data obtained in batch assays with
different initial HAc concentrations, we found that the maximum
specific rate of HPr consumption (qmp) was almost constant and
similar to that obtained from reactor continuous operation,
whereas the saturation constant obtained (KMp) increased with
the acetic acid concentration. This means that acetic acid acts
as a competitive inhibitor, parameter n ) 0 in eq 8. Using the
kinetic parameters for the Monod equation from continuous
operation (where no inhibition was detected), the kinetic
parameters m and HAc* for propionic acid degradation were
estimated.
On the other hand, the analysis of the data for HBut
degradation obtained in batch assays confirms the kinetics
parameters resulting from continuous operation, where the
Monod equation is valid.
Moreover, the acetic acid consumption is inhibited by high
substrate concentration. This process was usually described by
the Haldane equation.24,25 Here, the specific rate of HAc
consumption (qa) can be calculated by eq 9. In this work, the
inhibition constant, Kia, was obtained by nonlineal fitting of the
experimental data from batch assays, considering that the Monod
parameters previously obtained in continuous reactor operation
(qma and KMa) correspond to the values when no inhibition was
detected.
HAc
( )
qa ) qma (9)
HAc2
KMa + HAc +
Kia
Table 2 contains all of the kinetic parameters obtained for
both inhibition processes studied.
The kinetics parameters reported in the literature are quite
different. These differences are probably due to the inoculum,
system configurations, and operation modes employed.
The biomass used as inoculum affects the kinetic parameters
obtained from the type of microorganism that predominates in
a mixed culture. The influence of the retention time of the solids
as well as organic loading on the population is well-known for
the anaerobic systems. These operation parameters can deter-
mine the type of methanogens that is dominant in the culture:
Methanotrix at long SRT and low substrate loading or Metha-
nosarcina at short SRT and high substrate loading, like it was
shown by Vasiliev and Vavilin.26
Pavlostathis and Giraldo-Gomez27 reviewed the Monod
kinetic parameters for the anaerobic process; from this paper,
it is easy to note the great variability of the same parameter.
For acetate degradation: KM ) 11-421 mg of COD/L, µmax )
0.08-0.7 1/days, and YXS ) 0.01-0.045 g of VSS/g of COD.
For other VFA: KM ) 12-500 mg of COD/L, µmax ) 0.13-1.2
1/days, and YXS ) 0.025-0.047 g of VSS/g of COD.
Anyway, the yield on substrate for the mixed biomass (YX/S)
obtained in this study as well the Monod parameters are in the
range previously indicated.
With respect to the inhibition modeling, we found much more
variability considering the type of inhibition as well as the form
in which the inhibitor concentration was considered. For
works, the total acetate (ionized and unionized forms) was
considered as substrate,28,29 while, in others, only the unionized
acetate was assumed as the substrate.30,31
In the ADM1 model, all of the anaerobic oxidation processes
are subject to inhibition by hydrogen or free ammonia ac-
cumulations, as well as due to extreme pH. These effects on
the conversion rate were implemented by introducing in the
Monod type rate velocity equation several multiplier terms that
reflect noncompetitive inhibition. In general, the model tends
to overpredict VFA concentrations maybe due to the form in
which the inhibition is treated.
In this work, the product inhibition was treated in a similar
way, but the substrate inhibition was best accounted for using
a Haldane type expression, considering total acid concentration
(ionized and unionized forms).
Simulations of the experimental data of VFA consumption
are obtained using the kinetic parameters resulting in the kinetic
study presented. Also, the next stoichiometric coefficients for
acetic acid production were assumed: 0.802 g of COD HAc/g
of COD HBut and 0.575 g of COD HAc/g of COD HPr. Figure
4 shows the experimental (as symbols) and predicted values
(as lines) of each acid in two batch assays with 2682 and 5351
mg of COD/L as initial VFA total concentration. In these cases,
 Ind. Eng. Chem. Res., Vol. 47, No. 15, 2008 5343
Table 2. Kinetic Parameters Obtained for Acetic, Propionic, and Butyric Acids
continuous feeding batch assays
substrate qm (g of COD/g of VSS · d) KM (g of COD/L) Kia (g of COD/L) nb (g of COD/g of VSS · d) mb (g of COD/L) I*c (g of COD/L)
HAc 0.682 0.892 0.667
HPr 0.082 0.426 0 -6.405 4.633
HBut 0.239 0.055 0 0
soluble COD 0.751 2.077
a
Haldane model. b Monod modified. c Acetic acid concentration.

Figure 4. Experimental and predicted values for acetic (9), propionic (∆), and butyric (1) acids in two batch assays with the next total initial concentration:
(A) 2682 mg of COD/L; (B) 5351 mg of COD/L.

Figure 5. Experimental and predicted values for acetic (9), propionic (∆), and butyric (1) acids in two continuous feedings with the next organic loading
rate: (A) 0.151 mg of COD/mg of VSS · L; (B) 0.634 mg of COD/mg of VSS · L.
the biomass concentration (VSS concentration) was measured
only at the initial and final time of reaction due to the high
volumes that are necessary for their determination. Experimen-
tally, we probed that the VSS content was almost constant in
batch assays, probably due to the short time of reaction; for
this reason, the profiles of VFA were predicted using the kinetic
expressions 8 and 9.
From Figure 4, it is verified that all of the VFAs measured can
be simulated acceptably. In addition, propionic acid is the most
resistant to degradation, remaining in fermentation broth at low
levels starting off with lower concentration of the inhibitor (HAc).
On the other hand, Figure 5 presents the experimental and
predicted values for individual VFA in two continuous feedings
with an organic loading rate of 0.151 and 0.634 mg of COD/
mg of VSS · L. Here, the deviations from predicted values at
the initial time of organic loading changes are greater, but the
steady state concentration can be predicted accurately.
4.3. Application of Lineal Relations. According to the
described methodology, we can estimate biomass growth rates in
all experiments conducted in continuous operation mode, using
the correspondent lineal relation (set of eq 4) and the kinetic
parameters obtained (Table 2). The net growth rate was calculated
as the growth on the mixed substrate minus the decay rate.
In the same way, methane production was simulated, with
the correspondent linear relation obtained, as a function of each
5344 Ind. Eng. Chem. Res., Vol. 47, No. 15, 2008
Figure 6. Experimental and predicted values for acetic acid (0), biomass (O), and methane ([) in two continuous feedings with the next organic loading
rate: (A) 0.087 mg of COD/mg of VSS · d; (B) 0.151 mg of COD/mg of VSS · d.
fatty acid conversion rate and biomass production as well. Here,
we suppose that all of the hydrogen produced from oxidation
of propionic and butyric acids was converted for methane.
Figure 6 shows the experimental and predicted values
obtained in continuous operation for biomass and methane
production.
The main differences in methane estimation are observed at
an organic loading rate of 0.151 mg of COD/mg of VSS · L and
at the first moments of organic loading change, but this is
probably due to errors in gas collection.
In a general way, in accordance with Figure 4B, we observed
that the acetic acid tends to be accumulated preferably to
propionic and butyric acids. This situation indicates that the
higher VFAs continue producing acetic acids, although the
methanogenic step was inhibited. A similar behavior was
reported by Lin et al.32
Finally, the estimation of dependent velocities in the anaerobic
VFA degrading system could be down using the lineal relation-
ships derived by the black box description. This is especially
useful to estimate methane generation and biomass production,
but it is also possible to calculate some rates not usually
measured, namely, N-source consumption.
5. Conclusions
The methodology applied to approach the modeling of the
anaerobic degradation of VFAs in a more simplified manner
was successfully used for the modeling of many aerobic
microbial processes, but it always was used for pure cultures
of microorganisms growing on a pure substrate.
The more relevant results presented in this work can be
summarized as follows:
-The application of the black box description permits
simplifying the kinetic study for the system, because it is
necessary to provide only four kinetic expressions to describe
the VFA comsumption, biomass production, and methane
generation. The lineal relations obtained in this study are
suitable for VFA degradation only, due to the fact that it is
based on an important parameter, namely, Gibbs energy
dissipation per C-mol of biomass produced (Ds01/rAX), that
was calculated on the basis of the degree of reduction of
each volatile fatty acid and their initial proportion in the feed
of the bioreactor. We think the proportion of VFAs in the
mixed substrate has a minor importance in the final step of
estimation of each acid concentration change in the reactor
in the range of organic loading rates studied.
-The use of an inoculum developed and maintained under
controlled conditions in a laboratory reactor guarantees the
reproducibility of results in kinetic assays. This is normally
difficult in anaerobic digestion due to the fact that the biomass
used for experiments usually was taken from wastewater plants
or industrial treatment lines.
-In this study, a good number of kinetics parameters were
obtained, introducing an inhibition process in acetic acid and
propionic acid degradation. The principal limitation to the
applicability of these kinetic parameters for others studies is
the composition of the microorganism consortium, that must
be similar, as well as their potential methanogenic activity.
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ReceiVed for reView November 21, 2007
ReVised manuscript receiVed May 7, 2008
Accepted May 8, 2008
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