Process Biochemistry 39 (2004) 1257–1267

A pilot-scale study of total volatile fatty acids production by anaerobic

fermentation of sewage in fixed-bed and suspended biomass reactors
M.F. Colmenarejo a , E. Sánchez b , A. Bustos a , G. Garcı́a a , R. Borja b,∗
a Centro de Ciencias Medioambientals (CSIC), C/Serrano, 115-Duplicado, 28006 Madrid, Spain
b Instituto de la Grasa (CSIC), Avda. Padre Garcı́a Tejero 4, E-41012 Sevilla, Spain

Received 9 May 2003; accepted 26 June 2003

Abstract

A comparative study of a fermentation process for total volatile fatty acids (TVFA) production using pilot-scale fixed-bed (FAS) and sus-
pended biomass (FER) reactors in which similar operational conditions was carried out. The influence of the changes of ambient temperatures
at fixed operational conditions was also studied. Oxidation–reduction potential (ORP) increased and effluent pH decreased as the hydraulic
retention time (HRT) decreased, which was favourable for TVFA production. Equations describing the ORP and pH variations with the HRT
were obtained. ORP variation with HRT for FAS and FER reactors followed a logarithmic function with a regression coefficient, R2 , equal to
0.98. The variations of pH with HRT followed polynomial functions with regression coefficients of 0.96 and 0.98 for FAS and FER reactors,
respectively. Hydrolysis process increased with the experiment duration. At the beginning of the experiment, effluent soluble COD (SCOD)
decreased with respect to the influent but further effluent SCOD increased showing higher values compared to the influent. Cold temperatures
were more favourable than summer temperatures for the accumulation of TVFA at the liquid effluent. The FAS reactor was more effective in
the production of TVFA than the FER reactor. The maximum yields of TVFA were obtained at an organic volumetric loading rate (BV ) of
1.9 g COD/l per day, corresponding to an HRT of 3.4 h, for both reactors. A maximum increase of ammonia and phosphorus was observed at
the maximum value of HRT coinciding with an increase of pH and a decrease of ORP, as could be previously observed. The average P/SCOD
ratio for the influent and effluent were 0.06 and 0.05, respectively, for FAS and FER reactors. The average Ammonia/SCOD ratio for the
influent and effluent were 0.15 and 0.14, respectively. These results demonstrate that effluent quality was improved by the treatment employed
in case a further process of nutrient removal is carried out.
© 2003 Elsevier Ltd. All rights reserved.

Keywords: Hydrolysis; Fermentation; Total volatile fatty acids (TVFA); Fixed-bed reactor (FAS); Suspended biomass reactor (FER); Hydraulic retention time
(HRT); Nutrient biological removal; Organic volumetric loading rate; Ambient temperature

1. Introduction
Biological phosphorus removal (BPR) is related to the
storage capacity of this element by certain microorganisms
known as Poly-P organisms [1]. Despite the fact that biolog-
ical systems for phosphorus removal have been the subject
of investigation for over a decade and significant progress
has been obtained, an adequate mechanistic model has not
yet been developed [2–4]. The study of the fermentation
products generated (or added) at the anaerobic zone and
used as suitable substrate by Poly-P microorganisms has
demonstrated that among these products, volatile fatty acids
∗ Corresponding author. Tel.: +34-95-4689654; fax: +34-95-4691262.
E-mail address: rborja@cica.es (R. Borja).
(VFA), mainly acetic and isovaleric acids are the most influ-
ential and lead to a linear relationship between phosphorus
release during the anaerobic phase and its later capture un-
der aerobic conditions [5–11]. Unfortunately, not all urban
wastewater treatment plants receive wastewater which is eas-
ily biodegradable. Current methods of increasing the total
volatile fatty acids (TVFA) concentration are based on the
fermentation of primary sludge in a reactor designed for this
purpose located in the sludge line or the addition of prod-
ucts from anaerobic digestion [12–19]. With this purpose,
the anaerobic fermentation of sewage wastewater is one of
the most promising alternatives [20–22]. Colmenarejo et al.
[23] designed a pre-stage system to directly ferment the in-
fluent wastewater once screened and removed of grit. This
procedure involves pre-sedimentation of the wastewater at a
high upward velocity and its subsequent fermentation in an

0032-9592/$ – see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0032-9592(03)00253-X
1258 M.F. Colmenarejo et al. / Process Biochemistry 39 (2004) 1257–1267
up-flow, fixed-bed reactor which includes two areas in which
hydrolysis and acidogenesis reactions take place simultane-
ously. In one of the sections, which is packed with a specific
plastic material, microorganisms mainly act upon dissolved
organic matter and particulate matter. The microorganisms
present in the remaining section are able to use the settled
organic matter found in suspension. Both types of action su-
perimpose in the effluent from the fermenter. An increase
of 52.7% in acetate concentration was observed when ur-
ban wastewater was used to feed a laboratory-scale reactor
under a hydraulic retention time of 0.78 h.
The objective of this research was to carry out a com-
parative study of the fermentation process of sewage to
TVFA production at a pilot-scale using a fixed- and a
suspended-bed reactor operating at similar conditions and
also to study the effect of changes of the ambient tempera-
ture at fixed operational conditions.
2. Materials and methods
2.1. Description of the pilot plant
The pilot-scale plant was composed of three units:
1. A decanter.
2. A reactor with support media (FAS).
3. A reactor without support (FER).
The decanter consisted of a plastic cylindrical tank with
a sectional surface area of 0.39 m2 and an operational vol-
35
30
25
Temperature (ºC)
20
15
10
0
1 1 2 3
Fig. 1. Temperature distribution during the 12 months of the pilot-scale experiments.
ume of 0.67 m3 . The reactors with support media (FAS) and
without support (FER) consisted of cylindrical plastic tanks
with conical bottoms, and had sectional surface areas and
operational volumes of 0.71 m2 and 1.21 m3 , respectively.
The conical sections had volumes of 0.16 m3 . The support
of the reactor FAS, consisted of cubes of polyurethane foam
of 2 cm per side. The physical properties of the support ma-
terial were: 98% porosity; 35 × 103 m2 /m3 specific surface
area; and 0.2 g/ml density. The volume occupied by the sup-
port media was 1.07 m3 . The reactors were constituted by
two sections: a cylindrical one for the liquid fraction and a
conical one in which the sludge was hydrolysed.
The pilot plant was situated near of the Guardarrama ur-
ban wastewater purification plant (UWPP) at the vicinity of
Madrid City.
2.2. Pilot plant operation
The decanter was fed with raw sewage by using a pump
with variable flow-rates in the range of 0.5–5 m3 /h. The su-
pernatant was pumped to the reactors previously described
(FAS and FER) at flow-rates in the range of 0.2–1 m3 /h.
Both reactors were equipped with recycling connections, for
sludge recycling at flow-rates in the range of 0.4–1.5 m3 /h.
The experiment duration was 1 year. Fig. 1 shows the
variation of the ambient temperature during the 12 month
experiment. Three levels or ranges of temperature could be
appreciated according to the season: during winter season,
from November (month 11) to February (month 2), the
temperature ranged from a minimum of close to 0 ◦ C to a
Minimum
Medium
Maximum
4 5 7 8 9 10 11 12
Months
 M.F. Colmenarejo et al. / Process Biochemistry 39 (2004) 1257–1267
25
Average Ambient Temperature (ºC)
20
15
10
0
May
February
January
March
Jun
April
Months
Fig. 2. Average temperature for the different months of the experiment.
maximum of around 14 ◦ C with an average value of 7.3 ◦ C;
during the spring and autumn seasons, from March (month
3) to May (month 5) and October (month 10) to November
(month 11), respectively, the temperature ranged from a min-
imum of around 3 ◦ C up to a maximum of 20 ◦ C with an aver-
age value of 12.8 ◦ C; finally, during the summer period, from
June (month 6) to September (month 9), the temperature
varied between a minimum of around 13 ◦ C and a maximum
of 32 ◦ C with an average value of 22.5 ◦ C. Fig. 2 shows the
variation of the maximum, minimum and medium tempera-
tures with the operation time for each seasonal period of this
study.
The experiment was carried out at hydraulic retention
times (HRTs) in the range of 1.7–5.2 h, the influent strength
being the same for both reactors during the summer. The
effect of the ambient temperature was evaluated at HRTs
between 3.0 and 3.7 h.
2.3. Laboratory analysis and field determinations
Ambient temperature, pH and oxidation–reduction poten-
tial (ORP) were measured online in the pilot plant during
the experiment. The following parameters were analysed in
samples of the influent and effluents: total volatile fatty acids
(TVFA), soluble COD (SCOD), phosphorus (P), ammonia
nitrogen (NH4 + ) and total suspended solids (TSS). Analy-
1259
July
September
November
December
August
October
ses of TVFA and sludge solid fraction (SSF) were also de-
termined in the recycling, which corresponded to the sludge
section of the reactors. All analyses were carried out accord-
ing to the recommendations of the Standard Methods for the
Examination of Water and Wastewater [24].
3. Results and discussion
3.1. Effect of the HRT and ambient temperature
on ORP and pH
Oxidation–reduction potential is a measure of the degree
of metabolic reactions that take place in the reactor. Hydrol-
ysis stage is characterised by oxidation–reduction potential
(ORP) values of around −300 mV, while methanogenesis
takes place at ORP values lower than −300 mV and close
to −500 mV [25,26]. Fig. 3 illustrates the variation of ORP
at different HRT during the summer for the FAS and FER
reactors. The experimental points apparently show that val-
ues of ORP were not significantly influenced by the type of
reactor and both FAS and FER behaved in the same way.
The hydraulic retention time apparently had an strong in-
fluence on the ORP. Regression tests demonstrated that a
logarithmic correlation exists between HRT and ORP. The
relationship obtained between both the parameters is given
1260 M.F. Colmenarejo et al. / Process Biochemistry 39 (2004) 1257–1267
0
0 1
-100
-200
ORP (mv)
-300
-400
-500
-600
Fig. 3. Variation of the ORP with the HRT for FAS and FER reactors.
by the following equation:
ORP = −267.6[ln(HRT)] − 32.1
the regression coefficient, R2 , is 0.98.
ORP is the oxidation–reduction potential (mV) and HRT
the hydraulic retention time (h). The equation clearly de-
scribes that ORP decreases as the HRT increases favour-
ing the methanogenic stage. Hydrolysis, with the maximum
production of volatile fatty acids, could be enhanced at
HRTs <3 h. At HRT over 3 h, methane production could be
favoured for decreased ORP values.
Fig. 4 shows the variation of effluent pH with the HRT for
FAS and FER reactors under summer seasonal temperatures.
It was found that for low HRT values the pH decreased with
respect to the influent pH, but when HRT increased up to
values over 3 h, the effluent pH increased with respect to the
influent pH value. Values of effluent pH were slightly higher
for FAS than for FER reactor, probably because of the fact
that biomass attachment increased the microbial retention
time, favouring the development of methanogenic bacteria,
specially of acetate consumer microorganisms. Regression
curves fixed to the experimental data well, and polynomial
empirical equations appear to well describe the process be-
haviour in both reactors. The equations obtained for FAS
and FER reactors, respectively, were as follows:
pHFAS = 7.34 − 0.01(HRT)3 + 0.14(HRT)2 − 0.32(HRT)
pHFER = 7.34 − 0.02(HRT)3 + 0.02(HRT)2 −0.42(HRT)
the correlation coefficients, R2 , are 0.98 and 0.97 for FAS
and FER reactors, respectively.
The shape of the curves obtained corroborates that the
optimal conditions for TVFA formation were at HRT of
around 3 h.
The oxidation–reduction potential appears to be affected
by the variations of ambient temperatures at a range of
HRTs between 3.0 and 3.7 h. The minimum value of ORP
2 3 4 5 6
FAS
FER
HRT (d)
for FAS reactor during the summer was −397 mV, while
the maximum value during the winter was −374 mV. This
(1)
result corresponded with maximum and minimum values of
pHs at 7.31 and 6.49, respectively. In the case of the FER
reactor, the minimum and maximum values of ORP were
−387 and −442 mV for winter and summer, respectively,
corresponding to pH values of 7.28 and 6.97, respectively.
These results can be attributed more to hydrolytic bacteria
adapting to changes in temperature than methane forming
bacteria. The latter microorganisms are more affected by
the changes of temperature than the other groups of bac-
teria involved in the anaerobic decomposition of organic
matter.
3.2. Effect of the HRT and ambient temperature
on TVFA formation
Fig. 5 shows the variation of the concentration of TVFA
at the influent, the recycling and the effluent as a function
of the experiment duration. These results demonstrated that
TVFA formation was slightly higher in the support zone
of the FAS reactor than in the FER reactor. However, the
TVFA formation was more intense in the sludge zone of
FER reactor than that observed in the reactor FAS. This
phenomenon could be attributed to the utilisation of a sup-
port media which contributed to an increase in the rate of
organic matter assimilation and degradation, whereas in the
(2) case of the FER reactor (without support) the degradation is
limited by the contact between the microorganisms and the
substrate. Hence, the solids accumulated at the bottom of the
(3) FAS reactor were more stabilised than those accumulated at
the bottom of the FER reactor.
The production of TVFA appears to be maximum at the
lowest values of HRT. The maximum TVFA concentration
was observed in the recycling zone of the FER reactor at a
HRT of 1.7 h. For the FAS reactor, the maximum concen-
tration of TVFA was obtained at a HRT of 3.4 h.
At approximately constant HRT and variable ambient
temperature, it was observed that the concentration of TVFA
in the bulk liquid of the FAS and FER reactors increased
 M.F. Colmenarejo et al. / Process Biochemistry 39 (2004) 1257–1267
7,6
7,5
7,4
pH
7,3
7,2
7,1
7
0 1 2 3
HRT (h)
Fig. 4. Variation of the effluent pH with the HRT for FAS and FER reactors.
with decreased ambient temperature and the same behaviour
was observed in the recycling zone of both reactors, stress-
ing the fact that accumulation of TVFA was favoured by
the decrease of temperature.
3.3. Effect of HRT and ambient temperature on SCOD,
TSS and sludge concentration in the recycling
Fig. 6 shows the values of the influent and effluent SCOD
during the experimental period. As can be seen, the hydroly-
sis process increased with the experiment duration and with
the advance of the operation time. During the initial period
of the experiment, effluent SCOD decreased in relation to
the observed value for the influent, but in the last period
of the experimental time, effluent SCOD increased showing
higher values compared to those of the influent. This fact
shows an intensification of the hydrolysis process because
of the increase in total COD solubilisation. The concentra-
tion of effluent SCOD was higher in the FER than in the
1261
FAS (pH)
FER (pH)
4 5 6
FAS reactor for all the HRTs studied, except at 3.7 h (period
between 6th August and 13th August). These results can be
attributed to an increase of methanogenesis in the FAS reac-
tor, which agrees with previous observations in other works
reported in literature [17–20].
At HRTs in the range of 3.0–3.7 h and variable ambient
temperatures, the SCOD concentration observed in the ef-
fluents of FAS and FER reactors was maximum during the
summer and minimum at the beginning of the experiment.
Fig. 7 shows the variation of the values of TSS for influ-
ents and effluents of both reactors with the operation time
for the different HRTs studied during the experiment. As
can be seen, the effluent total suspended solids (TSS) con-
centrations were lower than the TSS concentrations for the
influent in both FAS and FER reactors. These results can
be explained by the solubilisation and degradation of partic-
ulate organic matter in both reactors. An increase of HRT
determined an increase of TSS removal which could be ob-
served during the periods included between 31 March–5 July
1262 M.F. Colmenarejo et al. / Process Biochemistry 39 (2004) 1257–1267

Fig. 5. Variation of the TVFA concentration and the HRT with the operation time for the liquid and recycling zones of the FAS and FER reactors.

Fig. 6. Variation of the influent and effluent SCOD concentrations and the HRT with the operation time for FAS and FER reactors.
 M.F. Colmenarejo et al. / Process Biochemistry 39 (2004) 1257–1267
Fig. 7. Variation of the influent and effluent TSS concentrations and the HRT with the operation time for FAS and FER reactors.
and 14 August–18 September. When the experiment dura-
tion was increased the reactors performance also increased,
improving the TSS removal in both cases. The initial con-
centration of TSS also influenced the percentage of TSS re-
moval, as could be observed during the operational period
between 31 March and 5 July. On comparing the perfor-
mance of FAS and FER reactors, lower values of effluent
TSS were obtained for FER at the beginning of the experi-
ment (periods between 31 March and 5 August) for the low-
est HRTs studied. However, the FAS performance was better
than FER during the periods ranging between 6 August and
3 December. These periods also corresponded with higher
SCOD removals in FAS compared to those obtained in the
FER reactor.
For HRT values in the range from 3.0 to 3.7 h, but by
varying ambient temperature, the concentration of effluent
TSS was maximum during the winter and minimum during
the summer, probably due to the higher concentration of
recycling sludge observed during the winter.
Fig. 8 illustrates the effects of the operation time and HRT
on the sludge concentration in the conical section expressed
as solids which can settle. The shape of this figure demon-
strates the influence of HRT on the sludge concentration.
An increase of HRT determined an increase of sludge con-
centration in the sludge section of both FAS and FER re-
actors. For an approximately constant value of HRT whilst
the sludge concentration remained constant for FER as the
operational period or experiment duration increased, an in-
crease of the sludge concentration was observed in the case
1263
of FAS reactor. As can be seen, the sludge concentration in
the recycling zone of the FER reactor was higher than that
observed in the FAS reactor during the entire experiment,
probably due to the higher SCOD and TSS removals ob-
tained in the FAS reactor, which can be observed in Figs. 6
and 7. By fixing the hydraulic retention time and varying the
ambient temperature, it was observed that the sludge con-
centration increased in winter as compared to summer and
spring. This fact could be explained by an increase of the
population of hydrolytic bacteria in relation to methanogenic
bacteria, which occurred when the temperature decreased,
as was previously described, causing the reduction of gas
emissions in the process.
3.4. Effect of HRT and ambient temperature on total
phosphorus, ammonia and on the (P/SCOD) and
(NH4 /SCOD) ratios
Fig. 9 shows the variation in phosphorus and ammonia
concentrations at the influent and effluents of FAS and FER
reactors with the operation time. Phosphorus concentration
in the effluents was higher compared to what was observed in
the influents between July (month 7) and December (month
12), probably as a consequence of the recycling, which was
able to increase the phosphorus concentration in the reactor.
The effluent phosphorus concentration was higher in the
reactor FER when compared to the reactor FAS. As can
be expected, ammonia nitrogen in the effluents was higher
compared to the influent concentration due to the microbial
1264 M.F. Colmenarejo et al. / Process Biochemistry 39 (2004) 1257–1267

Fig. 8. Variation of the solids content of the sludge and the HRT with the operation time for FAS and FER reactors.

Fig. 9. Variation of the influent and effluent phosphorus and ammonia concentrations and the HRT with the operation time for FAS and FER reactors.
 M.F. Colmenarejo et al. / Process Biochemistry 39 (2004) 1257–1267 1265

activity. The maximum values of ammonia and phosphorus Table 1

were observed at the maximum value of HRT, coinciding Summary of the experimental data of TVFA concentration in the FAS (2)
and FER (3) reactors
with an increase of pH and a decrease of ORP, previously
Parameter (units) 1 2 3 4 5
observed. Ammonia nitrogen was higher in the effluents of
HRT (h) 1.7 3 3.4 3.7 5.2
FER in all conditions studied except during the minimum BV (g COD/l per day) 3.83 2.17 1.91 1.76 1.25
HRT period, which was used between July (month 7) and S1 (g acetic acid/l) 0.049 0.041 0.047 0.048 0.048
August (month 8), in which the differences in both reactors S2 (g acetic acid/l) 0.050 0.050 0.061 0.051 0.057
were minimum and could be neglected. L2 (g acetic acid/l) 0.057 0.053 0.065 0.060 0.064
SE2 (g acetic acid/l) 0.051 0.050 0.062 0.052 0.058
At HRTs in the range of 3.0 to 3.7 h, ammonia and phos-
S3 (g acetic acid/l) 0.047 0.046 0.062 0.047 0.053
phorus concentrations in the liquid fractions were higher in L3 (g acetic acid/l) 80 57 81 71 68
the spring and summer, than in the winter. An increase of SE3 (g acetic acid/l) 51 48 65 50 55
ambient temperature was probably favourable for ammonia Values of S2 , L2 , SE2 , S3 , L3 , and SE3 are the averages of 150 determi-
and phosphorus production, due to the increase of methano- nations. The differences between the observed values were <5% in all
genesis activity at higher temperatures, as was previously cases. S1 , the TVFA concentration in the influent of both reactors; S2 ,
L2 , SE2 , the TVFA concentration in the liquid section, sludge section and
discussed.
effluent of the FAS reactor, respectively; S3 , L3 , SE3 , the TVFA con-
The average P/SCOD ratio for the influents was 0.06, centration in the liquid section, sludge section and effluent of the FER
while in the effluents this ratio was found to be 0.05 for FAS reactor, respectively.
and FER reactors. The average ratio of ammonia/SCOD for
the influents and effluents of both reactors were 0.15 and
0.14, respectively. These results demonstrate that effluent TVFA concentration in the effluent of the reactor for each
quality was slightly improved by the treatment employed for experiment was calculated considering the values of TVFA
a further process of nutrient removal. in the sludge and liquid zones of both reactors. Table 1 sum-
marises the experimental results obtained at different HRT
3.5. Determination of TVFA formation rate and organic volumetric loading rates (BV ). Values of TVFA
concentration at the influents (S1 ), in the liquid section of
With the aim of determining the TVFA formation rate, a the FAS reactor (S2 ), in the sludge section (L2 ), and in the
TVFA balance in the reactors was carried out. For this pur- effluent (SE2 ) are shown. The values of the TVFA concen-
pose, the experiments at variable HRT and almost constant tration were also measured in the FER reactor, (liquid sec-
ambient temperature were considered. The determination of tion of reactor (S3 ), sludge section (L3 ), and effluent (SE3 )).

Fig. 10. Variation of the TVFA yield with the organic volumetric loading rate (BV ) for FAS and FER reactors.
1266 M.F. Colmenarejo et al. / Process Biochemistry 39 (2004) 1257–1267
The corresponding values of TVFA for the effluents of FAS
and FER reactors were calculated following Eqs. (4) and
(5), respectively:
(VT2 − VC2 )S2 + VC2 L2
SE2 = (4)
VT2
(VT3 − VC3 )S3 + VC3 L3
SE3 = (5)
VT3
Eq. (4) allows the determination of the average value of
TVFA concentration in the effluent of FAS reactor (SE2 ) to
be calculated, where VT2 , is the operational total volume of
the FAS reactor and VC2 is the sludge section volume. In
the same way, Eq. (5) describes the determination of the
average value of TVFA concentration for FER reactor (SE3 )
considering that VT3 is the total operational volume of the
FER reactor, and VC3 the sludge section volume.
The concentration of TVFA in the sludge sections of
the reactors (L2 and L3 ) was higher than the concentration
of TVFA in the corresponding liquid sections, which was
previously discussed. Obviously, the increase of solids de-
termined an increase of organic matter concentration and,
therefore, the development of acid former microorganisms
was enhanced. The concentration of TVFA in the sludge
section of FER reactor was higher than that of FAS reactor
probably because in the former the sludge accumulated was
less mineralised than in the FAS reactor. Otherwise, the
concentration of TVFA in the liquid sections was higher
for FAS reactor compared to the concentration observed
in the FER reactor. Probably, the attachment or the use
of a support media was favourable for increasing the or-
ganic matter decomposition, as was reported in literature
[25,26].
Fig. 10 illustrates the variation of the TVFA yield, given
as g acetic acid/g COD added to the reactors, as a function of
the organic volumetric loading rate (BV ) for FAS and FER
reactors. The maximum TVFA yield for both reactors was
obtained at a BV of 1.91 g COD/l per day, corresponding to
a HRT of 3.4 h. TVFA yield was higher for FAS than for
FER reactor for all the BV values studied, except for a BV of
1.91 g COD/l per day, in which a higher yield was obtained
for FER reactor. For high BV values, low TVFA yields were
observed, which was caused by a low value of HRT and,
therefore, by an incomplete organic matter breakdown. On
the contrary, low values of BV and long HRTs favoured the
methanogenesis and, hence, the degradation of TVFA for
the production of methane.
Acknowledgements
The authors wish to express their gratitude to the Span-
ish “Comisión Interministerial de Ciencia y Tecnologı́a
(CICYT)”, Project PETRI no. 95-0118-0P, “Canal de Isabel
II”, and “Ministerio de Educación, Cultura y Deportes” for
providing financial support, and the Cuban “Ministerio de
Ciencia, Tecnologı́a y Medio Ambiente”.
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