Biochemical Engineering Journal 21 (2004) 183–191

Aerobic purification of dairy wastewater in continuous regime

Part I: Analysis of the biodegradation process in
two reactor configurations
F. Carta-Escobar, J. Pereda-Marı́n, P. Álvarez-Mateos∗ , F. Romero-Guzmán,
M.M. Durán-Barrantes, F. Barriga-Mateos
Department of Chemical Engineering, Group of Food Technology, University of Sevilla, C/Profesor Garcı́a González s/n, 41012 Sevilla, Spain

Received 10 October 2003; received in revised form 9 June 2004; accepted 18 June 2004

Abstract

The removal of organics and nitrogen species is postulated in many water quality regulations. In the dairy industry, milk, water and
detergents are discharged to wastewater treatment plants as a result of routine operations. In general, the treatment applied has been
aerobic. This work is focused on the study of the biodegradation of a synthetic dairy wastewater (4000 mg/L COD, COD/TKN ra-
tio of 4 and pH around 11.5) in continuous regime. Two different reactor systems thermostated at 30 ◦ C were used. The first sys-
tem was a single reactor with a working volume of 80 L. The second one was a three-stage reactor cascade with a total working
volume of 30 L. In general, high organic matter removals (exceeding 96.8%) were achieved with the two systems. However, the per-
formance of the systems showed much variability in relation to the biomass settling properties, resistance to filtration and tendency
to produce viscous bulking and foaming phenomena associated with the specific characteristics of the wastewater and operational
conditions.
© 2004 Elsevier B.V. All rights reserved.

Keywords: Aerobic processes; Biodegradation; Bioreactors; Mesophiles; Purification; Waste-water treatment

1. Introduction ated lagoons, or a combination of these. Moreover, a nat-

Wastewaters from the dairy industry are polluting chiefly
because of the organic matter they contain and they should
be treated before discharge into surface waters. These nutri-
ents feed algae blooms that deplete dissolved oxygen, dam-
age habitats for fish nurseries and threaten leisure activities.
Conventional treatment of dairy wastewater involves aero-
bic processes, since fats, lactose and proteins are all easily
degraded by bacterial populations [1]. The techniques used
are mainly activated sludge processes, trickling filters, aer-
∗ Corresponding author. Tel.: +349-545-57183; fax: +349-545-56447.
E-mail address: palvarez@us.es (P. Álvarez-Mateos).
ural aerobic biological treatment is also possible in which
wastewaters after sedimentation are directly used on agri-
cultural soils where the soil microorganisms remove organic
matter [2].
Today, there are many processes for the treatment of dairy
wastewaters. However, two trends are very clear. They are
based either on the recovery of valuable components, mainly
proteins and lactose, or on the degradation of substances that
can alter negatively the environmental quality of the water
courses [3].
This work is focused on the study of the degradation of an
artificial dairy wastewater with a chemical oxygen demand
of 4000 mg/L, a COD/TKN of 4 and with pH values around
11.5 in continuous regime. Two experimental reactor systems

1369-703X/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2004.06.007
184 F. Carta-Escobar et al. / Biochemical Engineering Journal 21 (2004) 183–191

Table 1
Characterization of global effluents from seven dairy industries
COD BOD Fats TN TP Ph TS VaS TSS VSS
1 4000 2160 NP 200 60 5-9 5100 4300 NP 500
2 2926 1580 294 36 21 6.7 2750 1880 NP NP
3 633 260 NP 106 NP 8.9 710 447 240 NP
4 2209 1112 60 NP NP 7.2 NP NP 278 NP
5 4500 2300 NP 56 33 7.2 2540 1093 816 NP
6 3190 1950 690 43 7 5–10 NP NP 820 NP
7 4000 2600 400 55 35 8–11 – – 675 635
Composition in mg/L with the exception of pH; NP: not published [3,4]. COD: chemical oxygen demand; BOD: biological oxygen demand; TN: total nitrogen;
TP: total phosphorus; TS: total solids; VS: volatile solids; TSS: total suspended solids; VSS: volatile suspended solids.

were used in order to achieve the best design and operational
conditions.
2. Materials and methods
2.1. Composition of synthetic wastewater and inocula
The influent consisted of mixtures of water and full-cream
milk with a pH around 11 and a chemical oxygen demand
of 4000 mg/L, characteristics very similar to some effluents
from dairy industries as is shown in Table 1 [3,4]. This ar-
tificial wastewater was stored in refrigerated tanks (to pre-
vent alterations) and was dosed into the reactors by means of
peristaltic pumps. The microorganisms were obtained from
a dairy wastewater mixed with those originating from a do-
mestic sewage treatment plant.
2.2. Analytical methods
The following parameters were analysed: pH (pHmeter
423), total chemical oxygen demand (TCOD) and soluble
chemical oxygen demand (SCOD) after filtering through a
0.45 ␮m pore diameter membrane filter (by the dichromate
reflux method 508), total solids (TS) (dried at 103–105 ◦ C,
2540 B), total suspended solids (TSS) (2540 D), volatile
suspended solids (VSS) (2540 E), dissolved oxygen (DO)
(membrane electrode method, 4500-0G), sludge volume in-
dex (SVI) (2710 D) and biomass nitrogen content (4500-Norg
Kjeldahl method). All analyses followed the procedures out-
lined in standard methods [5].
2.3. Equipment
Two experimental systems differing in reactor configura-
tions, thermostated at 30 ◦ C (mesophilic range), were used.

Fig. 1. Scheme of the system I for the wastewater treatment.

 F. Carta-Escobar et al. / Biochemical Engineering Journal 21 (2004) 183–191 185

2.4. Experimental conditions

2.4.1. System I
Six tests were programmed, varying the volumetric load
(CV ), defined as the amount of COD fed to the system per
reaction volume and time (Table 2). The duration of each test
was selected depending on its performance, with a minimum
time of 30 days. Once conditions were changed, at least 2
weeks were necessary for adaptation.

2.4.2. System II
Four tests carried out in duplicate were programmed
(Table 3). In this case, the minimum duration of each test
was 50 days in order to follow the nitrification process.

3. Results and discussion

Fig. 2. Scheme of the system II for the wastewater treatment.
2.3.1. Single reactor system (system I)
This comprised a reactor with a rectangular cross-section
and an effective volume of 80 l. The effluent was discharged
from the last zone. The other parts included a thermostat,
a refrigerator, feed tanks, a compressor, a peristaltic pump
and a collector. The air was introduced through a perfo-
rated tube from the bottom of the reactor (Fig. 1). The
samples were taken from the last zone. The dissolved oxy-
gen levels were measured at four points as is shown in the
figure.
2.3.2. Three-reactor cascade (system II)
Three thermostated borosilicate glass reactors were con-
nected in series by glass and flexible tubes forming a three-
stage reactor system (Fig. 2). The effective volume of each
reactor was 10 l. Two series of reactor systems were used.
The reactors were positioned at different levels so that a con-
stant sequential flow through the reactors was maintained by
gravity. Each reactor was provided with ceramic air diffusers
connected to an air blower. The air diffusers also served as
an excellent mixer, ensuring each reactor behaved as a con-
tinuous stirred tank reactor (CSTR). Samples were collected
from each reactor via a lateral outlet.
The discussion of the results will be made based on the
average values during the pseudo-stationary state, considered
the period twice the hydraulic retention time.
3.1. General description, treatment efficiencies
3.1.1. System I
3.1.1.1. Dissolved oxygen levels. Although all the tests were
carried out with air injection, performance depends on the
load applied. On the one hand, the dissolved oxygen levels
are considerably reduced during the first few days, and be-
come stabilized at 0.2 mg/L O2 in all parts of the bioreactor
at CV higher than 0.741 kg/m3 COD per day (Fig. 3a). On the
other hand, with lower loads (Fig. 3b), the oxygen profiles
are completely different, being characterized by a smaller de-
crease during the first 10 days, and by the presence of cyclic
fluctuations with an average period of 5 days. These fluctua-
tions are associated with a greater competition between mi-
crobial species and superior organisms, due to the increase in
the quality of the medium, and have already been observed
by other researchers [6–8]. As the organic load is reduced, the
sludge settleability is improved and solids are accumulated
inside the system. As a result, a final zone in the bioreactor
is characterised by a high solid deposition and low dissolved
oxygen concentrations (point 4 in Fig. 3b). Thus, the tests
will be differentiated in two groups: the first integrated by

Table 2
Working conditions and average values of the measured parameters when pseudo-stationary state was achieved in the tests carried out in system I
CV (kg/m3 COD per day) Initial conditions Pseudo-stationary state conditions ηSCOD (%)

COD0 (mg/L) pH0 pH TS (mg/L) TCOD (mg/L) SCOD (mg/L)

1.337 3566 11.37 8.02 2174 2280 – –
1.181 3842 12.31 7.96 2177 1757 – –
0.957 3866 11.42 8.07 2063 1683 228 94.1
0.741 3924 11.38 8.06 2214 1469 118 96.8
0.505 3957 11.33 8.18 5938 5375 55 98.4
0.326 4069 11.27 8.48 3886 2445 83 97.8
186 F. Carta-Escobar et al. / Biochemical Engineering Journal 21 (2004) 183–191

Table 3
Working conditions in the tests carried out in system II, considering it a bioreactor with a total volume of 30 litres
CV (kg/m3 ·per day) Initial conditions Pseudo-stationary conditions Efficiencies

pH0 COD0 (mg/L) TCOD (mg/L) SCOD (mg/L) ηTCOD (%) ηSCOD (%)
0.237 11.42 3745 710 38 81.0 99.0
0.237 1080 57 71.2 98.5
0.335 11.28 4018 438 55 89.1 98.6
0.335 543 41 86.5 99.0
0.381 11.57 3807 827 77 78.3 98.0
0.368 876 44 77.0 98.8
0.700 11.51 3714 2063 51 44.4 98.6
0.650 1252 52 66.2 98.6

the tests performed with loads higher than 0.741 kg/m3 COD
per·day, and the second with the tests carried out with loads
lower than that value.
3.1.1.2. Concentration of solids and purification efficiencies.
CV ≥ 0.741 kg/m3 COD per day.
The TCOD and TS average values are shown in Table 2.
The evolution of the two parameters is similar, as is observed
in Fig. 4a and b. The increase in the levels of TCOD and ST
is due to the improvement in the biomass settling properties.
The change in the biomass settleability took place at the end
of the test carried out with a volumetric load of 0.741 kg/m3
COD·per day. In this first group of tests, it is clear that the
treatment system was overloaded, as is reflected in the insuf-
ficient oxygen supply and high COD values.
CV ≤ 0.741 kg/m3 COD per day.
As in previous tests, the TCOD and TS levels show
the same trend (Fig. 5a and 5b). In these tests, the ef-
fluent SCOD was very much lower than the influent val-
ues and the efficiencies turned out to be higher than
98.6% (Table 1); levels high enough for an activated sludge
system.

Fig. 3. (a and b) DO levels in two tests carred out in system I.

 F. Carta-Escobar et al. / Biochemical Engineering Journal 21 (2004) 183–191
Fig. 4. (a and b) TCOD and TS levels in the tests carried out in system I at volumetric loding rates higher than 0.741 kg/m3 COD per day.
3.1.2. System II
An attempt was made with this system, we tried to
solve the difficulties found in the system I mainly regard-
ing the dissolved oxygen levels in the mixed liquor and solid
accumulation.
3.1.2.1. Dissolved oxygen levels. In this case, it was possible
to maintain higher oxygen concentrations in the three reactors
of the series. However, undesirable situations arose as well
(Fig. 6a and b). For instance, the R11 reactor during the test
at 0.335 kg/m3 COD per day showed very low oxygen levels
due to the formation of viscous biofilms on the air diffusers.
These biofilms were comprised mainly of exopolysaccha-
rides of bacterial origin associated with a medium enriched in
nutrients [9].
3.1.2.2. Treatment efficiencies. The average values of
TCOD, SCOD and treatment efficiencies are collected in
Table 3. In general, the chemical oxygen demand is reduced
as the effluent passes through the reactors; treatment efficien-
cies around 98–99% were obtained at the end of the series.
TCOD is reduced by 50% in the first reactors, whereas SCOD
is reduced by 70%. A good performance in COD removal was
obtained. The connection of a third reactor (or even a second)
would not be necessary in some cases to achieve a significant
improvement in the treatment efficiencies.
187
3.1.3. Comparison of the results obtained with the two
systems
A plot of the treatment efficiencies versus the volumetric
load, and considering the three-reactor cascade as three re-
actors of 10, 20 and 30 L, respectively, shows similar results
for the two experimental designs (Fig. 7). With reference to
SCOD values, the efficiencies are high enough. In general,
at loads lower than 1 kg/m3 COD per day, the minimum per-
centage of organic matter removal is 82%. Regarding TCOD,
the achieved treatment efficiencies are similar in the two sys-
tems, with a linear decrease as the volumetric load increases.
3.2. pH
3.2.1. System I
In general, and as a characteristic of the aerobic treat-
ment of dairy wastewaters, the pH levels were stable [10].
The average values are collected in Table 2. According
to the results, a volumetric load of 0.741 kg/m3 COD
per·day is again the transition value between the two per-
formances. As can be observed, the pH values are very sim-
ilar at higher loads. However, these values increase expo-
nentially as the organic load decreases. Again, the overload-
ing of the system at loads higher than 0.741 kg/m3 COD
is clear; the reduction in the stabilisation pH is associ-
ated with lower levels of dissolved oxygen (Fig. 3a and b)
188 F. Carta-Escobar et al. / Biochemical Engineering Journal 21 (2004) 183–191

Fig. 5. (a and b) TCOD and TS in the tests carried out in system I at volumetric loding rates lower than 0.741 kg/m3 COD per day.

and with the occurrence of fermentations on a smaller
scale.
3.2.2. System II
In these tests, the pH levels remain very stable in each
reactor. The average values are shown in Table 4. As the
effluent passes through the reactors, there is an increase in the
pH levels. This shows that the acidification associated with
the action of lactic acid bacteria [11], takes place mainly in
the first reactors, where the organic matter removal is also the
most noticeable.
3.2.3. Comparison of the results obtained with both
systems
A plot of the stabilisation pH versus the studied volu-
metric loads, shows the same trend for the two systems
(Fig. 8). Again, the levels are practically constant at loads
higher than 0.741 kg/m3 COD per day, and increase expo-
nentially as the organic load is reduced. In previous works
carried out in batch reactors with similar wastewaters and
operational conditions, the stabilisation pH was kept in the
range of 9.2–9.3, values towards which the system tends as
the volumetric load decreases.
3.3. Common operational problems
Generally, and in both systems, biomass settling proper-
ties, resistance to filtration and tendency to produce viscous
bulking and foaming phenomena showed much variability.
According to the literature, during the treatment of wastew-
ater from a powdered milk and butter factory, common phe-
nomena of bulking were observed at sludge volume index
(SVI) values higher than 600 mL/g [12]. Fig. 9 shows the

Table 4
Average pH levels reached in each reactor of the three-reactor cascade (system II)
CV (kg/m3 COD per day) R11 R12 R13 CV (kg/m3 COD per day) R21 R22 R23

0.237 8.16 8.77 8.97 0.237 8.41 8.94 9.04

0.335 7.74 8.24 8.71 0.335 8.39 8.62 8.85
0.381 8.21 8.15 8.44 0.368 8.22 8.73 8.72
0.700 7.47 7.81 8.12 0.650 7.30 8.03 8.25
 F. Carta-Escobar et al. / Biochemical Engineering Journal 21 (2004) 183–191 189

Fig. 6. (a and b) Dissolved oxygen levels in two tests carried out in system II.

TCOD efficiencies system II SCOD efficiencies system II

SCOD efficiencies system I TCOD efficiencies system I
120
Purification efficiencies (%)

100

80

60

40

20

0
0 0 .5 1 1 .5 2 2 .5
3
Cv (kgCOD/m ·d)

Fig. 7. Comparison of the treatment efficiencies achieved in both systems.

SVI values during a complete period. As is observed, the
achieved values are higher than 200 mL/g, which is the level
considered as the limit for defining bulking conditions [13].
Murthy et al. [14] found that when the ratio between mono-
valent and divalent ions exceeded two, a considerable resis-
tance to the filtration took place. This resistance was observed
in most of the tests, that ratio being close to 11 in the artificial
wastewater used as influent. Two important factors related
to the biomass settleability have to be taken into account: a
COD/TKN ratio close to one in the wastewater to be treated
and, volumetric loading rates between 0.51 and 0.67 kg/m3
COD per day [15]. In this work, the artificial wastewater had
a COD/TKN ratio of 4, four times higher than that recom-
mended for a good biomass sedimentation. With reference
190 F. Carta-Escobar et al. / Biochemical Engineering Journal 21 (2004) 183–191

Fig. 8. Comparison of the stabilization pH levels reached in the two system.

Fig. 9. Sluged volume indexes reached during tests carried out in system II.

to the volumetric loads, the sludge settleability improved no-
ticeably at loads lower than 0.741 kg/m3 COD per day, which
is very close to the range previously mentioned. Regarding
foaming phenomena, some events took place in tests carried
out in System II. They were characterised by the formation of
a thick and brown foam layer which was difficult to remove.
Moreover, an excessive production of extracellular biopoly-
meres took place under high organic loading conditions. This
increased the resistance to filtration and therefore, made the
determination of the solid content difficult. Richard et al.
[16] established that the overproduction of exopolysaccha-
rides produced a significant reduction in the nitrogen content
of the biomass. In fact, the analysis of sludge samples col-
lected in the tests carried out in system I, confirms this (Table
5). On other hand, there are references [17] concerning sys-
tems with high mass loads (0.6 kg COD/biomass per day)
and with retention times of 5 days and temperatures of 30 ◦ C,
that lead easily to bulking phenomena. This was associated
with an overgrowth of zooglea and an excessive production
of extracellular polysaccharides. In Table 5, the mass loads
(Cm ) and the hydraulic retention times for tests in system I
are collected. The volatile suspended solids were calculated
assuming a VS/TS ratio of 0.52 for these wastewaters [18]
and considering the volatile solids estimated as volatile sus-
pended solids. As can be seen, there are very similar aspects
between the applied conditions and those mentioned in the

Table 5
Nitrogen biomass content, studied masic loads (Cm ) and hydraulic retention times(θ H ) in the tests carried out in system I
CV (kg/m3 COD per day) VSS (mg/L) N (%, g/g dried matter) Cm (kg COD/kg VSS per day) θ H (◦ )

1.337 1130 4.53 1.183 2.7

1.181 1132 2.92 1.044 3.2
0.957 1072 4.88 0.892 4.0
0.741 1151 9.49 0.643 5.3
0.505 3088 8.36 0.163 8.0
0.326 2021 8.31 0.161 12.3
 F. Carta-Escobar et al. / Biochemical Engineering Journal 21 (2004) 183–191
literature, so the bulking phenomena is not surprising. The
removal of fats and proteins from these wastewaters before
the aerobic treatment, is recommended to avoid operational
problems [19].
4. Conclusions
The low oxygen levels, the high resistance to filtration
of the samples and the formation of viscous biofilms showed
that system I was overloaded at loads higher than 0.741 kg/m3
COD per day. However, the treatment efficiencies were higher
than 96.8% at loads lower. In system II, TCOD and SCOD
are reduced by 50 and 70%, respectively, in the first reac-
tors. With reference to SCOD values, the efficiencies are
high enough (>98.5%). Considering the two systems, the
results show that at loads lower than 1 kg/m3 COD per
day, the minimum percentage in organic matter removal is
82%.
Concerning pH values, the overloading of system I is re-
flected in a stabilisation at similar levels. Basically, as the or-
ganic load is reduced, the pH tends to stabilise at those levels
achieved in discontinuous tests treating similar wastewaters
with the same operational conditions.
In general, biomass settling properties, resistance of the
samples to be filtered and the trend to produce viscous bulking
and foaming phenomena showed much variability. This be-
haviour is related to the composition of the artificial wastew-
ater, the high COD/TKN ratio, the range of organic loads and
the retention times used. The removal of fats and proteins
before aerobic treatment is recommended in order to prevent
bulking and foaming phenomena.
Acknowledgements
The authors express their acknowledgements to Conse-
jerı́a de Educación y Ciencia de la Junta de Andalucı́a for
their financial support in conducting the research reported.
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