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Batch mixed culture of Chlorella vulgaris using settled and

diluted piggery waste

L. Travieso a , F. Benı́tez b , E. Sánchez a , R. Borja a,∗ , A. Martı́n c , M.F. Colmenarejo d

a Consejo Superior Investigaciones Cientı́ficas (CSIC), Instituto de la Grasa, Avda. Padre Garcı́a Tejero 4, 41012 Sevilla, Spain
b Ministerio de Ciencia, Tecnologı́a y Medio Ambiente (CITMA), Inversiones Gamma, División Ambiental, Calle 23, No. 802, Vedado,
Plaza, Apartado 10, 400 Ciudad de la Habana, Cuba
c Departamento de Ingenierı́a Quı́mica, Facultad de Ciencias, Campus Universitario de Rabanales, Edificio C-3,

Ctra. Madrid-Cádiz, Km 396, 14071 Córdoba, Spain

d Centro de Ciencias Medioambientales (CSIC), C/ Serrano, 115 duplicado, 28006 Madrid, Spain

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

Article history: A study of the effect of the initial concentration of settled piggery wastewater in the range
Received 20 March 2006 from 250 to 1100 mg COD/l on a mixed culture of Chlorella vulgaris and bacteria was car-
Received in revised form ried out using 1-l batch culture bottles. COD removals and microorganisms concentrations
25 May 2006 increased with the time in a range of initial substrate concentrations from 250 to 800 mg
Accepted 1 June 2006 COD/l. Nevertheless, when the initial substrate concentration increased to 1100 mg COD/l,
the COD removal and microorganism concentrations decreased considerably, showing the
occurrence of an inhibition phenomenon. The specific growth rates of the exponential
phases obtained for each initial concentration of substrate were determined and verified
Piggery waste by the utilization of a modified logistic model applied to total chlorophyll ( C) and volatile
Substrate suspended solids (VSS) concentrations, indirect measurements of the concentrations of
Chlorella vulgaris microalgae and total microorganisms, respectively. A strong relationship between the ratio
Kinetic parameters of the initial substrate concentration and the initial concentration of microorganisms S0 /X0

(BX ) and the specific growth rate expressed in
Batch culture
 C and VSS was found. Process inhibition was
observed for BX values of 100 mg COD/mg C and 4.8 mg COD/mg VSS, respectively. The
values of max were found to be 0.050 h−1 (1.20 d−1 ) for microalgae and 0.044 h−1 (1.06 d−1 ) for
total microorganisms. The maximum value of the observed yield (Yobs. max. ) was determined

with respect to microalgae and was equal to 0.088 mg C/mg COD.
© 2006 Elsevier B.V. All rights reserved.

1. Introduction with 25.6%, 17.3%, 14.1% and 9.5% of total heads, respectively.
Within the region of Andalucı́a, in the province of Sevilla, there
Piggery waste treatment and disposal is one of the most impor- are an estimated 700,000 heads producing a volume of piggery
tant environmental problems to be solved in many countries. waste close to 1.1 million tonnes per year (Boletı́n Mensual de
In Spain, there are approximately 25 million pigs concentrated Estadı́stica, 2003).
in reduced areas with inadequate systems of treatment and Due to the low cost of construction and operation, one
disposal. The Spanish regions with the highest percentages of the most widely used and attractive treatment systems is
of pigs are: Valencia, Aragón, Castilla-León, and Andalucı́a, the stabilization pond: no aeration equipment is used and

Corresponding author. Tel.: +34 95 4689654; fax: +34 95 4691262.
E-mail address: rborja@cica.es (R. Borja).
0925-8574/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
oxygen requirements are provided by natural surface aer- Monod model to describe the effect of substrate concentra-
ation and photosynthesis by microalgae. The bacteria con- tion.
sume the oxygen released by the microalgae to decompose Thus, based on the knowledge drawn from literature, the
the organic matter producing carbon dioxide, ammonia, and main objective of this work was to carry out a study of the
phosphates, which are assimilated by the microalgae (Oswald, effect of initial concentration of settled piggery wastewater
1955; Palmer, 1977; Travieso, 1979; Travieso and Benı́tez, 1982; on C. vulgaris cultivation and on substrate removal, as well as
Ramalho, 1983; Hamer, 1985; Lavoie and de la Noüe, 1985; to determine the kinetic parameters in batch experiments.
Finlayson et al., 1987; Lembi and Waaland, 1988; De la Noüe
and Prouix, 1988; Travieso et al., 1988; Phang, 1991; Mayo and
Noike, 1996; Lau et al., 1995, 1998; Bich et al., 1999; Babel 2. Materials and methods
et al., 2002; Valderrama et al., 2002; Kayombo et al., 2003;
Craggs et al., 2004; Grönlund et al., 2004). Microalgae har- 2.1. Equipment
vested in stabilization ponds constitute a valuable source of
proteins, vitamins, and other compounds for animal feed- Batch cultures were grown in 1-l glass bottles, 20 cm in height
ing and can be obtained at the same time, with a consid- and 8 cm internal diameter. The bottles were sealed with rub-
erable decrease of wastewater pollution (Richmond, 1986, ber caps with two holes; one for the introduction of air through
1988; Belcher and Swale, 1988; Borowitzka and Borowitzka, diffusers for mixing and the other for air outlet and for taking
1988; Lembi and Waaland, 1988; De la Noüe and Prouix, the samples.
1988; Grobbelaar et al., 1990; Phang, 1991; Bustos et al., 1992;
Ogbondeminu and Okoye, 1992; Vonshak, 1993; Hall and Rao, 2.2. Waste used as culture media of Chlorella vulgaris
1994; Vilchez et al., 1997; Kayombo et al., 2002, 2003; Fiedler
et al., 2003). One of the most common species found in sta- The experiments were carried out using piggery wastewater
bilization ponds is Chlorella vulgaris. Piggery waste is charac- from a farm near the laboratory as a substrate. The character-
terized by a high concentration of organic compounds and a istics and features of the raw wastewater are summarized in
good balance of carbon and nutrients, which may fulfil the Table 1. As can be seen, the value of BOD was approximately
requirements for a microalgae culture (Travieso, 1979; Travieso 40% lower than COD value as was previously reported in other
and Benı́tez, 1982; Finlayson et al., 1987; Borowitzka and papers (Travieso, 1979; Travieso and Benı́tez, 1982; Travieso et
Borowitzka, 1988; Travieso et al., 1988; Grobbelaar et al., 1990; al., 1988).
Phang, 1991; Sevrin-Reyssac, 1998). The kinetics of microal-
gae growth is similar to that observed for other microor- 2.3. Analytical techniques
ganisms, the difference being that the source of energy is
instantaneous and independent of the culture media (Palmer, The analyses carried out in the experiments were: chemical
1977; Lavoie and de la Noüe, 1985; Lembi and Waaland, 1988; oxygen demand (COD), pH, total suspended solids (TSS) and
Richmond, 1988; Ogbondeminu and Okoye, 1992; Vonshak, volatile suspended solids (VSS). All analyses were performed
1993; Kayombo et al., 2003). Light intensity, temperature, nutri- according to standard methods for the examination of waters
ents, and pH are the most important factors for optimising and wastewaters (APHA, 1992). The concentration of C. vul-
the microalgae culture (Travieso, 1979; Travieso and Benı́tez, garis was determined by the measurement of total chlorophyll

1982; Travieso et al., 1988; Lau et al., 1995; Ogbonna et al., 1995; ( C), using the spectrophotometry standard method (APHA,
Ogbonna and Tanaka, 1996; Sevrin-Reyssac, 1998; Martı́nez 1992).
et al., 1999; Zhang et al., 1999; Zonneveld, 1998; John and
Flynn, 2000; Carlozzi and Sacchi, 2001; Babel et al., 2002;
Kayombo et al., 2003; Tukaj et al., 2003). For almost con-
stant values of light intensity and temperature, the culture Table 1 – Characteristics of the settled piggery
depends mainly on the substrate concentration. Different wastewater used in the batch experimentsb
kinetics models for batch cultures growth have been devel-
Parameter (mg/l)a Value
oped. Martı́nez et al. (1999) studied the growth of Scenedesmus
obliquus using the Monod model in a mineral medium with COD 10189
BOD 4582
phosphorus concentrations of up to 372 ␮M and tempera-
TS 7210
tures in the range of 20–35 ◦ C. The inhibition constants due
VS 5122
to the presence of phosphorus increased when the temper- TSS 3076
ature rose from 20 to 30 ◦ C, and decreased at temperatures VSS 2650
higher than 30 ◦ C. The maximum specific growth rate was Total N 341
0.047 h−1 at 30 ◦ C, while the highest yields were achieved at PO3−
4 419
20 ◦ C. Zhang et al. (1999) used an inhibition model to describe pH 6.0

the heterotrophic growth of microalgae Chlamydomonas rein- a

COD: chemical oxygen demand; BOD: biological oxygen demand;
hardtii in batch culture. Wachenheim et al. (2003) and Liu TS: total solids; VS: volatile solids; TSS: total suspended solids;
et al. (2003) applied logistic functions to describe the micro- VSS: volatile suspended solids; Total N: total nitrogen; PO3− 4 :
bial growth pattern in batch conditions. Kayombo et al. (2003) orthophosphate.
used both a modified logistic model to describe the growth Average values of 15 samples, with a variance coefficient of less
of C. vulgaris and heterotrophic microorganisms and the than 5% in all cases.
Table 2 – Characteristics of the culture medium
Component Concentration (%)

(NH4 )NO3 22.4

(NH4 )2 SO4 0.8
MgSO4 ·7H2 O 49.1
NaK2 PO4 12.2
FeSO4 ·H2 O 3.3
CoCl2 ·6H2 O 0.10
CuSO4 ·5H2 O 0.4
CaCl2 2.06
H3 BO3 2.74
Zn SO4 ·7H2 O 1.15
Na2 MoO4 0.20
Urea 4.80

2.4. Chlorella vulgaris

A strain of bacteria-free C. vulgaris was used as inoculum. The

culture of microalgae was previously carried out in a synthetic
medium until the required amount for carrying out the batch
experiments was obtained. The composition of the culture
Fig. 1 – Variation of the Napierian logarithm of the quotient
medium is shown in Table 2.
between the total chlorophyll concentrations at any time
and the beginning of the experiment (ln(X/X0 )) with time
2.5. Ambient conditions 
for microalgae growth ( C).

The experiments were carried out during summer, using natu-

ral light. Temperatures ranged from 27 to 32 ◦ C during the day COD/l. The initial concentration of VSS also increased when
and from 21 to 25 ◦ C during the night, while the solar radiation the initial COD increased, and the differences between the
was in the range of 0.60–0.75 W/cm2 d during this season. final and initial VSS concentration values rose as the initial
substrate concentration increased from 250 to 800 mg COD/l,
2.6. Experimental procedure achieving a drastic reduction for the highest initial substrate
concentration studied. This result shows the occurrence of an
The piggery wastewater used in the batch experiments was inhibition of all population of microorganisms present at an
previously settled for 1 h and further diluted with distilled initial substrate concentration of 1100 mg COD/l. This inhibi-
water up to COD concentrations of 250, 400, 520, 650, 800 tion process might be caused by a reduction of the dissolved
and 1100 mg/l, respectively. Three culture bottles were used
for each initial substrate concentration. The content of the
18 bottles was mixed by bubbling filtered and sterilized air at
a flow-rate of 30 l h−1 . The experiment lasted 190 h. Thirteen
samples of 20 ml were taken in each of the culture bottles for
determination of the parameters described in Section 2.3. The
initial volume was maintained by the addition of an equal
volume of piggery wastewater at the corresponding concen-

3. Results and discussion

3.1. Experimental results

As can be seen in Figs. 1 and 2, the chlorophyll and VSS concen-

trations (measurements of C. vulgaris and total microorgan-
isms concentration, respectively) increased with the operation
time for the different initial substrate concentrations stud-
ied, achieving maximum values at the end of the experiment.
Additionally, the concentration of total chlorophyll at the end Fig. 2 – Variation of the Napierian logarithm of the quotient
of the experiment increased as the initial concentration of between the VSS concentrations at any time and the
substrate increased from 250 to 800 mg COD/l, and finally, beginning of the experiment (ln(X  /X0 )) with time for total
decreased suddenly for an initial concentration of 1100 mg microorganisms growth (VSS).

of the total chlorophyll ( C) and, again, three different stages
of the batch culture may be defined: lag, exponential growth,
and stationary phases.

3.2. Kinetic model

In order to know what the characteristics of the culture growth

at different initial concentrations of settle piggery wastewater
are and to study the influence of the initial substrate con-
centration on the process performance, a kinetic model was
developed and applied to the experimental results obtained.
The most widely unstructured models used for describing cell
growth are the Monod, logistic, Haldane and Zwitering mod-
els among others (Hao et al., 2002; Liu et al., 2003; Kayombo
et al., 2003; Wachenheim et al., 2003). The logistic model is a
substrate independent equation and can accurately describe
the inhibition of biomass growth in different cultures, which
occurs in many batch fermentations (Liu et al., 2003; Kayombo
Fig. 3 – Variation of substrate concentration (mg COD/l) et al., 2003; Wachenheim et al., 2003). According to this model,
during the experiment for different initial concentrations. the microbial growth could also be expressed as a sinusoidal
curve by a modified logistic function as follows:

ln X = ln X0 + ln(Xm /X0 ){exp[−exp(e /ln(Xm /Xe ))(L − t) + 1]}

oxygen concentration. In addition, the increase in the concen-
tration of suspended solids also reduces the light penetration (1)
causing a decrease in the concentration of available nutrients
for microalgae. The results obtained were comparable to those where L is the duration of the lag phase, Xm the concentra-
obtained by other authors using a mixed culture of microalgae tion of microorganisms at an operation time equal to infinite,
and bacteria (Vonshak, 1993; Zonneveld, 1998). and e is the specific growth rate of the microorganisms. A
Fig. 3 shows the variation of COD with the time during the plot of the data of ln(X/X0 ) versus time allows determination
experiment. The concentration of COD decreased with time of the values of ln(Xm /X0 ), e and L for each initial substrate
due to the uptake of organic matter by the mixed culture. The concentration studied (Figs. 1 and 2). In order to obtain the
percentages of COD removed at 190 h were 88.0%, 57.5%, 55.6%, e and L values for each initial substrate concentration (S0 )
56.5%, 60.6% and 20.6% for initial COD concentrations of 250, studied, the pair values, ln(X/X0 ) versus time, corresponding
400, 520, 650, 800 and 1100 mg/l, respectively. These results to the exponential growth phase were plotted. Straight lines
show that substrate removal efficiency decreased significantly were obtained with slopes equal to e and intercepts equal to
at initial COD of 1100 mg/l due to the process inhibition. It L, for each set of experiments carried out. Finally, the value of
was found that organic matter removal increased with the ln(Xm /X0 ) for each initial substrate concentration studied may
microorganism concentration in a range of initial substrate be obtained by the plot of the values of ln(X/X0 ) correspond-
concentrations from 250 to 800 mg COD/l, but decreased when ing to the stationary phase versus the inverse of time. The
the initial substrate concentration increased to 1100 mg COD/l. intercept at 1/t = zero might be considered as ln(Xm /X0 ) (Hao
Fig. 1 shows the plot of the Napierian logarithm of the (X/X0 ) et al., 2002; Kayombo et al., 2003). Table 3 shows the calculated
ratio versus time (h) for different initial substrate concen- values of L, e and Xm for C. vulgaris and total microorgan-

tration (S0 ) studied, where the parameter (X) represents the isms measured as total chlorophyll ( C) and VSS, respec-

concentration of total chlorophyll ( C) at any time and X0 tively. Regression coefficients of the straight lines obtained in

represents the value of the chlorophyll concentration ( C) the exponential growth phases ranged from 0.91 to 0.98 for a
at the beginning of the experiment. The curves obtained are probability level of 95% (p ≤ 0.05). The straight lines obtained
classical growth curves corresponding to biological processes by plotting the values of ln(X/X0 ) corresponding to the sta-
carried out in batch conditions. In those plots, three phases tionary phase versus the inverse of time gave values of the
of the culture may be clearly appreciated: lag, exponential regression coefficient in the range of 0.85 to 0.97 (p ≤ 0.05).
growth, and stationary phases. The maximum concentration Table 3 shows that the values of e increased as the ini-
of C. vulgaris was achieved for an initial substrate concentra- tial concentration of substrate increased both for microalgae
tion of 800 mg COD/l and the minimum one for initial substrate and total microorganisms, except at a substrate concentration
concentrations of 250 and 1100 mg COD/l, respectively. of 1100 mg COD/l, for which the values decreased suddenly,
Fig. 2 shows the variation of the Napierian logarithm of the showing the occurrence of a clear inhibition process, as was
X /X0 ratio with the operation time, for the six initial substrate previously mentioned. The values of e obtained were compa-
concentrations studied. In this case, X and X0 , represent the rable to those obtained by other authors in batch and mixed
VSS concentrations at any time and at the beginning of the cultures of algae and bacteria (Zhang et al., 1999; Valderrama
experiments, respectively. The pattern of the curves obtained et al., 2002) when complex substrates were used. The values

was very similar and comparable to that obtained in the case of the specific growth rates obtained for microalgae ( C) and
Table 3 – Summary of the kinetic parameters of the batch culture at different initial substrate concentrations obtained
from the data plotted in Figs. 1 and 2
S0 (mg COD/l) Chlorella vulgaris Total microorganisms

L (h) e (h−1 ) Xm (mg C/l) L (h) e (h−1 ) Xm (mg VSS/l)

250 49.7 0.011 32.4 62.8 0.016 272

400 59.1 0.015 32.9 55.6 0.016 374
520 59.6 0.020 34.0 50.3 0.018 490
650 49.9 0.020 46.0 59.9 0.021 544
800 53.8 0.024 51.0 66.0 0.023 554
1100 13.6 0.008 36.4 59.2 0.011 445

total microorganisms (VSS) were very close, indicating that the The relationship between the specific growth rate and spe-
overall growth might have been controlled by microalgae. In cific load may be expressed, according to a Monod type equa-
order to verify the validity of the model proposed for describ- tion, as follows (Zhang et al., 1999; Liu, 2000):
ing the growth of microalgae and total microorganisms, the
experimental and theoretical values of X both for microalgae e = max [BX /(K + BX )] (2)

( C) and total microorganisms (VSS), respectively, were plot-
ted versus time (Figs. 4 and 5). As can be seen, the differences where BX is the ratio: S0 /X0 , max the maximum specific
between the experimental values and the theoretical ones growth rate, and K is a saturation constant equivalent to
(curves) obtained by Eq. (1) were lower than 5% in all cases. the value of BX when e is equal to 1/2 max. . By lineariza-
The small deviations obtained in both cases suggest that the tion of Eq. (2), it is possible to determine the values of K
proposed model predicts the kinetics of microalgae and total and max as follows: a plot of 1/e versus 1/BX should give
microorganism growth very accurately and that parameters a straight line of the slope equal to K/max and intercept equal
obtained represent the activity of both, microalgae and total to 1/max. Fig. 6 illustrates the calculation of these parameters
microorganism, affecting the batch aerobic degradation of this for microalgae growth. In this case, the intercept and slope

waste. values were found to be 19.96 h and 1619 mg C/mg COD,
The results obtained also show that the value of e incre- respectively. The regression coefficient was R2 = 0.98 (p ≤ 0.05)
ased when the initial substrate concentration (S0 ) increased showing that the model can be applied to the experimental
from 250 to 800 mg COD/l and decreased for a S0 value of data. Hence, the values of max and K were calculated from
1100 mg COD/l. Liu et al. (1998) and Liu (2000) emphasized the these values and were found to be 0.05 h−1 (1.2 d−1 ) and 81 mg

importance of the S0 /X0 ratio in batch cultures of microorgan- COD/mg C, respectively. Fig. 7 shows the plot of 1/e versus
isms for activated sludge processes. In addition, this parame- 1/BX for total microorganisms growth, based on VSS mea-
ter also plays an important role in batch cultures of algae and surements. As can be seen, a straight line was also obtained
bacteria. Therefore, the S0 /X0 ratio defined as the specific load with a regression coefficient R2 = 0.85. The value of the inter-
(BX ) was used, instead of S0 , to evaluate the influence of the cept and the slope were 22.9 h and 110.37 mg VSS/mg COD,
initial substrate concentration on the specific growth rate (e ). respectively, with a probability level of 90% (p ≤ 0.10). From the

Fig. 4 – Comparison of the total chlorophyll concentration Fig. 5 – Comparison of the experimental values of the VSS

values ( C) and the theoretical curves obtained by Eq. (1), concentrations and the theoretical curves obtained by Eq.
for some influent substrate concentrations (S0 ) in the range (1), for some influent substrate concentrations (S0 ) in the
of 250–1100 mg COD/l (p ≤ 0.05). range of 250–1100 mg COD/l (p ≤ 0.05).
Fig. 6 – Variation of the inverse of the specific Fig. 8 – Variation of the observed microalgae yield (1/Yobs. )
microorganism growth rate (1/e ) as a function of the as a function of the inverse of the specific load (1/BX ).
inverse of the specific load (1/BX ) for microalgae growth.

gae yield given in mg C/mg COD (Yobs. ) were obtained by the
quotient between microalgae growth and the substrate con-
values of the intercept and the slope the corresponding val-
sumed during the exponential growth phase. Therefore:
ues of max and K were calculated and found to be 0.044 h−1
(1.056 d−1 ) and 4.85 mg COD/mg VSS, respectively. The values
Yobs. = (X2 − X1 )/[− (S2 − S1 )] (3)
of max obtained both for microalgae and total microorgan-
isms growth were very similar, emphasizing the fact that the 
where Yobs. is the observed microalgae yield (mg C pro-
overall process was controlled by the microalgae growth. The
duced/mg COD consumed), X2 and X1 the concentration of
numerical values of max obtained for both cases were approx- 
total chlorophyll ( C) at the end and at the beginning of the
imately half those obtained by Kayombo et al. (2003) for settled
exponential growth phase, respectively, S2 and S1 are the COD
sewage and as high as those obtained for complex substrates
concentration at the end and at the beginning of the expo-
(Zhang et al., 1999; Valderrama et al., 2002).
nential growth phase, respectively. Once the Yobs. values were
Based on the data of microbial growth and substrate uptake
calculated, it was observed that these values appear to be
in the exponential phase, the values of the observed microal-
related to BX , by a relationship similar to the Eq. (2). There-

Yobs. = Yobs. max . BX /(KY + BX ) (4)

where KY is the value of BX for which the value Yobs. is equal

to (1/2) Yobs. max. . According to Eq. (4) a plot of the values of
1/Yobs. versus 1/BX should give a straight line with intercept
equal to 1/Yobs. max and slope equal to KY /Yobs. max. . As can
be seen in Fig. 8, a plot of the above mentioned pair values
gave a straight line from which the values of the constants

Yobs. max. and KY were calculated to be 0.088 mg C/mg COD

and 6.35 mg COD/mg C, respectively. The value of the linear
regression coefficient obtained was R2 = 0.88 with a probabil-
ity level of 95% (p ≤ 0.05). The value of the observed yield may
be estimated in terms of dry matter of microalgae, considering

that C is 11.7% of the cell weight (Rioboo et al., 2002). There-
fore, the maximum observed yield is 0.75 mg of dry weight/mg

Fig. 7 – Variation of the inverse of the specific 4. Conclusions

microorganism growth rate (1/e ) as a function of the
inverse of the specific load (1/BX ) for total microorganisms Mixed culture of C. vulgaris grew satisfactorily at batch condi-
growth. tions, using settled and diluted piggery wastewater as culture
media. Inhibition occurred at an initial substrate concentra- Fiedler, E., Juanico, M., Shelef, G., 2003. Simulation model of
tion of 1100 mg COD/l. waste stabilization reservoirs. Ecol. Eng. 20 (2), 121–145.
Chemical oxygen demand (COD) removal efficiency was Finlayson, M., Chick, A., von Oertzen, I., Mitchel, D., 1987.
Treatment of piggery effluents by an aquatic plant filter. Biol.
maximum (88.0%) for an initial COD concentration of 250 mg/l
Wastes 19, 179–196.
and minimum (20.6%) for an initial COD concentration of Grobbelaar, J.U., Soeder, C.J., Stengel, H., 1990. Modelling algal
1100 mg/l, being practically constant (50–60%) in the range of productivity in large outdoor cultures and wastewater
400–800 mg/l. systems. Biomass 21, 297–314.
A kinetic model based on a modification of the logistic func- Grönlund, E., Klang, A., Falck, S., Hanaeus, J., 2004. Sustainability
tion was capable of describing the pattern of C. vulgaris and of wastewater treatment with microalgae in cold climate,
bacteria growth at batch conditions. evaluated with energy and socio-ecological principles. Ecol.
Eng. 22 (3), 155–174.
Specific growth rates for microalgae and overall microor-
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The authors wish to express their gratitude to the Ministerio Kayombo, S., Mbwette, T.S.A., Mayo, A.W., Katima, J.H.Y.,
Jorgensen, S.E., 2002. Diurnal cycles of variation of
de Educación, Cultura y Deportes (Spanish Government), the
physical–chemical parameters in waste stabilization ponds.
program of Scientific Cooperation with Iberoamérica (Spanish
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Foreign Ministry) and Consejerı́a de Educación y Ciencia of the Kayombo, S., Mbwette, T.S.A., Katima, J.H.Y., Jorgensen, S.E., 2003.
Junta de Andalucı́a for providing financial support. We also Effect of substrate concentration on the growth of
wish to thank the Ministerio de Ciencia Tecnologı́a y Medio heterotrophic bacteria and algae in secondary facultative
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