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BatchMixed Culture of C. Vulgaris Using Settled and Diluted

BatchMixed Culture of C. Vulgaris Using Settled and Diluted

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Batch mixed culture of Chlorella vulgaris using settled and diluted piggery waste

´ L. Travieso a , F. Ben´tez b , E. Sanchez a , R. Borja a,∗ , A. Mart´n c , M.F. Colmenarejo d ı ı
Consejo Superior Investigaciones Cient´ficas (CSIC), Instituto de la Grasa, Avda. Padre Garc´a Tejero 4, 41012 Sevilla, Spain ı ı ´ Ministerio de Ciencia, Tecnolog´a y Medio Ambiente (CITMA), Inversiones Gamma, Division Ambiental, Calle 23, No. 802, Vedado, ı Plaza, Apartado 10, 400 Ciudad de la Habana, Cuba c Departamento de Ingenier´a Qu´mica, Facultad de Ciencias, Campus Universitario de Rabanales, Edificio C-3, ı ı ´ ´ Ctra. Madrid-Cadiz, Km 396, 14071 Cordoba, Spain d Centro de Ciencias Medioambientales (CSIC), C/ Serrano, 115 duplicado, 28006 Madrid, Spain
b a

a r t i c l e
Article history:

i n f o

a b s t r a c t
A study of the effect of the initial concentration of settled piggery wastewater in the range from 250 to 1100 mg COD/l on a mixed culture of Chlorella vulgaris and bacteria was carried out using 1-l batch culture bottles. COD removals and microorganisms concentrations increased with the time in a range of initial substrate concentrations from 250 to 800 mg 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

Received 20 March 2006 Received in revised form 25 May 2006 Accepted 1 June 2006

Keywords: Piggery waste Substrate Chlorella vulgaris Kinetic parameters Batch culture

phases obtained for each initial concentration of substrate were determined and verified by the utilization of a modified logistic model applied to total chlorophyll ( C) and volatile suspended solids (VSS) concentrations, indirect measurements of the concentrations of microalgae and total microorganisms, respectively. A strong relationship between the ratio of the initial substrate concentration and the initial concentration of microorganisms S0 /X0 (BX ) and the specific growth rate expressed in observed for BX values of 100 mg COD/mg values of
max

C and VSS was found. Process inhibition was C and 4.8 mg COD/mg VSS, respectively. The

were found to be 0.050 h−1 (1.20 d−1 ) for microalgae and 0.044 h−1 (1.06 d−1 ) for C/mg COD.

total microorganisms. The maximum value of the observed yield (Yobs. max. ) was determined with respect to microalgae and was equal to 0.088 mg © 2006 Elsevier B.V. All rights reserved.

1.

Introduction

Piggery waste treatment and disposal is one of the most important environmental problems to be solved in many countries. In Spain, there are approximately 25 million pigs concentrated in reduced areas with inadequate systems of treatment and disposal. The Spanish regions with the highest percentages ´ ´ of pigs are: Valencia, Aragon, Castilla-Leon, and Andaluc´a, ı

with 25.6%, 17.3%, 14.1% and 9.5% of total heads, respectively. Within the region of Andaluc´a, in the province of Sevilla, there ı are an estimated 700,000 heads producing a volume of piggery waste close to 1.1 million tonnes per year (Bolet´n Mensual de ı Estad´stica, 2003). ı Due to the low cost of construction and operation, one of the most widely used and attractive treatment systems is 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. doi:10.1016/j.ecoleng.2006.06.001

The maximum specific growth rate was 0. 1993. All analyses were performed according to standard methods for the examination of waters and wastewaters (APHA. TSS: total suspended solids. 1995. PO3− : 4 orthophosphate. De la Noue and Prouix. 1996. 1992. as well as to determine the kinetic parameters in batch experiments. Mart´nez ı et al. while the highest yields were achieved at 20 ◦ C. Ogbondeminu and Okoye. Phang. 1992. Lembi and Waaland. Belcher and Swale. the difference being that the source of energy is instantaneous and independent of the culture media (Palmer. 1988. BOD: biological oxygen demand. ı 1982. 2004). 1998). 1982. which are assimilated by the microalgae (Oswald. ¨ 1988.. 1996. 2002. 1988. Kayombo et al. Kayombo et al. . Kayombo et al. Lembi and Waaland. 1998. ammonia. Mayo and Noike. ¨ Finlayson et al. 1992. Carlozzi and Sacchi. 2001. 1987. 2003). and other compounds for animal feeding and can be obtained at the same time. Babel et al.. 1991. 1995. pH.. 2. and phosphates.. VS: volatile solids. 1990.. Kayombo et al. 1994.... 1979. Ogbonna et al. total suspended solids (TSS) and volatile suspended solids (VSS). 1998. As can be seen. with a considerable decrease of wastewater pollution (Richmond. 2. 1985. Finlayson et al. (1999) studied the growth of Scenedesmus ı obliquus using the Monod model in a mineral medium with phosphorus concentrations of up to 372 M and temperatures in the range of 20–35 ◦ C. Borowitzka and Borowitzka. Hall and Rao. 1982. Phang. using the spectrophotometry standard method (APHA. the culture depends mainly on the substrate concentration. ı ¨ Ramalho.. Richmond. Wachenheim et al. 1982. 1992). 1990. Average values of 15 samples. 1998. TS: total solids. 1979. 1988. 1988. For almost constant values of light intensity and temperature. 1988. Different kinetics models for batch cultures growth have been developed. 1999. The concentration of C. 2003. 2003).. 2.3. 1992). Travieso and Ben´tez.. (2003) used both a modified logistic model to describe the growth of C.oxygen requirements are provided by natural surface aeration and photosynthesis by microalgae. 1987. 1999. 1988. 1983. 1979. VSS: volatile suspended solids. 1979. Lavoie and de la Noue. Travieso et al. based on the knowledge drawn from literature. 1955. 1997.2. and decreased at temperatures higher than 30 ◦ C. Phang.1.. 2004. Mart´nez et al.. Travieso. 1986. one for the introduction of air through diffusers for mixing and the other for air outlet and for taking the samples. 1991.... Bich et al. 1985. Travieso and Ben´tez.. Vilchez et al. 2003.047 h−1 at 30 ◦ C.0 b COD: chemical oxygen demand. Borowitzka and ı Borowitzka. Vonshak. vulgaris cultivation and on substrate removal.. Ogbonna and Tanaka. 1991. 1995. vulgaris was determined by the measurement of total chlorophyll ( C). 2003. (1999) used an inhibition model to describe the heterotrophic growth of microalgae Chlamydomonas reinhardtii in batch culture. The kinetics of microalgae growth is similar to that observed for other microorganisms. Total N: total nitrogen. Kayombo et al. Vonshak. 1988).. Tukaj et al. Fiedler et al. 1977. vitamins. De la Noue and Prouix. 2002. 1993.. Materials and methods Equipment Batch cultures were grown in 1-l glass bottles. Zhang et al. Travieso and Ben´tez. 20 cm in height and 8 cm internal diameter. Gronlund et al. Light intensity. Ogbondeminu and Okoye. Microalgae harvested in stabilization ponds constitute a valuable source of proteins. Lau et al. Travieso and Ben´tez. temperature. (2003) applied logistic functions to describe the microbial growth pattern in batch conditions. Sevrin-Reyssac. 2002. Analytical techniques The analyses carried out in the experiments were: chemical oxygen demand (COD). One of the most common species found in stabilization ponds is Chlorella vulgaris.. Hamer. Waste used as culture media of Chlorella vulgaris The experiments were carried out using piggery wastewater from a farm near the laboratory as a substrate. Lembi and Waaland. The bacteria consume the oxygen released by the microalgae to decompose the organic matter producing carbon dioxide. 2003). The characteristics and features of the raw wastewater are summarized in Table 1. Table 1 – Characteristics of the settled piggery wastewater used in the batch experimentsb Parameter (mg/l)a COD BOD TS VS TSS VSS Total N PO3− 4 pH a Value 10189 4582 7210 5122 3076 2650 341 419 6. ¨ Craggs et al. 2. John and Flynn. 1999. Babel et al. Lau et al. Bustos et al. with a variance coefficient of less than 5% in all cases. Zhang et al.. 1985. Lavoie and de la Noue. 1988. Thus. 1988. Grobbelaar et al. Zonneveld. 1988. (2003) and Liu et al. 1988.. Valderrama et al. 1988. Travieso et al. Sevrin-Reyssac. and pH are the most important factors for optimising the microalgae culture (Travieso. the value of BOD was approximately 40% lower than COD value as was previously reported in other papers (Travieso. nutrients. the main objective of this work was to carry out a study of the effect of initial concentration of settled piggery wastewater on C. ¨ 1977.. vulgaris and heterotrophic microorganisms and the Monod model to describe the effect of substrate concentration... 2002. Palmer. Grobbelaar et al. Travieso et al. The bottles were sealed with rubber caps with two holes. Piggery waste is characterized by a high concentration of organic compounds and a good balance of carbon and nutrients.. Travieso et ı al. which may fulfil the requirements for a microalgae culture (Travieso. The inhibition constants due to the presence of phosphorus increased when the temperature rose from 20 to 30 ◦ C. 2000. 1988.

Results and discussion Experimental results As can be seen in Figs. respectively) increased with the operation time for the different initial substrate concentrations studied. 1 and 2. . and the differences between the final and initial VSS concentration values rose as the initial substrate concentration increased from 250 to 800 mg COD/l. Three culture bottles were used for each initial substrate concentration. Chlorella vulgaris A strain of bacteria-free C.3 0. 520. Additionally.15 0. 1 – Variation of the Napierian logarithm of the quotient between the total chlorophyll concentrations at any time and the beginning of the experiment (ln(X/X0 )) with time for microalgae growth ( C).4 0. 2. The content of the 18 bottles was mixed by bubbling filtered and sterilized air at a flow-rate of 30 l h−1 . achieving maximum values at the end of the experiment. COD/l. the concentration of total chlorophyll at the end of the experiment increased as the initial concentration of substrate increased from 250 to 800 mg COD/l.80 2.20 4.60–0. respectively. The culture of microalgae was previously carried out in a synthetic medium until the required amount for carrying out the batch experiments was obtained.75 W/cm2 d during this season. and finally.Table 2 – Characteristics of the culture medium Component (NH4 )NO3 (NH4 )2 SO4 MgSO4 ·7H2 O NaK2 PO4 FeSO4 ·H2 O CoCl2 ·6H2 O CuSO4 ·5H2 O CaCl2 H3 BO3 Zn SO4 ·7H2 O Na2 MoO4 Urea Concentration (%) 22. This inhibition process might be caused by a reduction of the dissolved 3.1 12. 3. 400. Experimental procedure The piggery wastewater used in the batch experiments was previously settled for 1 h and further diluted with distilled water up to COD concentrations of 250. The initial concentration of VSS also increased when the initial COD increased. while the solar radiation was in the range of 0. The experiment lasted 190 h. vulgaris was used as inoculum. using natural light. Ambient conditions Fig. 2. 800 and 1100 mg/l.10 0.4 2.8 49. Temperatures ranged from 27 to 32 ◦ C during the day and from 21 to 25 ◦ C during the night. Thirteen samples of 20 ml were taken in each of the culture bottles for determination of the parameters described in Section 2. 650. This result shows the occurrence of an inhibition of all population of microorganisms present at an initial substrate concentration of 1100 mg COD/l. decreased suddenly for an initial concentration of 1100 mg Fig. The initial volume was maintained by the addition of an equal volume of piggery wastewater at the corresponding concentration.06 2. The composition of the culture medium is shown in Table 2.6.2 3. achieving a drastic reduction for the highest initial substrate concentration studied.5.1. vulgaris and total microorganisms concentration.3. 2 – Variation of the Napierian logarithm of the quotient between the VSS concentrations at any time and the beginning of the experiment (ln(X /X0 )) with time for total microorganisms growth (VSS). the chlorophyll and VSS concentrations (measurements of C.74 1. The experiments were carried out during summer.4.

A plot of the data of ln(X/X0 ) versus time allows determination of the values of ln(Xm /X0 ). the increase in the concentration of suspended solids also reduces the light penetration causing a decrease in the concentration of available nutrients for microalgae. respectively. 2 shows the variation of the Napierian logarithm of the X /X0 ratio with the operation time.6% for initial COD concentrations of 250. respectively. The most widely unstructured models used for describing cell growth are the Monod.. The percentages of COD removed at 190 h were 88. e and L for each initial substrate concentration studied (Figs. In addition. Wachenheim et al. which occurs in many batch fermentations (Liu et al. Fig. 2002. for which the values decreased suddenly. Wachenheim et al. It was found that organic matter removal increased with the microorganism concentration in a range of initial substrate concentrations from 250 to 800 mg COD/l. the pair values. 2002) when complex substrates were used. 650. Finally. In this case. Liu et al. represent the VSS concentrations at any time and at the beginning of the experiments. 1 shows the plot of the Napierian logarithm of the (X/X0 ) ratio versus time (h) for different initial substrate concentration (S0 ) studied. 2003.. and stationary phases.85 to 0. Regression coefficients of the straight lines obtained in the exponential growth phases ranged from 0.91 to 0. but decreased when the initial substrate concentration increased to 1100 mg COD/l. 1998). The pattern of the curves obtained was very similar and comparable to that obtained in the case (1) where L is the duration of the lag phase. 2002. Valderrama et al. 3.05). The concentration of COD decreased with time due to the uptake of organic matter by the mixed culture. three different stages of the batch culture may be defined: lag... 60. logistic.. 800 and 1100 mg/l. 2003).0%. Straight lines were obtained with slopes equal to e and intercepts equal to L. respectively. 520.5%. 1999. Fig.05).. for each set of experiments carried out. According to this model. respectively. a kinetic model was developed and applied to the experimental results obtained. Xm the concentration of microorganisms at an operation time equal to infinite. 1 and 2). corresponding to the exponential growth phase were plotted. 57. 2003.. Fig. In order to obtain the e and L values for each initial substrate concentration (S0 ) studied.5%. 2003).. 2003). 2003. 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 concentration on the process performance. again. 3 shows the variation of COD with the time during the experiment. except at a substrate concentration of 1100 mg COD/l. Zonneveld. ln(X/X0 ) versus time. The straight lines obtained by plotting the values of ln(X/X0 ) corresponding to the stationary phase versus the inverse of time gave values of the regression coefficient in the range of 0.. Kayombo et al. The curves obtained are classical growth curves corresponding to biological processes carried out in batch conditions. and e is the specific growth rate of the microorganisms.. Table 3 shows the calculated values of L. Kinetic model Fig. Kayombo et al. showing the occurrence of a clear inhibition process. Haldane and Zwitering models among others (Hao et al.98 for a probability level of 95% (p ≤ 0. the value of ln(Xm /X0 ) for each initial substrate concentration studied may be obtained by the plot of the values of ln(X/X0 ) corresponding to the stationary phase versus the inverse of time. In those plots. exponential growth. for the six initial substrate concentrations studied. 56. X and X0 . The logistic model is a substrate independent equation and can accurately describe the inhibition of biomass growth in different cultures.6%. 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.97 (p ≤ 0. vulgaris and total microorganisms measured as total chlorophyll ( C) and VSS. 1993. The values of the specific growth rates obtained for microalgae ( C) and . The results obtained were comparable to those obtained by other authors using a mixed culture of microalgae and bacteria (Vonshak. Kayombo et al. 3 – Variation of substrate concentration (mg COD/l) during the experiment for different initial concentrations. vulgaris was achieved for an initial substrate concentration of 800 mg COD/l and the minimum one for initial substrate concentrations of 250 and 1100 mg COD/l. exponential growth. e and Xm for C. three phases of the culture may be clearly appreciated: lag. 2003. 400.6% and 20.. as was previously mentioned. The maximum concentration of C. 55.2. The values of e obtained were comparable to those obtained by other authors in batch and mixed cultures of algae and bacteria (Zhang et al. Table 3 shows that the values of e increased as the initial concentration of substrate increased both for microalgae and total microorganisms. where the parameter (X) represents the concentration of total chlorophyll ( C) at any time and X0 represents the value of the chlorophyll concentration ( C) at the beginning of the experiment. These results show that substrate removal efficiency decreased significantly at initial COD of 1100 mg/l due to the process inhibition.of the total chlorophyll ( C) and. The intercept at 1/t = zero might be considered as ln(Xm /X0 ) (Hao et al. and stationary phases.

9 h and 110.018 0. 6 illustrates the calculation of these parameters for microalgae growth. indicating that the overall growth might have been controlled by microalgae. this parameter also plays an important role in batch cultures of algae and bacteria.6 49.Table 3 – Summary of the kinetic parameters of the batch culture at different initial substrate concentrations obtained from the data plotted in Figs. The regression coefficient was R2 = 0.4 (h−1 ) Xm (mg VSS/l) 272 374 490 544 554 445 0. (1) were lower than 5% in all cases. max the maximum specific growth rate. Fig.9 34. The small deviations obtained in both cases suggest that the proposed model predicts the kinetics of microalgae and total microorganism growth very accurately and that parameters obtained represent the activity of both.020 0. The relationship between the specific growth rate and specific load may be expressed. In order to verify the validity of the model proposed for describing the growth of microalgae and total microorganisms.4 32. the differences between the experimental values and the theoretical ones (curves) obtained by Eq.1 59. 7 shows the plot of 1/ e versus COD/mg 1/BX for total microorganisms growth. By linearization of Eq.. respectively. for some influent substrate concentrations (S0 ) in the range of 250–1100 mg COD/l (p ≤ 0.3 59.6 Chlorella vulgaris e Total microorganisms C/l) L (h) 62.05).021 0. respectively. As can be seen. affecting the batch aerobic degradation of this waste.015 0. respectively. (1). Therefore. The results obtained also show that the value of e increased when the initial substrate concentration (S0 ) increased from 250 to 800 mg COD/l and decreased for a S0 value of 1100 mg COD/l.0 59. it is possible to determine the values of K and max as follows: a plot of 1/ e versus 1/BX should give a straight line of the slope equal to K/ max and intercept equal to 1/ max. 5 – Comparison of the experimental values of the VSS concentrations and the theoretical curves obtained by Eq.05) showing that the model can be applied to the experimental data. to evaluate the influence of the initial substrate concentration on the specific growth rate ( e ). In this case. 2000): e = max [BX /(K + BX )] (2) where BX is the ratio: S0 /X0 .7 59.10). (1).0 46. 4 – Comparison of the total chlorophyll concentration values ( C) and the theoretical curves obtained by Eq.37 mg VSS/mg COD.8 13. (1998) and Liu (2000) emphasized the importance of the S0 /X0 ratio in batch cultures of microorganisms for activated sludge processes. a straight line was also obtained with a regression coefficient R2 = 0.2 e (h−1 ) Xm (mg 32. Liu et al. and K is a saturation constant equivalent to the value of BX when e is equal to 1/2 max.024 0. instead of S0 . 4 and 5). 1 and 2 S0 (mg COD/l) L (h) 250 400 520 650 800 1100 49.0 36.8 55.96 h and 1619 mg C/mg COD. Fig.85.9 53. In addition.016 0.016 0.6 50. the experimental and theoretical values of X both for microalgae ( C) and total microorganisms (VSS). microalgae and total microorganism. were plotted versus time (Figs. 1999.98 (p ≤ 0. respectively.008 0. the S0 /X0 ratio defined as the specific load (BX ) was used. the intercept and slope values were found to be 19. Fig. the values of max and K were calculated from these values and were found to be 0.05). Liu. .023 0. . The value of the intercept and the slope were 22.0 51.020 0. according to a Monod type equation. (2). Hence.011 0. As can be seen.011 total microorganisms (VSS) were very close. From the Fig. for some influent substrate concentrations (S0 ) in the range of 250–1100 mg COD/l (p ≤ 0.9 66.2 d−1 ) and 81 mg C.05 h−1 (1. with a probability level of 90% (p ≤ 0. based on VSS measurements. as follows (Zhang et al.

emphasizing the fact that the overall process was controlled by the microalgae growth. .088 mg C/mg COD and 6. by a relationship similar to the Eq.. (2003) for settled sewage and as high as those obtained for complex substrates (Zhang et al. .75 mg of dry weight/mg COD. ) as a function of the inverse of the specific load (1/BX ).. The values of max obtained both for microalgae and total microorganisms growth were very similar. respectively. X2 and X1 the concentration of total chlorophyll ( C) at the end and at the beginning of the exponential growth phase.05). Therefore. Once the Yobs. the values of the observed microal- gae yield given in mg C/mg COD (Yobs.88 with a probability level of 95% (p ≤ 0. a plot of the above mentioned pair values gave a straight line from which the values of the constants Yobs.7% of the cell weight (Rioboo et al. and KY were calculated to be 0. is the observed microalgae yield (mg C produced/mg COD consumed). The value of the observed yield may be estimated in terms of dry matter of microalgae. using settled and diluted piggery wastewater as culture . BX /(KY + BX ) (4) where KY is the value of BX for which the value Yobs. respectively.056 d−1 ) and 4. 8 – Variation of the observed microalgae yield (1/Yobs. 7 – Variation of the inverse of the specific microorganism growth rate (1/ e ) as a function of the inverse of the specific load (1/BX ) for total microorganisms growth. ) were obtained by the quotient between microalgae growth and the substrate consumed during the exponential growth phase. it was observed that these values appear to be related to BX .Fig. 2002). 2002). Valderrama et al. The numerical values of max obtained for both cases were approximately half those obtained by Kayombo et al. Fig.. considering that C is 11. respectively. versus 1/BX should give a straight line with intercept equal to 1/Yobs. Conclusions Mixed culture of C. Therefore: Yobs. (4) a plot of the values of 1/Yobs.044 h−1 (1. = Yobs. 6 – Variation of the inverse of the specific microorganism growth rate (1/ e ) as a function of the inverse of the specific load (1/BX ) for microalgae growth. (2). S2 and S1 are the COD concentration at the end and at the beginning of the exponential growth phase. vulgaris grew satisfactorily at batch conditions. 4. max. = (X2 − X1 )/[− (S2 − S1 )] (3) where Yobs. is equal to (1/2) Yobs. max. respectively. the maximum observed yield is 0.85 mg COD/mg VSS. Based on the data of microbial growth and substrate uptake in the exponential phase. The value of the linear regression coefficient obtained was R2 = 0. max . 8. According to Eq. max and slope equal to KY /Yobs.35 mg COD/mg C. values of the intercept and the slope the corresponding values of max and K were calculated and found to be 0. As can be seen in Fig. Therefore: Yobs. 1999. Fig. max. values were calculated.

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