Aerobic biological treatment of black table olive washing wastewaters: effect of an ozonation stage

Process Biochemistry 35 (2000) 1183 – 1190 www.elsevier.com/locate/procbio

Aerobic biological treatment of black table olive washing wastewaters: effect of an ozonation stage Jesus Beltran-Heredia *, Joaquin Torregrosa, Joaquin R. Dominguez, Juan Garcia Departamento de Ingenierı´a Quı´mica y Energe´tica, Facultad de Ciencias, Uni6ersidad de Extremadura, A6da. de El6as s/n, 06071 Badajoz, Spain Received 28 June 1999; accepted 5 March 2000

Abstract The present work is a study of oxidative degradation of the organic matter present in the washing waters from the black table olive industry. Pollutant organic matter reduction was studied by an aerobic biological process and by the combination of two successive steps: ozonation pretreatment followed by aerobic biological degradation. In the single aerobic biological process, the evolution of biomass and organic matter contents was followed during each experiment. Contaminant removal was followed by means of global parameters directly related to the concentration of organic compounds in those effluents: chemical oxygen demand (COD) and total phenolic content (TP). A kinetic study was performed using the Contois model, which applied to the experimental data, provides the specific kinetic parameters of this model: 4.81 × 10 − 2 h − 1 for the kinetic substrate removal rate constant, 0.279 g VSS g COD − 1 for the cellular yield coefficient and 1.92 × 10 − 2 h − 1 for the kinetic constant for endogenous metabolism. In the combined process, an ozonation pretreatment is conducted with experiments where an important reduction in the phenolic compounds is achieved. The kinetic parameters of the following aerobic degradation stage are also evaluated, being 5.42 × 10 − 2 h − 1 for the kinetic substrate removal rate constant, 0.280 g VSS g COD − 1 for the cellular yield coefficient and 9.1 × 10 − 3 h − 1 for the kinetic constant for the endogenous metabolism. © 2000 Elsevier Science Ltd. All rights reserved. Keywords: Aerobic biological degradation; Black-olive wastewater; Kinetics; Ozonation pretreatment

1. Notation

qmax

BOW biological experiments of preozonated wastewater BW direct biological experiments COD chemical oxygen demand, measurement of substrate concentration (mg COD l−1) K kinetic rate constant defined by the ratio qmax/KS (h−1) kd kinetic rate constant for the biomass death phase (h−1) KS Contois saturation constant (mg COD g VSS−1) pO3 ozone partial pressure (kPa) q specific substrate removal rate (mg COD g VSS−1 h−1)

S t TP VSS X XCOD Xmax XTP YX/S m

maximum specific substrate removal rate (mg COD g VSS−1 h−1) biodegradable substrate concentration (mg COD l−1) bioreaction time (h) total phenolic content (mg caffeic acid l−1) volatile suspended solids (mg VSS l−1) biomass concentration (g VSS l−1) total COD removal (%) maximum biomass concentration (g VSS l−1) total phenolic compounds removal (%) cellular yield coefficient (g VSS g COD−1) specific biomass growth rate (h−1)

2. Introduction * Corresponding author. Tel.: + 34-24-289385; fax: + 34-24271304. E-mail address: [email protected] (J. Beltran-Heredia)

The world production of table olives is estimated to surpass a million tons per year [1], with the Mediter-

0032-9592/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 0 ) 0 0 1 6 0 - 6

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ranean countries being the main producers. There are three types of table olive: green, black, and black through oxidation. This last type of olive passes through a series of treatments to remove bitterness. In the first stage, the olives are stored and conserved in a sodium chloride solution. They are then darkened by aeration and successive sodium hydroxide treatments followed by intermediate washing of the fruit with water, thereby giving rise to two types of wastewater: alkali and wash-waters. The olives are finally immersed in a ferrous gluconate or lactate solution to fix the colour and then canned or bottled and sterilized. The whole process requires large volumes of water, mainly for the washing stages. In Spain, for instance, some 108 l year − 1 are consumed in this process. The treatment of each kg of olives gives rise to 8.5 l of wastewater, 1.5 l of which are alkali and 7 l washwaters. In most cases, these waters are dumped into the environment untreated. In other cases, the commonest treatment is to retain them in evaporation ponds. This procedure, however, causes bad smells and the possibility of polluting surface and ground waters. The process therefore constitutes a major environmental problem due to the high organic load of this wastewater and the large volume generated. Since the setting up of more stringent regulations concerning public waste disposal, there is a growing interest in the development of new technologies and procedures for the purification of this waste. Among these procedures, biological methods have been recognized as a viable possibility for the degradation of these wastewaters. These include aerobic degradation systems [2] or anaerobic digestions [3,4]. However, the presence of some substances, especially phenolic compounds which in general are toxic to biological treatments [5,6] inhibit the efficiency of these processes. For these situations, some chemical pretreatments which remove phenolic compounds and facilitate the latter biological treatments have been investigated. Among these, ozonation is a promising technology since ozone has many of the oxidizing properties desirable for wastewater treatments [7] and consequently is increasingly used because of its characteristics: it is a powerful oxidant capable of oxidative degradation of many organic compounds, readily available, soluble in water and leaves no by-products that need to be removed [8]. As little research has been reported with regard to the application of purification technologies to this residue [9], the degradation of washing wastewater from the black olive industry has been studied in the present work. The process is carried out in a first phase by means of a single aerobic degradation; and later in a second phase, by means of a combined treatment, with an ozonation pretreatment followed by an aerobic

degradation stage. In both cases, the objectives are to report data for the removal of the pollutant organic matter, represented by the decrease in the chemical oxygen demand (COD) and the total phenolic content (TP). Furthermore, as the design of equipment requires a knowledge of the kinetics of the processes which take place, a study of the biological stages is conducted according to the Contois model, with the aim to provide kinetic rate constants useful for the design of bioreactors where these aerobic stages are carried out in wastewater treatment plants.

3. Materials and methods

3.1. Washing wastewaters Wash-waters were obtained from the industrial plant ‘San Mer S.A.’ (Cabezuela del Valle, Extremadura Community, south-west Spain). The average results obtained after three analyses for each parameter of the physico-chemical composition of the wastewater were as follows: COD 6000995 mg l − 1; BOD5 50109153 mg l − 1; polyphenolic content 1809 21 mg l − 1; Kjeldahl nitrogen 2593 mg l − 1; total solids 57209210 mg l − 1; total dissolved solids 55209 195 mg l − 1 and volatile dissolved solids 21009 102 mg l − 1. These analyses were performed according to procedures described in Standard Methods [10]. The total phenolic content was determined by the Folin–Ciocalteau method [11] and is expressed as mg caffeic acid l − 1.

3.2. Aerobic degradation experiments The experiments were conducted in a completely mixed batch reactor. This was a 1200 cm3 cylindrical Pyrex glass vessel, provided with a cover containing inlets for bubbling the air feed and stirring, and outlets for sampling and venting. The reactor was submerged in a thermostatic bath with the necessary elements to maintain the temperature constant at 28°C within 9 0.5°C. An air stream was introduced into the medium through a bubble gas sparger with a constant flow rate of 125 l h − 1 at room conditions. Initially, the biological reactor was inoculated with activated sludge taken from a municipal wastewater treatment plant and several previous experiments were carried out for the acclimatization of the biomass to this substrate. For this purpose an initial volume of wastewater was fed to the digester, which was stirred and aerated for 6 days. Once the experiment had finished and after a settlement period, the biomass was separated by filtration from the liquid and introduced again into the reactor. This procedure was repeated in a similar way in the following experiments, with successive additions to the biomass of increasing volumes of

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Table 1 Experimental conditions, COD and total phenolics conversions in the single aerobic biological process Exp.

BW-1 BW-2 BW-3 BW-4 BW-5 BW-6

X0 (mg VSS l−1) 1210 460 168 359 140 765

COD0 (mg COD l−1) 5644 5508 3582 1602 1950 1950

XCOD (%) 81 79 75 76 74 73

wastewater, equivalent to gradually increasing concentrations of substrate. The acclimatization was considered to be achieved when a similar removal of COD was obtained after three experiments with the highest volume of wastewater loaded into the reactor. When the acclimatization period was finished, the aerobic degradation experiments were conducted by introducing into the biological reactor the amount of biomass required to obtain the desired initial concentration; and by loading 1000 ml of diluted wastewater in order to attain the predesignated initial concentration of substrate to be degraded in each experiment. These experiments lasted between 4 and 6 days, and periodically samples were withdrawn to analyse the substrate (expressed as mg COD l − 1), the biomass concentrations (expressed as mg VSS l − 1), and the total content of polyphenolic compounds (expressed as mg caffeic acid l − 1).

3.3. Ozonation experiments Ozonation experiments were conducted in a completely mixed chemical batch reactor (a 750 cm3 cylindrical Pyrex glass vessel) provided with a cover containing inlets for bubbling the gas feed and stirring and outlets for sampling and venting. The reactor was immersed in a thermostatic bath with the necessary elements to maintain the temperature constant within 9 0.5°C. For ozone generation, oxygen taken from a commercial cylinder was dried with silica gel traps and introduced into an ozone generator (Sander, model 301.7). The ozonation experiments of the combined process started when the ozone partial pressure, temperature and pH were adjusted to the desired value. The ozone– oxygen stream was then fed to the ozonation reactor and several samples were taken periodically to analyse the COD concentration and total polyphenolic content. When the experiments were finished, a volume of this ozonated wastewater was loaded into the bioreactor and an aerobic degradation experiment performed in the same way as described previously. The final concentration of substrate from the ozonation stage was the initial substrate concentration for the biological stage.

TP0 (mg TP l−1)

XTP (%)

168 165 102 49 53 50

50 45 48 49 51 44

COD0/X0 (g COD g VSS−1)

Xmax/X0

4.7 11.9 20.8 4.5 13.8 2.6

1.14 1.94 3.26 1.56 2.23 1.28

4. Results and discussion

4.1. Single aerobic biological degradation process For aerobic biological degradation an experimental series was carried out by varying the initial substrate concentration (COD0 between 5600 and 1600 mg l − 1) and the initial microorganism concentration (X0 between 140 and 1200 mg l − 1). Table 1 lists the values of these variables in each experiment, together with the final conversions obtained in the removal of COD. These conversions were obtained as defined in Eq. (1): XCOD =

(COD0 − CODf) × 100 COD0

(1)

In those experiments with similar initial substrate concentrations (COD0 = 5600 mg l − 1, experiments BW-1, BW-2), the initial concentration X0 of microorganisms had practically no effect on COD and total polyphenols reductions. With respect to experiments with similar initial concentration of microorganisms (X0 = 150 mg l − 1, experiments BW-3, BW-5), the different initial concentration of substrate did not affect the COD and total polyphenol conversions. During an experiment, the concentration of the substrate COD, biomass X, and total phenolic compounds TP was followed. As an example, Table 2 shows the values obtained for these parameters in experiment Table Evolution of the parameters in experiment BW-2 Time (h) 0 12 16 20 24.5 37.5 43 48 69 89.5 133.5

X (mg VSS l−1) 460 688 695 724 900 808 696 632 304 284 280

COD (mg COD l−1) 5508 4148 3910 3230 2329 1980 1902 1737 1515 1302 1167

TP (mg TP l−1) 165 163 157 144 122 121 108 104 98 94 89

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Fig. 1. Biomass evolution in experiments where the initial biomass X0 was varied.

parameters that adjusted all experimental points (including those corresponding to the series BOW) by least square regression analysis and gave a value for the intercept of 0.976 and for the slope of 0.095 g VSS g COD − 1, with a determination coefficient (r 2) of 0.924. The content of phenolic compounds TP also decreased continuously with time (see Table 2 for example experiment BW-2), and the removal obtained XTP (defined in a similar form as for substrate removal, Eq. (1)) in each experiment is shown in Table 1. As can be observed, an important level (approx. 50%) for this parameter was reached in all cases. From an industrial point of view, the most interesting period in the growth cycle of a batch cultivation is the exponential growth phase, when the population of microorganisms is perfectly adjusted to the environment. During this period, the rate of production of biomass may be described by a first-order kinetic equation [12]: dX = mX dt

(2)

where m is the specific growth rate of biomass. Simultaneously to the production of the cells, the substrate is decomposed and this rate is also proportional to the mass of cells present, according to the expression: −

Fig. 2. Xmax/X0 dependence with COD0/X0 relation.

BW-2, with similar trends being observed in the remaining runs. A continuous decrease in COD with time was observed (Table 2). The variation in biomass in experiment BW-2 is shown in Table 2 and follows the typical growth-cycle phases for batch cultivation [12]. After an acclimatization period (lag phase), the population of microorganisms was well adjusted to its new environment. The cells then multiplied rapidly and an important increase in the biomass concentration was observed (exponential growth phase), until the maximum size of population was reached (the stationary phase), Xmax. Finally, a decline in the cell number took place (death phase of microorganisms). This process can also be observed in Fig. 1 which represents the biomass concentration with time in a group of experiments in which the initial biomass X0 was varied (expts. BW-1, BW-2 and BW-3). There was also a positive influence of the COD0/X0 relation (initial substrate concentration per initial biomass concentration) on the Xmax/X0 relation (maximum biomass concentration per initial biomass concentration) (Table 1). Fig. 2 shows a plot of Xmax/X0 versus COD0/X0. There was a linear relationship between both

dS = qX dt

(3)

where q is the specific substrate removal rate, a single parameter which characterizes the degradation process. The literature provide several expressions which relate the specific rates (m and q) to substrate concentration [12,13]. Among these, the Contois model [14] provides excellent fits to the experimental results. In the case of the specific removal rate, this model proposes the following equation for q as a function of substrate concentration: q = qmax

S KSX +S

(4)

where qmax represents the maximum rate of substrate removal and KS is the Contois saturation constant. In order to obtain the specific kinetic parameters for this model, qmax and KS or a ratio between them, which constitutes the objective of the present kinetic study, Eq. (4) can be linearized in the form: 1 1 KX = + S q qmax qmaxS

(5)

According to Eq. (5), a plot of 1/q versus X/S must lead to a straight line for every experiment conducted. The intercept and slope will be 1/qmax and KS/qmax, respectively. For this purpose, the specific rate q must be previously evaluated for each time of bioreaction, by transforming its definition expression (Eq. (3)):

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Table 3 Evolution of the q and m parameters with time (Exp. BW-3) Time (h)

COD (mg COD l−1)

0 4.5 8 20 25 30.5 46 56.5 73 95.5

3582 3486 2874 1842 1656 1578 1308 1170 1146 868

q =−

dS X dt

X (mg VSS l−1) 168 168 264 520 548 504 460 428 420 328

−dS/dt (mg COD l−1 h−1) 133 107 93 51 40 30 14 8.5 3.8 1.3

(6)

For this evaluation, the term dS/dt was calculated by fitting the experimental data (S, t) to a polynomical equation by least-square regression analysis and dividing by the biomass concentration X at any time. It must be noted that S represents the biodegradable substrate concentration, which in this research was determined by subtracting the non-biodegradable concentration from the COD, i.e. the COD measured at the end of each experiment, when no more removal of substrate was observed. Table 3 shows the values calculated for − dS/dt and q, respectively in experiment BW-3 using this procedure. Once the specific rates q are known, Eq. (5) can be used as described before. Fig. 3 shows the general plot with all the values of the experiments depicted in Table 1. Points lie satisfactorily around a straight line, which confirms that the selected Contois model is adequate to correlate the experimental system studied. After least square regression analysis, the slope and intercept were determined, which provide values of 20.78 h for KS/qmax, and 7× 10 − 4 g VSS h mg COD − 1 for 1/qmax. being the determination coefficient of 0.877. This small value for 1/qmax suggests a very high value for qmax that cannot be calculated and reported accurately in this system from the regression analysis. Therefore, the term 1/qmax can be eliminated in Eq. (5), and Eq. (4) can be transformed into: q = qmax

S S =K KSX X

q (g COD g VSS−1 h−1)

dX/dt (mg VSS l−1 h−1)

m (h−1)

0.788 0.638 0.352 0.097 0.073 0.066 0.028 0.020 0.089 0.004

22.8 19.4 16.6 7.9 4.9 2.6 −2.8 −4.6 −4.9 −5.1

0.1355 0.1153 0.0631 0.0151 0.0089 0.0052 −0.0061 −0.0107 −0.0116 −0.0155

lar yield coefficient YX/S and the kinetic constant kd for the biomass decrease during the death phase of microorganisms. The first is defined as the ratio of g biomass produced per g substrate consumed, and can be expressed by the equation: YX/S = −

dX dS

and taking into account the definition Eqs. (2) and (3), it can be written: m= YX/Sq

(9)

However, this expression only holds for the exponential growth phase. For the whole growth cycle of microorganisms, the death phase must also be taken into account, when the decline in the cell number takes place. As Bailey and Ollis pointed out [12], relatively few studies have been made on this phase, because many industrial batch microbiological processes are terminated before the death phase begins. Usually the death rate of the microorganisms population during this period is assumed to follow first-order kinetics: −

dX = kdX dt

(10)

where kd is the previously mentioned kinetic constant for endogenous metabolism. Therefore, the global expression of the specific growth rate m must be:

(7)

This is a particular situation of the Contois model [14], when KSXˆS and the specific substrate removal rate follows first-order kinetics. In the present system, these conditions are fulfilled and the value obtained for the kinetic constant K is 4.81× 10 − 2 h − 1. Other kinetic parameters useful in the design of bioreactors are related to biomass evolution during the cycle of batch cultivations [12]. These include the cellu-

(8)

Fig. 3. Contois model plot in BW experiments.

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Table 4 Experimental conditions, COD and total phenolic conversions in the ozonation process Exp. OA-1 OA-2 OA-3 OA-4 OA-5

T (°C) 20 30 10 20 20

pH 6.5 6.5 6.5 9 4

pO3 (kPa) 4.45 4.48 4.48 4.36 4.51

COD0 (mg COD l−1) 6100 5930 6530 6220 5970

CODf (mg COD l−1)

XCOD (%)

4200 4000 4700 4730 4020

31 33 28 24 33

TP0 (mg TP l−1) 179 180 178 179 173

XTP (%) 30 58 46 67 55

Table 5 Experimental conditions, COD and total phenolic conversions in the aerobic biological process of preozonated wastewaters Exp. BOW-1 BOW-2 BOW-3 BOW-4

X0 (mg VSS l−1) 162 325 240 227

m = YX/Sq− kd

COD0 (mg COD l−1) 4535 4280 3755 1875

XCOD (%) 74 74 70 74

TP0 (mg TP l−1) 54 54 50 32

(11)

According to Eq. (11), a plot of m values versus q values in the experiments must lead to a straight line whose slope and intercept will be YX/S and kd, respectively. For this purpose, the specific growth rate m must also be calculated at any time of each experiment, in a similar way as the specific substrate removal rate q was evaluated. That is, the term dX/dt was determined by fitting the experimental data (X, t) to a polynomical equation by means of a regression analysis; and later, this term was divided by the biomass concentration X at any time. Table 3 also shows the values obtained for both parameters in experiment BW-3, showing the decreasing values of dX/dt and m with reaction time during the exponential growth phase and their negative values during the death phase. After least squares regression analysis was carried out, the cellular yield coefficient YX/S was determined as 0.279 g VSS g COD − 1 and for the kinetic constant for the death phase of microorganisms kd, a value of 1.92×10 − 2 h − 1 was obtained with a determination coefficient (r 2) of 0.892.

4.2. Combined process: ozonation followed by aerobic degradation The degradation of black table olive wastewaters was performed by the combined process constituted by an ozone oxidation followed by an aerobic biological degradation. In the ozonation stage, five experiments were conducted in the reactor described above, by varying the temperature and pH. COD removal and total phenolic content were followed during ozonation. Table 4 depicts the initial values for COD and total

XTP (%) 45 43 50 59

COD0/X0 (g COD g VSS−1)

Xmax/X0

28.0 13.1 15.6 8.3

3.4 2.1 2.8 1.8

polyphenolic compounds in these ozonation experiments, as well as the final conversions obtained for both parameters. The COD conversions after 3 h of reaction lay between 24 and 33% depending on the operating conditions. These moderate XCOD conversions can be attributed to the generally high reactivity of organic compounds to ozone, leading to intermediates consisting of smaller and less pollutant molecules. These still had a moderate reducing character in the COD test, in which they were oxidized to their final oxidation states. The mean conversions of total polyphenols were around 50%. There was a mild positive influence of temperature on COD removal (experiments OA-3, OA1, and OA-2) (Table 4). This mild effect may be due to two counteracting factors, as the temperature rose, the kinetic constant also rose, but the solubility of ozone in the wastewater decreased. pH appeared to have a slight negative effect on COD conversion. This low conversion during an ozonation treatment of wastewaters with high organic pollutant load has been observed in previous investigations [15]. A significant removal of phenolic compounds was observed. As mentioned previously, the phenolic compounds present in these types of effluents constitute an important limiting factor when using biological treatments of these residues, since they are toxic to some types of bacteria [6,16]. It can therefore be expected that their elimination in this ozonation pretreatment should reduce the global toxicity of the effluent for later biological treatment. Once the ozonation pretreatment was completed, four aerobic degradation experiments were conducted. In these experiments, the evolution of COD, biomass and total phenolic compounds was similar to the trends

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observed in the aerobic degradation of the non-pretreated wastewaters. Table 5 depicts the initial biomass X0, initial concentrations of substrate COD0 and phenolic compounds TP0, and the removal obtained for these parameters XCOD and XTP. In this stage the reductions of COD were in the range 70 – 74%, slightly lower than those obtained for non-pretreated experiments (73 –81%). This was probably due to the removal reached in the previous ozonation stage. For total phenolic compounds the reductions now had similar values to those obtained with non-pretreated experiments, around 50%. In the experiments with similar initial substrate concentrations (COD0 =4400 mg l − 1, experiments BOW-1, BOW-2), the initial concentration X0 of microorganisms did not affect the elimination of COD or total polyphenols. With respect to those experiments with similar initial concentrations of microorganisms (X0 = 235 mg l − 1, experiments BOW-3, BOW-4), the different initial concentration of substrate had practically no effect on the elimination of COD or total polyphenols. From the results reported in Tables 4 and 5, the global reductions obtained for both parameters after the two stages of the combined process can be evaluated, being the mean values of these removals: XCOD = 82% and XTP = 76%. These are higher than the average values obtained in any single treatment as would be expected, especially for the elimination of polyphenolic compounds. This demonstrates the efficiency of the combined process in the removal of organic matter of the wastewater generated in the black olive industry. There was a positive influence of COD0/X0 relation on the Xmax/X0 relation (Table 5). As was described previously, the experimental points for both series BW and BOW are located on the same straight line (Fig. 2). In addition, the kinetic study of the aerobic biological stage in this combined process was also performed in a similar way to that conducted in the single aerobic treatment, by applying the Contois model to the experimental data. For this purpose, the parameters dS/dt, q, dX/dt and m were also evaluated in these experiments.

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Then, according to Eq. (5), a plot of 1/q values against X/S was conducted; and according to Eq. (11) a plot of m against q was also performed (Fig. 4). By regression analysis in both cases, the following kinetic parameters were determined: K= 5.42× 10 − 2 h − 1 (r 2 = 0.874); YX/ S= 0.280 g VSS g COD − 1 and kd = 9.1× 10 − 3 h − 1 (r 2 = 0.905). The value of K was 13% higher than that obtained in the other experiment without a preozonation stage; that is, the kinetic rate constant for substrate removal increased. In both cases, the value of cellular yield coefficient was similar and the kinetic rate constant of the death phase of microorganisms decreased with preozonation treatment. This suggests that ozone reduced effluent toxicity, and so enhanced the later aerobic biological degradation due to the removal of an important amount of phenolic compounds which potentially inhibited biological oxidation.

5. Conclusions In the single aerobic biological degradation of black table olive washing wastewaters, the average removal of COD and polyphenols are 75 and 50%, respectively. A kinetic study applying the Contois model leads to the following values: K= 4.81×10 − 2 h − 1, YX/S = 0.279 g VSS g COD − 1, and kd = 1.92× 10 − 2 h − 1, respectively for the kinetic constant of COD reduction, cellular yield coefficient and kinetic rate constant of the death phase of microorganisms. In the combined process (ozonation pretreatment plus aerobic biological degradation), global removals of 82 and 76% were obtained for COD and total phenolic content, respectively, higher than those obtained in the single aerobic process. A similar kinetic study performed in the biological stage of this combined process leads to the following kinetic parameters: K=5.42× 10 − 2 h − 1; YX/S = 0.280 g VSS g COD − 1, and kd = 9.1× 10 − 3 h − 1. Therefore, the results obtained indicate a slight improvement in the kinetic parameters for the aerobic biological treatment when a preozonation stage is applied.

Acknowledgements

Fig. 4. Determination of cellular yield coefficient in BOW experiments.

This research has been supported by the ‘Comision Interministerial de Ciencia y Tecnologia’ (CICYT) of Spain, under Project AMB 97-0339, ‘Plan Nacional I+ D’ of Spain, under Project Programa FEDER 1FD97-1866, and by Junta de Extremadura under Grant IPR 98A014. Joaquı´n R. Domı´nguez Vargas wishes to thank Ministerio de Educacio´n y Cultura for the financial support to his Ph.D. Grant.

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