Biochemical Engineering Journal 9 (2001) 211219

Biodegradation of phenol by Pseudomonas putida DSM 548 in a trickling bed reactor

C.S.A. S1 , R.A.R. Boaventura
Laboratory of Separation and Reaction Engineering (LSRE), Department of Chemical Engineering, Faculty of Engineering, University of Porto, Rua Dos Bragas, 4050-123 Porto, Portugal Received 6 October 2000; accepted after revision 16 August 2001

Abstract A trickling bed biolm reactor packed with PORAVER particles was used to evaluate phenol and total organic carbon (TOC) removal efciencies by Pseudomonas putida DSM 548. As reported by other authors, it was observed that the average residence time in the reactor is a function of the hydraulic loading rate and the column depth. After the start-up period, the bioreactor was operated at high hydraulic loading rates (16.1 to 32.2 m3 m2 d1 ). Assuming a constant biomass concentration along the reactor, the overall reaction kinetics follows a pseudo rst-order model. The obtained biokinetic constants at 25 C were K25 C (phenol) = 287.6 d1 and K25 C (TOC) = 294.8 d1 . It was also observed that metabolites were not consumed along the reactor. The average biolm thickness was 1.6 mm and the wet and dry biomass densities were 1.04 and 0.17 g cm3 , respectively. Phenol and TOC removal efciencies, in the temperature range 1930 C, were expressed as a function of the lter depth and hydraulic loading rate. 2001 Elsevier Science B.V. All rights reserved.
Keywords: Phenol; Biodegradation; Kinetics; Trickling bed reactor; Pseudomonas putida

1. Introduction Phenolic compounds and their derivatives are present in the wastewater of many industries, including oil reneries, chemical plants, explosives manufacture and coke ovens. They are also used in the preparation of antiseptics, dyes, antirust products, synthetic resins, biocides, photographic chemicals, inks, varnishes, etc. [14]. In the last years, as a consequence of a great industrial growth, with special relief for the chemical industries, the presence of phenolic pollutants in the aquatic environment had a big increment. Phenol is toxic to many biochemical functions and to sh life [5]. It is water soluble and highly mobile, and so it is likely to reach drinking water sources downstream from discharges, where, even at low concentrations, it can cause severe odor and taste problems and pose risks to populations. Various regulatory water authorities have imposed strict limits to phenol concentration in industrial discharge streams
Corresponding author. Present address: Department of Chemical Engineering, Faculty of Engineering, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal. Tel.: +351-22-508-1683; fax: +351-22-508-1674. E-mail address: bventura@fe.up.pt (R.A.R. Boaventura). 1 Present address: Department of Chemical Engineering, High Engineering Institute of Porto, Rua Dr. Ant nio Bernardino de Almeida, 431 o 4200-072 Porto, Portugal.

[6]. The Portuguese legislation establishes a phenol concentration of 0.5 mg l1 as the limit for wastewater discharge into natural water bodies, land or municipal sewerage systems. For drinking waters, it has been prescribed a guideline concentration of 1 g l1 [7]. Phenolic compounds have traditionally been removed from industrial efuents by costly physical-chemical processes, although biological methods have also been applied, offering reduced capital and operating costs [8]. In the absence of high concentration of toxic substances, or in instances of their successful prior removal, biological treatment is widely employed for treatment of wastewater containing intermediate phenol levels (5500 mg l1 ) [3]. Phenol biodegradation by pure cultures of bacteria has been adequately described by substrate inhibition models [912]. Otherwise, when using mixed cultures there is no general agreement about the kinetic model but phenol is often considered inhibitory at high concentrations [1315]. At low substrate concentration, however, as inhibition is negligible, the model proposed by Monod may be used to describe the biodegradation process by either pure or mixed cultures. Furthermore, if the substrate concentration is much lower than the half-velocity constant, then a rst-order kinetic model often applies. Activated sludge reactors have been widely used for phenol removal from industrial wastewater, but immobilized cell

1369-703X/01/$ see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S 1 3 6 9 - 7 0 3 X ( 0 1 ) 0 0 1 4 9 - 8

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Nomenclature c D k Kphenol K KTOC L n Q S Se So tr T V X hydrodynamic constant (m0.15 d0.15 ) lter depth (m) specic biokinetic constant (m3 kg1 TSS d1 ) model constant (m0.17 d0.83 ) biokinetic constant (d1 ) model constant (m0.15 d0.85 ) hydraulic loading rate (m3 m2 d1 ) hydrodynamic constant ow rate (ml min1 ) substrate concentration (mg l1 ) outlet phenol or TOC concentration (mg l1 ) inlet phenol or TOC concentration (mg l1 ) average residence time (d) temperature ( C) volume (cm3 ) average biomass concentration (g TSS l1 )

Mikroorganismen und Zellkulturen) were used as inoculum for the reactor start-up. Non-acclimated Pseudomonas putida grows very slowly in contact with phenol as the oxi-degradation enzymes are of inductive nature. So, the microorganisms were rst grown in an easily degradable substrate (2000 mg l1 glucose) and acclimated to increasing phenol concentrations up to 500 mg l1 . In 3 weeks, the glucose concentration was gradually reduced to zero; every decrease of 400 mg l1 glucose was replaced by 20 mg l1 phenol. 2.2. Nutrient mineral medium The required nutrients for the growth of the microorganisms were provided by a mineral medium with the following composition per l of solution: 69.6 mg CaCl2 2H2 O, 8 mg NaCl, 103 mg KNO3 , 698 mg NaNO3 , 100 mg MgSO4 7H2 O, 100 mg NTA, 2 mg FeSO4 7H2 O, 0.1 mg ZnSO4 7H2 O, 0.043 mg MnSO4 5H2 O, 0.3 mg H3 BO3 , 0.24 mg CoSO4 7H2 O, 0.01 mg CuSO4 5H2 O, 0.02 mg NiSO4 7H2 O, 0.03 mg NaMoO4 2H2 O, 0.5 mg Ca(OH)2 , 5 mg EDTA, 544.4 mg KH2 PO4 , 2148.9 mg Na2 HPO4 , 30 mg (NH4 )2 SO4 . Although the mineral medium composition was based in literature data, the authors dened it from preliminary batch growth studies. 2.3. Analytical procedures Phenol was determined by HPLC (Shimadzu chromatograph, model LC-4A), using a Hypersil Green Env column (5 m average size particles) and a UV detector ( = 270 nm). The eluent was methanolwater mixture (60:40 (v/v)) pumped at a ow rate of 1.5 ml min1 . The total organic carbon (TOC) was measured using a DC-190 Dohrmann analyzer. The suspended biomass growth was followed up by optical density measurement (UVVIS spectrophotometer, PHILIPS 8620) at 610 nm. pH was measured using a CRISON pH-meter, model 2002. Volatile suspended solids (VSS) were determined according to standard methods [24]. 2.4. Experimental set-up The experimental set-up is schematically shown in Fig. 1. The trickling bed reactor is a 100 cm height and 9.24 cm internal diameter cylindrical Plexiglas column. At the bottom there is a 10 cm height section with six holes regularly distributed 5 cm below the biomass support. Ventilation occurs by natural convection, since the recirculated stream is previously heated in a thermostatic bath to obtain at least a temperature difference of 2 C relative to the ambient atmosphere, as proposed by Muoz [25]. Values of dissolved oxygen (DO) measured at the column outlet were always higher than 2 mg l1 . At the top of the reactor, phenol contaminated water is continuously distributed through a rotary

Greek letters removal efciency average biolm thickness (mm) constant density (g cm3) reactors offer several advantages over suspended cell reactors [16]. These include higher biomass concentrations, allowing higher loading rates, and resistance to shock loading of inhibitory compounds, then requiring less time to revert to normal operation. Fixed biolm reactors are receiving increasing interest in wastewater treatment [1719]. Phenol uptake by Pseudomonas putida cultures has been reported by several authors [11,16,2023]. In this work, Pseudomonas putida was also chosen as inoculum for phenol biodegradation in a trickling bed reactor. The main purposes of this investigation are as follows. 1. To understand the reactor behavior under different operating conditions and t a kinetic model to phenol removal efciency. 2. To characterize the biolm in terms of some physical properties, biological yield and cells activity. 3. To evaluate the inuence of temperature on the reactor performance.

2. Materials and methods 2.1. Inoculum Pure cultures of Pseudomonas putida DSM 548 (ATCC 17514) from DSMZ (Deutsche Sammlung von

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Fig. 1. Schematic representation of the experimental set-up.

distributor. Along the bed there are ve sampling ports, located at 10, 30, 50, 70 and 87 cm from the top. The biomass support is a siliceous granular material (PORAVER, from Dennert PORAVER GmbH, Germany), 24 mm average diameter, chemically inert, light and porous, rather similar to the common pumice stone. Feed solutions were prepared from tap water previously dechlorinated by air bubbling for about 15 h. Mineral and phenol solutions are pumped to the rotating distributing arm through peristaltic pumps (WatsonMarlow model MHRE 200). The efuent recirculation stream is added to the feed stream also using a WatsonMarlow peristaltic pump. 2.5. Hydraulic loading rates The reactor feed ow rates were calculated in order to operate at high hydraulic loading rate. Although there is not a general agreement about limits for operating in this regime, it has been considered that supercial velocities between about 10 and 40 m3 m2 d1 ) are characteristic of high loading rate conditions [26]. A hydraulic loading rate of 16.1 m3 m2 d1 , which corresponds to a ow rate of 75 ml min1 was selected for this work. This value is close to the lower limit of the range and so less mechanical efforts are exerted and a high hydraulic residence time is achieved in the reactor. The reactor performance in steady-state conditions was evaluated for four hydraulic loading rates: 16.1, 21.5, 26.9 and 32.2 m3 m2 d1 .

2.6. Phenol loading rate From studies carried out in batch reactors using Pseudomonas putida as inoculum it was concluded that the maximum cell growth rate occurs for phenol concentrations about 400 mg l1 [27]. In a continuous process, concentrations must be lower to avoid clogging risks. So, the phenol concentration at the reactor inlet decreased from 55 mg l1 , at the beginning of the operation, to about 30 mg l1 at the end of the start-up stage. Once the pseudo steady-state conditions were achieved, as indicated by the overall TOC and phenol removal efciencies practically constant, the phenol concentration at the reactor inlet varied between 16 and 34 mg l1 . To operate with a recirculation ratio of 4 and a 75 ml min1 ow rate at the reactor inlet, the feed ow rate was adjusted to 15 ml min1 . Assuming that the BOD5 of phenol solutions is approximately 1.5 g O2 per g phenol, the organic loading rate based on the combined ow (feed + recirculation) ranged between 258 and 548 g BOD5 m3 d1 . 2.7. Residence time The mean hydraulic residence time in the reactor was determined by the tracer injection technique. A 5 ml volume of 1% KCl solution was injected on the top of the trickling lter and the conductivity was measured at the outlet using a conductivity meter (Tacussel, model CD 78). The

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experimental response curves were recorded with an analogic recorder (Linseis, model L6512-1). 2.8. Biolm cells activity The activity of the cells from the biolm matrix was determined in samples removed from the upper, middle and lower sections of the reactor, respectively. The material was placed in Erlenmeyer asks, incubated at 25 C and vigorously stirred to homogenise the suspension for 1 min. Then the DO was measured for periods between 4 and 13 h, depending on the biolm activity. A DO electrode was immersed in the suspension and the DO decay recorded in the course of time. Before adding the carbon source, the DO consumption rate, caused by the biomass endogenous respiration, was determined. After that, a given volume of phenol solution was added to the suspension in order to get a concentration of 25 mg l1 and, after a short period for the electrode stabilization, the DO concentration was recorded again. In this second step, the increase in the DO consumption rate is due to the phenol oxidation + respiration. 2.9. Biolm density and amount of biomass in the reactor

Assuming pseudo rst-order kinetics and plug ow, the substrate removal rate is given by dS = K S dt or dS = kSX dt (2)

where K is the biokinetic parameter (kX), S the substrate concentration, k the specic biokinetic constant, t the time and X is the biomass concentration Assuming X constant and equal to the average biomass concentration in the reactor, as proposed by Eckenfelder [28] and Ramalho [29], K becomes constant and the integration of Eq. (2) from t = 0 to t = t r leads to Se = ekXtr So Substituting tr by its value given by Eq. (1) we get Se n = e(KD/L ) So (4) (3)

where K = ckX. Varying the hydraulic loading rate L and measuring the outlet substrate concentration, Se , constants K and n can be experimentally determined.

4. Results and discussion The volume of the wet biomass into the reactor was determined by measuring the volume of water necessary to ll the empty spaces between the support particles, before and after the biomass attachment. At the end of the experimental phase, samples of support particles covered with biomass were removed from each section of the reactor and placed in stainless steel trays. The water content of the biomass was calculated from the difference of weights before and after drying the particles at 105 C. Finally, the biomass was washed out by immersing the particles in 10% NaOH solution. The dry biomass was calculated as the difference of weights of the dry biomass covered particles and the clean particles. From the volume and weight of the wet biomass, the wet biomass density was calculated. 4.1. Reactor hydrodynamics The conductivity responses to the KCl injections, proportional to the deviation of the recorder pen, are presented in Fig. 2 for ow rates of 75, 100, 125 and 150 ml min1 . Experiments were carried out by percolating tap water through the packed bed after the pseudo steady-state, in terms of biomass concentration (average = 36.4 g TSS l1 ), was achieved. The mean residence times, calculated for each hydraulic loading rate are shown in Table 1. The model constants, c = 0.034 m0.15 d0.15 and n = 0.85, were determined by tting the experimental values of tr and L to the Eq. (1). 4.2. Reactor start-up 3. Theoretical background As proposed by Eckenfelder [28], the mean residence time of the uid in the reactor, tr , is related to the lter depth, the hydraulic loading rate and the nature of the support: tr = cD Ln (1) The start-up operation was carried out in a closed circuit (100% recycle ratio), using a batch culture of Pseudomonas putida at the end of the exponential growth phase as inoculum. After running the reactor for 3 weeks, a thin biolm covering the particles could be observed. Then the operation was shifted to continuous mode.
Table 1 Average residence time (tr ) as a function of the feed ow rate Flow rate (ml min1 ) Hydraulic loading rate (m3 m2 d1 ) tr (min) 75 16.1 4.65 100 21.5 3.91 125 26.9 2.73 150 32.2 2.42

where D is the lter depth, L the hydraulic loading rate, c and n are the constants that depend on the biomass support nature and the specic surface area. Calculating tr from the residence time distribution for different values of L, the constants c and n can be determined by tting the experimental data to Eq. (1).

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Fig. 2. Conductivity proportional responses to an injection of KCl solution at the bioreactor inlet.

The ow rate in continuous operation was Q = 75 ml min1 and the recycle ratio r = 4. The reactor performance was checked by measuring phenol and TOC concentrations, pH and temperature at the reactor inlet and outlet. Two months later, a pseudo steady-state was achieved at all sampling points. Fig. 3 shows the evolution of the TOC and phenol removal efciencies (based on combined feed) across the trickling bed. After operating the reactor during 65 days, those efciencies reached 53 and 43%, respectively. If we consider the phenol and TOC concentrations in the feed stream, before the dilution in the recirculation stream, 90 and 117.5 mg l1 , respectively, the overall efciencies increase to 90% for phenol and 85% for TOC. In these conditions, phenol degradation rate is about 0.25 g l1 d1 , higher than the value obtained [30] in a stirred-tank bioreactor (0.2 g l1 d1 ). Nevertheless, it is lower than the values corresponding to packed and uidizedbed bioreactors (3.1 and 4.3 g l1 d1 , respectively). In

these last two reactor congurations, the carrier is immersed in the liquid phase, which explains the difference. The TOC removal due to the organic metabolites (TOCmetabolites ) produced during phenol biodegradation was calculated as the difference between measured TOC and TOC corresponding to phenol. The respective values at the reactor inlet after dilution can be observed in Fig. 4. It can be noticed that TOCmetabolites is 10 mg l1 , after 20 days operation. The TOCmetabolites at the reactor outlet and the consumption along the reactor were also calculated. With the exception of a 5 days initial period, there was no evidence for metabolite consumption along the reactor. 4.3. Biolm The biolm cells activity, expressed as the oxygen consumption rate due to the phenol oxidation, was calculated at the different sections of the trickling bed reactor. The

Fig. 3. Phenol and TOC removal efciencies during the start-up period.

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C.S.A. S , R.A.R. Boaventura / Biochemical Engineering Journal 9 (2001) 211219 a Table 3 Wet and dry biomass in the reactor Reactor section Upper Middle Lower Total Mwet biomass (g) 782.99 642.82 60.39 1486.2 Mdry biomass (g) 152.60 79.82 11.39 243.81 X (g TSS l1 ) 68.3 35.7 5.1 Water content (%) 81 88 81 84

Table 4 Average phenol concentration (Se ) as a function of the lter depth (D) Fig. 4. Time evolution of TOC due to the presence of metabolites at the reactor inlet. D (m) Flow rate (ml min1 ) 75 0.00 0.10 0.30 0.50 0.70 0.87 1.00 Se 36.21 33.08 27.44 22.74 19.73 15.58 12.42 (mg l1 ) 29.05 24.81 23.12 20.07 16.88 14.90 13.46 23.25 21.48 18.62 16.18 15.03 13.34 11.21 25.16 22.54 20.61 18.25 16.74 15.64 13.71 100 125 150

experimental results, obtained in duplicate, as well as the average values in each section are presented in Table 2. The biomass activity follows the phenol concentration prole along the reactor: a higher activity is observed at the upper section, decreasing along the column depth, in an approximately linear mode. The wet and dry biolm densities were calculated as the ratio between the amount of biomass (wet and dry basis) and the biolm volume. The biomass weight, expressed as total suspended solids (TSS), was determined by adding the values obtained in the three different sections mentioned before (Table 3) and the biolm volume was calculated by the expression Vwet biolm = Vlter bed Vparticles Vvoids The lter bed volume is 6700 cm3 . As the average particle volume is 7.24 cm3 and the reactor is packed with 451 particles, V particles = 3265 cm3 . Considering the experimentally determined bed porosity (30%), 64 days after the reactor start-up, V voids = 2010 cm3 . Then, biolm densities are wet biolm = 1.04 g cm3 and dry biolm = 170 mg cm3 , respectively. Dry and wet biolm densities obtained by Monteiro [31] using a uidized-bed bioreactor were 1.8571.3 mg cm3 and 11.2 g cm3 . The dry biolm density in this work is largely higher, which can be explained by the reactor conguration and the composition of the mineral medium. The volume of wet biomass is 3.295 cm3 per particle. Assuming that clean and biomass covered particles are

spherical and taking into account that the average volume of clean particles is 7.24 cm3 , the biolm thickness is, on average, = 1.6 mm. 4.4. Model parameters (K and n) for phenol removal Phenol concentrations (Se ) as a function of the column depth (D) for various ow rates are presented in Table 4. The inlet phenol concentration (So ) is the value corresponding to D = 0. As ln(Se /So ) varies linearly with the reactor depth (D), K/Ln may be determined as the linear tting slope for each L value. The obtained K/Ln slopes for the four hydraulic loading rates considered in this study are presented in Table 5. The linear tting of ln(K/Ln ) as a function of ln L led to n = 0.83, as shown in Fig. 5. This value is very close to 0.85, calculated from the reactor hydrodynamics. The parameter K was also determined by linear tting of ln(Se /So ) versus D/Ln , as presented in Fig. 6. The slope of the straight line is K = 9.8 m0.17 d0.83 .

Table 2 Biolm cells activity (expressed as O2 consumption rate referred to the homogenate volume) Upper section O2 consumption rate (mg l1 h1 ) (endogenous respiration) O2 consumption rate (mg l1 h1 ) (endogenous respiration + metabolism) O2 consumption rate (mg l1 h1 ) (metabolism) Average VSS (mg l1 ) O2 specic consumption rate (mg O2 mg1 VSS d1 ) Average O2 specic consumption rate (mg O2 mg1 VSS d1 ) 0.897 1.179 0.282 100 0.068 0.070 0.585 0.883 0.298 100 0.072 Middle section 0.242 0.368 0.126 62 0.049 0.050 0.328 0.461 0.133 62 0.051 Lower section 0.430 0.541 0.111 96 0.028 0.031 0.190 0.320 0.130 96 0.033

C.S.A. S , R.A.R. Boaventura / Biochemical Engineering Journal 9 (2001) 211219 a Table 5 Values of K/Ln as a function of L L (m3 m2 d1 ) 16.1 21.5 26.9 32.2 K/Ln 1.010 0.918 0.623 0.621

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model parameters K and n are close to those found for phenol removal: K = 10.0 m0.15 d0.85 and n = 0.85. A similar expression for TOC removal efciency may be written as TOC (25 C) = 1 Se 0.85 = 1 e10.0(D/L ) So

Average deviations between calculated and experimental TOC removal efciencies are in the range 13%. 4.6. Biokinetic constants As K = ckX, the specic biokinetic constants, kphenol (25 C) and kTOC (25 C), were determined from K and c, previously calculated, and the average biomass concentration, X = 36.4 g TSS l1 . The following values were obtained: kphenol (25 C) = 7.9 m3 kg1 TSS d1 and kTOC (25 C) = 8.1 m3 kg1 TSS d1 . So, biokinetic constants are Kphenol (25 C) = 287.6 d1 and KTOC (25 C) = 294.8 d1 . 4.7. Biological yield
Fig. 5. Linear regression for the determination of the model parameter n (phenol removal).

In these conditions, the phenol removal efciency at 25 C, is given by the expression phenol (25 C) = 1 Se 0.83 = 1 e9.8(D/L ) So

The calculated efciencies using the kinetic model are compared with the experimental values in the Fig. 7. As can be observed, a reasonable agreement was achieved, with the highest deviations occurring near the column inlet for L = 32.2 m3 m2 d1 . 4.5. Model parameters (K and n) for TOC removal The same procedure adopted for phenol was applied to the TOC experimental values. As could be expected, the

The biomass generated per day was calculated from the VSS concentration in the nal efuent and the sludge retained in the bottom of the secondary settling tank. In two 20 days operation periods, the excess of biomass eliminated from the reactor was 1.257 and 1.339 g VSS d1 , respectively, which corresponds to an average value of 1.298 g VSS d1 . In the same periods, the phenol removal in the reactor was 1.62 g d1 . Then, the average biological yield was 0.8 g biomass produced per g phenol removed. 4.8. Effect of temperature on phenol removal The phenol and TOC removal efciencies for three different temperatures are presented in Table 6. The high difference between phenol and TOC removal efciencies at 30 C is probably due to the higher production of metabolites at this temperature. This means an increase in the TOC content at the reactor outlet. Considering, as usually mentioned

Fig. 6. Linear regression for the determination of the model parameter K (phenol removal).

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Fig. 7. Phenol removal efciency along the reactor: comparison between experimental and model data.

Table 6 Temperature inuence on TOC and phenol removal efciency () Temperature ( C) Phenol (mg l1 ) Inlet 19 25 30 17.15 30.12 19.5 Outlet 10.76 14.48 4.81 37 52 75 phenol (%) TOC (mg l1 ) Inlet 33.42 27.38 28.67 Outlet 21.28 14.92 15.01 36 46 48 TOC (%)

in the literature, that the removal efciency, , varies with temperature, T, in accordance with the expression (1 /2 ) = (T1 T2 ) , the constant was determined from the experimental results and we got phenol = 1.067 and TOC = 1.025. Then, within the temperature range 1930 C, phenol and TOC removal efciencies can be adequately described by the expressions phenol (T ) = 1 Se So = (1 e9.8(D/L
0.83 )

5. Conclusions The average residence time, tr , in the trickling bed reactor is a function of the hydraulic loading rate, L, and the column depth, D. For the experimental conditions used in this study, tr is tr = 0.034D L0.85

1.067 TOC (T ) = 1 Se So

(T 25)

T
0.85 )

= (1 e10.0(D/L

) 1.025(T 25)

The variation of the biokinetic constants with temperature is given by Kphenol (T ) = Kphenol (25 C) 1.11(T 25) and KTOC (T ) = KTOC (25 C) 1.04(T 25) .

The main biolm characteristics are: percent water content = 84%, wet density = 1.04 g cm3 , dry density = 170 mg VSS cm3 , thickness = 1.6 mm and cells activity, expressed as O2 consumption mg VSS d1 , varying between 0.07 and 0.03, at the reactor top and bottom, respectively. Except for a 5 days initial period, no perceptible metabolite consumption is observed along the reactor. The phenol and TOC biokinetic constants at 25 C, assuming a pseudo rst-order kinetics, are Kphenol (25 C) = 287.6 d1 and KTOC (25 C) = 294.8 d1 . The calculated average biological yield during the pseudo steady-state operation period is 0.8 g of biomass produced per g of phenol removed.

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Acknowledgements The nancial support from FCT, the Portuguese Foundation for Science and Technology (research fellowship BTI/12041 and research project PEAM/SEL/522/95) is gratefully acknowledged. References
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