Ind. Eng. Chem. Res. 2007, 46, 6681-6687
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Biodegradation of Polyethoxylated Nonylphenols in Packed-Bed Biofilm Reactors Lorenzo Bertin,† Diana Di Gioia,† Claudia Barberio,‡ Laura Salvadori,‡ Leonardo Marchetti,† and Fabio Fava*,† Department of Applied Chemistry and Material Science, UniVersity of Bologna, Viale Risorgimento 2, 40136 Bologna, Italy, and Department of Animal Biology and Genetics, UniVersity of Firenze, Via Romana 17, 50125 Firenze, Italy
The opportunity to apply an immobilized-cell biotechnological process in the remediation of polyethoxylated nonylphenol (NPnEO) contaminated water was studied in this work. To this purpose, three identically configured aerobic column reactors packed with different materials (silica beads, granular activated carbon, or glass spheres) were developed, inoculated with the NPnEO-degrading Pseudomonas sp. strain BCb 12/3, and compared for their ability to biodegrade the two NPnEO industrial mixtures Igepal CO-520 and CO-210 fed in synthetic wastewater at concentrations in the range 30-90 mg/L. The three biofilm reactors, tested under batch conditions, showed comparable degradation capabilities and specificities, being able to remove from 77 to 99% of the total Igepal mixtures supplied after 9 days of batch treatment. NPnEOs were mostly removed through biodegradation, as suggested by the accumulation of two metabolites typical of NPnEO aerobic biodegradation, such as phenol and 4-nonylphenol, and by the low NPnEO amounts recovered from the reactors at the end of the study. Fluorescent in situ hybridization and DAPI staining performed at the end of the study showed that the bacterial biofilm was well and homogenously developed on the packed beds of the three reactors and that it was mostly composed by bacteria belonging to Gammaproteobacteria, i.e., the Proteobacteria class which includes the genus Pseudomonas. Introduction Ethoxylated nonylphenols (NPnEOs, where n is the number of ethoxy units in the molecule) are synthetic nonionic surfactants largely utilized for both domestic and industrial purposes because of their excellent deterging and emulsifying properties and low cost. NPnEOs have been reported as endocrine disrupters,1 and there is clear evidence of their toxicity toward aquatic organisms.2 NPnEO-contaminated wastewaters are usually sent to conventional activated sludge treatment plants, where they are partially degraded thus originating several bioconversion products, such as shorter ethoxy chain congeners (n ) 1, 2, or 3) and 4-nonylphenol (4-NP), which are discarded through the plant effluents.3,4 Thus, a tertiary treatment is often needed to reach the required effluent quality. Ozonation is one of the processes most employed for such a purpose.5 However, its high cost and low biocompatibility progressively limit its application on a large scale. Several physical-chemical alternatives have been proposed, such as a Fenton’s reagent pretreatment,6 NPnEO photodecomposition with TiO2 immobilized fiberglasses,7 activated clay adsorption processes,8 and NPnEO degradation by ultraviolet B irradiation,9 but none of these technologies has provided a definitive and environmentally safe solution to the problem. Biological treatments might represent an alternative costeffective and environmentally sustainable solution for removing nonionic surfactants and related compounds persisting in the effluents of conventional biological plants fed with NPnEOcontaminated wastewaters. Among the possible technologies proposed and applied in the wastewater treatment, those relying on specialized biomass passively immobilized on suitable organic or inorganic carriers inside packed-bed reactors are the * To whom 2093212. Fax: † University ‡ University
correspondence should be addressed. Tel.: +39 051 +39 051 2093220. E-mail:
[email protected]. of Bologna. of Firenze.
most promising ones,10-14 especially with pollutants displaying the same high recalcitrance and poor bioavailability exhibited by NPnEOs.15 Despite this, no attempts to apply packed-bed biofilm reactors (PBBRs) in the decontamination of NPnEOcontaminated waters have been reported so far in the literature. In recent studies, some aerobic bacterial cultures able to grow on and biodegrade NPnEOs have been isolated from an acclimated active sludge and characterized in our laboratory from a taxonomic point of view and for their degradation capabilities.16,17 The objective of this work was to explore the possibility of applying one of these cultures in the development of a biotechnological immobilized-cell process for the treatment of NPnEO-contaminated effluents resulting from wastewater activated sludge treatment plants. The strain Pseudomonas sp. BCb 12/3,16 showing particularly interesting degradation performances, was chosen as the biocatalyst and allowed to be immobilized in three identically configured aerobic PBBRs fed with water artificially contaminated with NPnEO and operating under batch conditions. Materials and Methods Chemicals. Culture media components, two industrial mixtures of NPnEOs, i.e., Igepal CO-520 and Igepal CO-210, and frosted glass spheres (GSs) used as immobilization supports (diameter 7 mm) were purchased from Sigma-Aldrich, Milan, Italy. GSs (diameter 8 mm) were frosted and washed with concentrated HNO3 before being used. Analytical-grade and HPLC-grade solvents were from Baker, Deventer, The Netherlands. Granular activated carbon (GAC) (CP4-60 product, consisting of cylinders of 3 mm diameter and 10 mm length) and silica beads (SBs) (Celite R-635 product, consisting of cylinders of about 5 mm diameter and 10 mm length) were kindly supplied by Chemviron Carbon (Feluy, Belgium) and World Minerals (Santa Barbara, CA), respectively. Microbial Culture and Media. The strain Pseudomonas sp. BCb 12/3 was isolated from the activated sludge of the
10.1021/ie061663d CCC: $37.00 © 2007 American Chemical Society Published on Web 03/30/2007
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Figure 1. Scheme of the batch PBBRs employed in this study.
Baciacavallo (Prato, Italy) treatment plant by using a commercial mixture of NPnEO having an average ethoxylation degree of 2 as the sole carbon and energy source.17 Minimum mineral medium (MMM) and Tryptic Soy Agar medium (TSA) prepared according to Fava et al.18 were used to grow the strain. Packed-Bed Biofilm Reactors. A schematic drawing of the three identically configured aerobic PBBRs developed in this study is shown in Figure 1. The reactors consisted of a glass column (diameter 5 cm; height 45 cm) having an empty volume of about 0.65 L with an external jacket in which water at 30 °C was continuously recycled. The inlet line for fresh medium and the line for supplying 0.22 µm filter sterilized air were at the bottom of the column, whereas the outlet line for the exhaust air was placed on a small reservoir located at the top of the reactor. An air trap, constituted by a 250-mL Erlenmeyer flask containing 200 mL of distilled water, was inserted in the exhaust air line in order to trap the NPnEOs eventually leaving the bioreactors through that line as a foam. The reactors were operated according to an upflow scheme. A recycle line continuously carried wastewater from the loop located at the top part of the reactors to the bottom part of them. A probe for dissolved oxygen (Model 97-08, ATI-Orion, Boston, MA) and a probe for pH (Model 81-04, ATI-Orion, Boston, MA) were placed on the same loop of the reactors. A feeding trap, consisting of a small glass column (internal volume 30 mL) packed with acid-washed glass beads (diameter 3 mm) on which preestablished amounts of NPnEO mixtures were injected and dispersed, was inserted in the recycle line of each reactor at the beginning of each experiment. The bioreactor systems were sterilized by recycling an aqueous ethanol solution (70% v/v) containing HCl (1% v/v) for 2 days, washed with sterile water, and then packed with 258.76, 296.6, or 756.32 g (dry weight) of SBs, GAC, or frosted GSs, respectively, previously sterilized in an autoclave (110 °C per 30 min). The capacities of the whole empty systems, the volumes of their packed beds, and the volumes of those occupied by the air along with the actual reactor working volumes (calculated by considering the medium displacement due to the supports and the supplied air) are reported in Table 1. Biofilm was generated in each reactor by recycling a high-density BCb 12/3 strain cell suspension for 2
weeks at the flow rates reported in Table 1, calculated so that the ratio between the actual working volumes and the recycling flow rates was 0.5 h for each reactor. Experiments of Bioremediation of NPnEO-Contaminated Waters. The ability of the Pseudomonas sp. BCb 12/3 biofilm to biodegrade NPnEOs was studied by introducing either Igepal CO-520, having an average ethoxylation degree of 5, or Igepal CO-210, having an average ethoxylation degree of 1.5, through the feeding trap in each bioreactor operating in batch mode. Four 9-day batch experiments, each performed in duplicate, were carried out. Three experiments were performed with Igepal CO520, which was supplied to the bioreactors in order to have it at an initial theoretical concentration of 30, 60, or 90 mg/L (i.e., assuming Igepal constituents to be completely soluble in the water phase); the forth experiment was carried out with Igepal CO-210 at the initial theoretical concentration of 60 mg/L, assuming it to be completely soluble in the water phase. Two milliliter samples of recycled medium were periodically taken through the sampling port and analyzed for the concentration of NPnEOs and their aromatic metabolites (via high-performance liquid chromatography-diode array detector, HPLCDAD). The same samples were also utilized to monitor bacterial cell concentration and purity through plate counting on MMM and TSA plates and through fluorescent in situ hybridization (FISH). At the end of each experiment, the reactor media along with the feeding and the air traps were removed and analyzed for NPnEO concentrations. The PBBRs were washed twice with sterile MMM and then refilled with the same medium. Before new surfactant was introduced in the reactors, the reactor media were analyzed by HPLC to verify the absence of NPnEO. The Igepal mixture removal percentages were calculated with respect to the amount introduced in the columns from the feeding traps according to the following equation:
Igepal removal % )
(Ii - Ir(ft)) - (Ir(rw) + Ir(at)) Ii - Ir(ft)
× 100
where Ii ) Igepal initially introduced in the feeding traps and Ir ) Igepal recovered at the end of the experiments from the feeding traps (Ir(ft)), the recycled waters (Ir(rw)), or the air traps (Ir(at)). 4-NP, also detected in the different system compartments at the end of each experiment, was included in the mass balances by calculating the amount of Igepal corresponding to the detected metabolite and then adding the obtained values to the recovered Igepal. To this aim, 4-NP detected amounts were multiplied by Igepal CO-520 or Igepal CO-210 molecular weights (calculated by considering the average ethoxylation degree of NPnEO composing the two mixtures) and divided by the 4-NP molecular weight. The Igepal concentrations thus obtained were corrected considering the recovery efficiency of the NPnEO extraction procedures, which were 80% and 75% from water phases (recycled broth and air trap) and solid phases (injection traps and supports), respectively. At the end of the last experiment, the three packed-bed reactors were opened and 3 g carrier samples were collected in triplicate at 5, 18, and 36 cm of height (from the bottom) of the columns and subjected to solvent extraction to determine the adsorbed NPnEO amounts. To this aim, the carriers were washed with water and then vigorously stirred and sonicated in 10 mL of diethyl ether, and the resulting organic phase was collected. The procedure was repeated and the organic phases were joined and evaporated, and the resulting solid phase was suspended in 2 mL of exhane and subjected to HPLC-DAD analyses. The amount of NPnEO recovered from the reactor packed beds was
Ind. Eng. Chem. Res., Vol. 46, No. 21, 2007 6683 Table 1. PBBR Volumes and Related Recycling Flow Rates
SB-PBBR GAC-PBBR GS-PBBR
empty system vol, mL
packed-bed vol, mL
air vol, mL
actual working vol, mL
recycling flow rate, mL/h
686 666 696
346 379 273
20 17 25
320 270 398
640 540 796
included in the mass balances performed on the last experiment with Igepal CO-210. Biofilm Characterization. Other 3 g samples of biofilmcovered carriers were collected at the end of the last experiment at 5, 18, and 36 cm of height (from the bottom) of the columns and analyzed for immobilized biomass content. To perform this analysis, the procedure reported by Bertin et al. ,13 based on protein concentration measurements, was applied. To observe biofilm establishment on the different carriers, samples of the supports were taken from the same three regions of the reactors and subjected to fluorescent microscopy observation after staining with the fluorescent dye 4′,6-diamidino-2-phenylindole (DAPI).17,19 To this purpose carriers, 20 for each sample, were suspended in 5 mL of 4% (v/v) paraformaldehyde (PF) and fixed according to Amann et al.20 The fixed samples were vortexed; 3 µL of this suspension was spotted on a glass slide where it was mixed with 1 µL of DAPI solution (1 µg/µL in doubledistilled water) and 5 µL of washing buffer.20 After 5 min incubation at room temperature, the glasses were washed with distilled water and air-dried. FISH was performed on samples of the recycled medium after its fixing in 4% (v/v) paraformaldehyde (PF) according to Salvadori et al.17 PF-fixed cells of Pseudomonas strain BCb 12/3 were used as a positive control in each hybridization experiment. The probes used were EUB33821 and GAM42a,22
capable of hybridizing with most bacteria and Gammaproteobacteria (Pseudomonas included), respectively. Hybridizations with probe GAM42a were performed in the presence of BET42a unlabeled competitor probe according to Manz et al.22 DAPI staining was performed on the same samples to evaluate the presence of cells not capable of hybridizing with the fluorescent probe. Analytical Methods. HPLC-DAD analyses were performed as described by Salvadori et al.17 Igepal concentrations were calculated as the sum of the concentrations of the single NPnEO mixture congeners. Both MMM agar plates containing Igepal CO-520 (100 mg/L) as the sole carbon source and TSA plates were employed for plate counting, to determine Pseudomonas sp. BCb 12/3 cell concentration and to evaluate possible contamination of the reactor, respectively. Results Biodegradation of Igepal CO-520 in the Bioreactors. The time course of the experiments performed with Igepal CO-520 fed with initial theoretical NPnEO concentrations of 30, 60, and 90 mg/L in the three packed-bed biofilm reactors are presented in Figure 2. The concentration of NPnEO detected in the recycle phase was well below the overall added amount in all three reactors and was in the range 3-8 mg/L (Figure 2A-a, B-a,
Figure 2. Concentration of Pseudomonas sp. strain BCb 12/3 (a) and Igepal CO-520 (b) as a function of the time in the SB-PBBR (4), GAC-PBBR (9), and GS-PBBR (O) during the experiment with Igepal CO-520 applied at the initial theoretical concentrations of 30 (A), 60 (B), and 90 mg/L (C). Each point represents the average of duplicate experiments.
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Table 2. Removal Percentages of Igepal CO-520 with Respect to the Amount of Overall Pollutants Transferred in the Reactors through the Feeding Traps at Initial Theoretical Igepal CO-520 Concentrations of 30 (A), 60 (B), and 90 mg/L (C)a recovered from added to feeding trap, mg
recycled water, mg
feeding trap, mg
air trap, mg
removal with respect to amt transferred in reactor, %
SB-PBBR GAC-PBBR GS-PBBR
10.7 9.00 13.3
(A) 0.63 ( 0.03 6.23 ( 0.09 0.00 ( 0.00 0.52 ( 0.02 6.74 ( 0.51 0.00 ( 0.00 0.78 ( 0.07 3.89 ( 0.40 0.00 ( 0.00
85.99 77.17 91.71
SB-PBBR GAC-PBBR GS-PBBR
21.3 18.0 26.6
(B) 1.31 ( 0.14 0.10 ( 0.01 0.09 ( 0.01 0.57 ( 0.07 0.12 ( 0.01 1.25 ( 0.15 1.56 ( 0.10 1.00 ( 0.08 0.01 ( 0.00
93.39 89.80 93.86
SB-PBBR GAC-PBBR GS-PBBR
32.0 27.0 39.8
(C) 1.18 ( 0.12 0.32 ( 0.09 0.48 ( 0.05 0.23 ( 0.02 0.20 ( 0.01 0.15 ( 0.03 1.31 ( 0.03 0.26 ( 0.04 1.69 ( 0.40
94.76 98.57 92.41
a Recovered amounts also include Igepal CO-520 theoretically converted into 4-NP. The results represent the average of two identical 9-day batch experiments.
and C-a), regardless of the Igepal amount injected into the feeding trap, but in agreement with the water solubility of NPnEOs.23 Two aromatic metabolites were detected in all reactors through the experiments; on the basis of their HPLC relative retention times and UV spectra they were characterized as phenol and 4-nonylphenol (4-NP). In particular, phenol transiently accumulated in all the PBBRs (maximum concentration achieved was 0.1 mg/L) during the first 4 days of the experiments. Conversely, 4-NP, which appeared after the second or third day of treatment in the presence of Igepal CO-520, was found to persist in all reactors by generally reaching concentrations in the range 0.1-0.2 mg/L at the end of the experiments. The mass balance of NPnEO fate in the three reactors is presented in Table 2. The final removal percentages were high in all PBBRs, and they were found to increase markedly with the mass of pollutants introduced in the reactors (Table 2). The concentration of freely suspended cells of Pseudomonas sp. BCb 12/3 available in the recycle medium, monitored through plate cell counts, was comparable and constant throughout time in the three reactors at all Igepal CO-520 concentrations evaluated (Figure 2A-b, B-b, and C-b). Colonies obtained on both TSA and MMM added with Igepal CO-520 were similar in number and generally had the morphology typical of Pseudomonas sp. strain BCb 12/3 (data not shown). FISH analyses performed on samples of the recycled medium showed that the majority of the cells hybridizing with EUB338 probe also hybridized with GAM42a probe; in addition, the same amount of cell was stained with the DAPI dye. As an example, the results obtained for the SB support in the experiment performed with 60 mg/L Igepal CO-520 are presented in Figure 4A. Biodegradation of Igepal CO-210 in the Bioreactors. The last experiment was performed in duplicate by feeding (through the trap) the three reactors with Igepal CO-210 at the theoretical concentration of 60 mg/L. Figure 3 shows that Igepal CO-210 concentration was below 2 mg/L in all reactors, in agreement with the extremely low water solubility of the low ethoxylated congeners composing this mixture.23 The amount of NPnEOs adsorbed on the supports was also determined on the same packed samples. NPnEO congeners recovered from these samples could be ascribed to Igepal CO210, and their overall amounts were 0.28, 2.03, and 1.15 mg in
Figure 3. Concentration of Pseudomonas sp. strain BCb 12/3 (a) and Igepal CO-210 (b) as a function of time in the SB-PBBR (4), GAC-PBBR (9), and GS-PBBR (O) during the experiment with Igepal CO-210 applied at the initial theoretical concentration of 60 mg/L. Each point represents the average of duplicate experiments.
the SB, GAC, and GS reactors, respectively. The mass balance of the experiment performed with Igepal CO-210 was redetermined on the basis of these amounts (Table 3). The results evidenced that the most efficient reactor, in terms of Igepal CO210 removed through biodegradation, was the SB one. The same aromatic metabolites already evidenced in the Igepal CO-520 experiments were detected. Phenol concentration was below 0.1 mg/L in all reactors throughout the 9-day experiments, whereas 4-NP reached a maximum of 0.2 mg/L after 5 days in the GAC reactor (data not shown). Concentrations of 4-NP around 0.15 mg/L were present in all reactors at the end of the experiments. Pseudomonas sp. BCb 12/3 concentration was constant in all three reactors throughout the experiment. The results of in situ hybridization performed on the recycle medium samples were similar to those obtained for Igepal CO-520 (data not shown). Biofilm Features. The total biomass immobilized on SB, GAC, and GS carrier samples collected from the top, the middle, and the bottom of the related packed beds at the end of the study was (expressed as mg of dried biomass/g of dried support) 0.023, 0.030, and 0.026; 0.045, 0.047, and 0.042; and 0.025, 0.028, and 0.037, respectively. Considering the dry weight of the support which constituted the SB, GAC, and GS packed beds (258.76, 296.60, and 756.32 g, respectively) and the average immobilized dried biomass recovered from each reactor, the total immobilized biomass available in the SB-, GAC-, and GS-PBBRs was 6.73, 13.35, and 26.47 mg, respectively (on dry weight basis). Such values were compared to the amounts of biomass freely suspended in the reactor media at the end of the last experiment, which were 0.09, 0.14, and 0.32 mg in the SB-, GAC-, and GS-PBBRs, respectively (on dry weight bases). Therefore, the percentage of the freely suspended biomass was almost 1% with respect to the total one in all the reactors. DAPI staining performed on the supports recovered at the three levels of the reactors showed that almost all cells were stained, thus indicating that they were highly viable, and that bacteria were extensively distributed on the immobilization supports. The
Ind. Eng. Chem. Res., Vol. 46, No. 21, 2007 6685 Table 3. Percentages of Removal and Biodegradation of Igepal CO-210 in the Three Reactors at Initial Theoretical Concentration of 60 mg/La
SB-PBBR GAC-PBBR GS-PBBR a
added to feeding trap, mg
recycled water, mg
21.3 18.0 26.6
0.13 ( 0.03 0.07 ( 0.01 0.03 ( 0.00
recovered from feeding trap, mg 1.00 ( 0.21 0.02 ( 0.00 2.42 ( 0.42
air trap, mg
removal with respect to amt transferred in reactors, %
adsorbed on immobilization supports, mg
biodegraded, %
0.40 ( 0.03 0.29 ( 0.04 0.43 ( 0.02
97.40 97.98 98.10
0.28 ( 0.02 2.03 ( 0.11 1.15 ( 0.10
96.02 86.70 93.35
Recovered amounts also include Igepal CO-520 converted into 4-NP. The results represent the average of two identical 9-day batch experiments.
Figure 4. FISH analysis and DAPI staining. (A) SB recycled medium: the same microscopic field showing, from top to bottom, cells hybridizing with EUB338 probe, with GAM42a probe, stained with DAPI. (B) Biofilm fragments detached from SB carriers taken from top to bottom of the bioreactor (shown in the same direction), stained with DAPI. The bar in each picture is equivalent to 10 µm.
dimension of the immobilized cell aggregates detached from the bottom and medium sections were similar in size, whereas those detached from the top section were of smaller size. As an example, fluorescent microscope pictures taken from samples withdrawn from the SB reactor are shown in Figure 4B. Discussion NPnEOs are toxic surfactants widely distributed in the environment, in which they are mainly introduced through the effluents or sludges of wastewater treatment plants fed with nonionic surfactant contaminated industrial and domestic wastewaters.2 Such pollutants can be removed from the effluents by using specialized microrganisms in a tailored bioreactor system. This remediation strategy would be much more sustainable than the chemical alternatives currently applied on the large scale. Thus, in this research, a preliminary study on the feasibility of a biological NPnEO posttreatment in a dedicated immobilizedcell packed-bed bioreactor system was performed at the laboratory scale. To this aim, three identically configured aerobic PBBRs packed with different cell immobilization carriers were developed and compared for their ability to biodegrade two of
the most used mixtures of NPnEOs under batch conditions. Immobilized-cell technology was chosen as it is generally able to offer important advantages, such as high stability, robustness, and biodegradation performances generally higher than those displayed by conventional freely suspended cell bioreactors.10-14 Different cell immobilization support materials were chosen as it is known that their nature and structure can have a marked influence on the final activity of the biofilm and therefore on the efficiency of the treatment.18 Very high and comparable NPnEO removal efficiency were displayed by the three reactor systems fed with the Igepal mixtures (Table 2). In general, over 75% of the pollutants introduced in the PBBRs were removed after 9 days of batch treatment. The performances increased proportionally with the mass of pollutants introduced in the reactors and lowering the average number of ethoxylated residues (Table 2). Considering the data obtained after the last experiment, where the amount of NPnEO adsorbed on the carriers was also measured, only a low amount of overall pollutants (ranging from 1 to 10%) was physically adsorbed onto the biofilm carriers (Table 3). The majority of NPnEO introduced in the reactors was therefore
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depleted mostly through microbial degradation processes as supported by the following findings: (i) the persistence of high concentrations of NPnEO biodegrading dispersed cells in the recycled lines of the reactors (Figures 2 and 3), (ii) the occurrence of relevant amounts of immobilized specialized biomass in the reactor packed beds at the end of the experiments, and (iii) the generation of small but detectable amounts of NPnEO degradation metabolites such as phenol and 4-NP in all reactors along the experiments. The accumulation of 4-NP in the reactors indicated that NPnEOs were biodegraded through one of the most common pathways,24,25 i.e., by shortening of the ethoxylic chain. 4-NP was probably further metabolized through biodegradation of the alkylic chain, giving rise to the few milligrams per liter phenol that was detected. Phenol is not a common metabolite of NPnEO biodegradation, but there are reports in the literature that describe the release of the alkylic chain from the aromatic ring of 4-NP giving rise to the corresponding nonylic alcohol.26,27 A similar degradation mechanism can give rise to phenol, but this hypothesis needs to be confirmed. Liquid batch cultures of Pseudomonas sp. strain BCb 12/316 displayed lower NPnEO biodegradation performances with respect to those observed by BCb 12/3 biofilm, thus evidencing that strain biodegradation potential was enhanced upon immobilization. Significant increases in the degradation capabilities upon immobilization have been observed for a number of different microorganisms and substrates.10-14,18 A higher NPnEO bioavailability, due to the efficient pollutant supply system used and to an enhanced pollutant molecular diffusion determined by the supports, as well as a higher biomass availability, may have been responsible for the enhanced pollutant degradation observed in the bioreactors. DAPI staining analyses showed that biofilm was well established and extensively distributed on the three supports (Figure 4) and bacteria, either immobilized or on the recycle medium, were viable. In addition, the positive response of the cells present on recycle samples to FISH, which is a technique working efficiently with cells having a high protein synthesis activity, indicated that cells were viable and metabolically active. These findings indicate the suitability of the utilized strain for the packed-bed technology developed in this study. Despite the significant differences occurring between the three PBBRs in terms of content of immobilized biomass and ability to physically adsorb NPnEOs, all PBBRs exhibited a comparable bioremediation potential and microbial stability and also the same NPnEO biodegradation pathway (Tables 2 and 3, Figures 2 and 3). These findings, taken together, are a strong indication of the feasibility and real potential of the biofilm technology developed in this study in the bioremediation of NPnEOcontaminated wastewaters. In conclusion, Pseudomonas sp. strain BCb 12/3 is capable of an effective NPnEO biodegradation in the physiological conditions of immobilized cells. GSs, GCA, and SBs were suitable to produce stable and efficient biofilm reactor systems. To the best of our knowledge, this is the first report in which a similar technology was proposed in the bioremediation of Igepal-contaminated wastewaters. We believe that this study provides experimental evidence of the real possibility of using BCb 12/3 strain as immobilized cells in a fixed bed bioreactor system for the continuous treatment of NPnEO-contaminated wastewaters. Further studies will be aimed at evaluating the PBBR potential on real NPnEO contaminated effluents. Acknowledgment The authors wish to thank GIDA S.p.A., Prato, Italy, for partially funding the project.
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ReceiVed for reView December 22, 2006 ReVised manuscript receiVed February 23, 2007 Accepted February 26, 2007 IE061663D
