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ARTICLE Elimination of Hydrophobic Volatile Organic Compounds in Fungal Biofilters: Reducing Start-Up Time using Different Carbon Sources Alberto Vergara-Fernańdez,1,2 Sergio Hernańdez,1 Sergio Revah3 1

Departamento de Ingenierıá de Procesos e Hidraúlica (IPH), Universidad Autońoma Metropolitana-Iztapalapa, Me´xico DF, Mexico 2 Escuela de Ingenierıá Ambiental, Facultad de Ingenierıá, Universidad Cato´lica de Temuco, Temuco, Chile 3 Departamento de Procesos y Tecnologıá, Universidad Autońoma Metropolitana-Cuajimalpa, Artificios 40, Col. Miguel Hidalgo, Delegacioń A´lvaro Obregoń, Mexico; telephone: þ52-55-2636-3801; fax: þ52-55-2636-3800 (ext. 3832); e-mail: [email protected] Received 5 August 2010; revision received 24 October 2010; accepted 26 October 2010 Published online 12 November 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bit.23003

ABSTRACT: Fungal biofilters have been recently studied as an alternative to the bacterial systems for the elimination of hydrophobic volatile organic compounds (VOC). Fungi foster reduced transport limitation of hydrophobic VOCs due to their hydrophobic surface and extended gas exchange area associated to the hyphal growth. Nevertheless, one of their principal drawbacks is their slow growth, which is critical in the start-up of fungal biofilters. This work compares the use of different carbon sources (glycerol, 1-hexanol, wheat bran, and n-hexane) to reduce the start-up period and sustain high n-hexane elimination capacities (EC) in biofilters inoculated with Fusarium solani. Four parallel experiments were performed with the different media and the EC, the n-hexane partition coefficient, the biomass production and the specific consumption rate were evaluated. Biofilters were operated with a residence time of 1.3 min and an inlet n-hexane load of 325 g m3reactor h1. The time to attain maximum EC once gaseous n-hexane was fed was reduced in the three experiments with alternate substrates, as compared to the 36 days needed with the control where only n-hexane was added. The shortest adaptation period was 7 days when wheat bran was initially used obtaining a maximum EC of 160 g m3reactor h1 and a critical load of 55 g m3reactor h1. The results were also consistent with the pressure drop, the amount of biomass produced and its affinity for the gaseous n-hexane, as represented by its partition coefficient. Biotechnol. Bioeng. 2011;108: 758–765. ß 2010 Wiley Periodicals, Inc.

Correspondence to: S. Revah Contract grant sponsor: Direccioń de Investigacioń de la Universidad Cato´lica de Temuco, Chile Contract grant sponsor: Conacyt (Mexican Council for Science and Technology) Project Contract grant number: SEMARNAT-00120

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Biotechnology and Bioengineering, Vol. 108, No. 4, April, 2011

KEYWORDS: fungal biofilter; fusarium solani; n-hexane; biofiltration; biofilter start-up

Introduction Biological technologies for air pollution control are environmentally sound and economic alternatives and are being increasingly used in industry. Biofiltration involves passing waste air through a packed reactor containing active microorganisms capable of degrading pollutants (Shareefdeen and Singh, 2005). In general, biofilters achieve the highest rates of removal when compounds that are treated are water soluble and biodegradable (Deshusses and Johnson, 2000; Miller and Allen, 2004). The low solubility of hydrophobic molecules in the aqueous biofilm is one of the major problems for their treatment in biofilters. However, this obstacle may be reduced using fungi as the biological agent (Davison et al., 2000; Kennes and Veiga, 2004; Vergara-Fernańdez et al., 2006). Fungi have several advantages for the abatement of hydrophobic volatile organic compounds (VOC) in gas phase biofilters including their ability to degrade a large number of VOCs (Qi et al., 2002), their resistance to low humidity and pH, their capacity to colonize unoccupied space with the aerial hyphae and to penetrate the solid support increasing the availability of nutrients. On the other hand, they grow slower than bacteria, thus requiring longer times for the start-up, their filamentous growth promotes increased pressure drop and under some conditions may produce spores that could present some health hazard if not contained (Prenafeta-Boldu´ et al., 2006). ß 2010 Wiley Periodicals, Inc.

One of the first studies using fungi for the treatment of VOCs in biofilters was done by Cox et al. (1996) who reported an elimination capacity (EC) of 79 g m3reactor h1 for the gaseous styrene degradation with the fungus Exophiala jeanselmei. Garcıá-Penã et al. (2001) studied the effect of humidity and the compaction of the bed on the performance of the biofilter and its EC using the fungus Paecilomyces variotii (formerly Scedosporium apiospermun TB1), for toluene elimination with a mixture of vermiculite and activated carbon as support. Garcıá-Penã et al. (2001) observed EC close to 260 g m3reactor h1 with removal efficiency (RE) of 98%, within 30 days of start-up. Woertz et al. (2001) also used a fungal biofilter for toluene elimination, which was used as a carbon source and energy for the aerobic elimination of nitric oxide in the gas phase, obtaining RE of 93% with inlet concentration of 250 ppmv of nitric oxide, and a residence time of 1 min for a toluene load of 90 g m3reactor h1. The EC observed for toluene in steady state was 270 g m3reactor h1, after 50 days of operation. Aizpuru et al. (2005) reported a toluene EC of 290 g m3reactor h1, reached after a start-up period of 30 days, in a biofilter packed with porous ceramic Rasching rings inoculated with the fungus Paecilomyces variotii. Although a dense mycelium was formed, pressure drop was limited by the open and rigid structure of the ceramic rings. For a-pinene, Jin et al. (2007), reported EC higher than 100 g m3reactor h1 with a fungus (Ophiostoma sp.), these values are higher than those found with bacterial biofilters. For this system, the fungus required several weeks to colonize the biofilter and attain its steady conditions. Spigno and De Faveri (2005) obtained an n-hexane average EC of 150 g m3reactor h1 after 50 days of operation in steady-state conditions with a fungal biofilter inoculated with Aspergillus niger. Arriaga and Revah (2005a,b) obtained n-hexane ECs around 150 g m3reactor h1 in a fungal biofilter inoculated with Fusarium solani, using perlite as support. In both works the steady state was reached in over 30 days. In general, start-up takes long time in fungal biofilters as these microorganisms have relatively slow growth rates which is further reduced by the low bioavailability of the substrate due to their relatively low concentrations in air and to their limited solubility in the aqueous biological active phase. The slow attainment of the steady-state condition implies that the biofilter has to be operated with low efficiencies during this period. In this work, the use of three different carbon sources was studied to increase the initial development of fungal biomass to generate a shorter startup and an increased EC in a biofilter inoculated with Fusarium solani for gaseous n-hexane degradation.

Materials and Methods Microorganisms and Inoculum Fusarium sp. was isolated as described by Arriaga and Revah (2005a) and was classified as Fusarium solani CBS 117476

by the Centraalbureau voor Schimmelcultures, The Netherlands. Its preservation, cultivation conditions and spore production was realized according to Garcıá-Penã et al. (2001). The biofilter was inoculated with a mineral medium solution containing 2 107 spores mL1.

Carbon Sources and Mineral Medium The substrates used were: glycerol (Baker, 99.8%), 1-hexanol (Merck, >98%), wheat bran, and n-hexane (Baker, 98.5%). The mineral medium used in the biofiltration experiments was reported previously by Arriaga and Revah (2005a) had an initial pH of 4.0.

Biofilter Systems The gas phase biofilters were similar to those reported previously by Vergara-Fernańdez et al. (2008) and consisted of a 1 m cylindrical glass column with an inner diameter of 0.07 m (Fig. 1). They were packed with 250 g of dry perlite (2.4 L, bed void fraction of 68% corresponding to void reactor volume of 1.63 L and particle size between 3.4 and 4.8 mm) mixed with the mineral medium, carbon sources (glycerol, 1-hexanol, or wheat bran), and the spore suspension. The biofilters were incubated at 30 (3) 8C. Hexane-saturated air was mixed with moistened air (using a humidification column) and introduced at the top of the biofilter with a flow rate of 1.2 L min1 (hydraulic retention time, HRT of 1.3 min) corresponding to a superficial velocity of 27 m h1. The n-hexane inlet concentration was 7.4 g m3 corresponding to an inlet load of 325 g m3 1 reactor h . A mixture of the generated leachate and water were sprayed periodically to control moisture during the time of operation. In all experiments, chloramphenicol (20 g m3) was added to the mineral medium to limit bacterial growth. Previous studies reported the use of chloramphenicol as an effective strategy for controlling the bacterial population in biofilters (Garcıá-Penã et al., 2001). The predominance of the fungus was confirmed by microscopic observations at the end of each experiment. Pressure drop was measured online with a pressure transducer. Four start-up and growth conditions were evaluated in the biofilters. In the control experiment biofilter B1, the fungus was grown with gaseous n-hexane as only carbon source (Vergara-Fernańdez et al., 2008), in B2 it was initially grown with glycerol (10 g L1 in the liquid medium), in B3 with 1-hexanol (10 g L1 in the liquid medium), and in B4 with a mixture of wheat bran (30% w/w) and perlite. Given the nature of wheat bran it was not possible to establish equivalence with other carbon sources. In experiments B2, B3, and B4, n-hexane feed was initiated when the fungus had entered the stationary phase as seen by the CO2 production. This strategy was followed after preliminary tests showed that the presence of a more available substrate inhibited the uptake of n-hexane (Vergara-Fernańdez et al. (2006). To

Vergara-Fernańdez et al.: Fungal Biofiltration of Hydrophobic VOCs Biotechnology and Bioengineering

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Figure 1.

Schematic diagram of the laboratory-scale biofilter.

evaluate the influence of n-hexane load, different concentrations of n-hexane between 0.5 and 12 g m3 were tested, maintaining for 3 days a constant gas flow rate. Results from the biofiltration experiments are expressed in terms of n-hexane inlet load (L, g m3reactor h1), biofilter EC (g m3reactor h1), and RE (%RE) according to: L¼

EC ¼

Q Sin Vr

(1)

Q ðSin -Sout Þ Vr

(2)

Sin -Sout 100 Sin

(3)

%RE ¼

where, Sin and Sout are inlet and outlet n-hexane concentration, respectively (g m3), Q is air flow (m3 h1), and Vr is the void reactor volume (m3 reactor).

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Batch Experiments Glass bottles of 125 mL (microcosms), stoppered with Mininert Teflon valves (VICI Precision Sampling, Baton Rouge, LA), were used to determine the consumption rate of n-hexane. Five grams of the biofilter medium were added to the bottles with 20 mL of mineral medium, then 3 mL n-hexane were injected to obtain an initial headspace concentration of approximately 15 g m3. In all experiments, a bacterial inhibitor (20 mg L1 chloramphenicol) was included in the mineral medium. Headspace n-hexane consumption was followed until exhaustion. The maximum specific 1 n-hexane consumption rate, Vmax, (mghexane g1 biomass h ) was obtained by dividing the maximum volumetric rates, calculated with the integrated Gompertz model (Acunã et al., 1999), by the initial biomass present in the sample. Hexane partition coefficient (HPC), defined as the mass of hexane adsorbed per mass of biomass, (ghexane g1biomass), was evaluated in microcosms with samples drawn from each biofilter. HPC determination was performed using the

whole biomass sample including the support as described previously by Vergara-Fernańdez et al. (2006). The cellular yield was evaluated in 500 mL microcosms stoppered with Mininert valves. In each bottle 80 mL of mineral medium and carbon source to a concentration of 1 g L1 (glycerol and 1-hexanol) were added. The final biomass was evaluated by dry weight. Glycerol and 1-hexanol consumption was determined by CO2 production.

Analytical Methods Hexane concentration was measured with FID-GC and CO2 production by TCD-GC, according to Arriaga and Revah (2005a). The fungal biomass in the perlite was measured as volatile solids with a thermogravimetric analyzer according to Arriaga and Revah (2005b), except for biofilter B4. Humidity of the biofilter medium and the biomass concentration in liquid medium was estimated by dry weight methods. Measurements were done in triplicate. Elemental analysis was determined in samples of Fusarium solani grown on 1-hexanol (liquid medium) to evaluate the cellular yield. The fungus was found to contain 48.01 (0.88)% carbon and 6.47 (0.16)% nitrogen using an elemental analyzer (CHNS/O Analyzer, Series II, 2400 Perkin Elmer, Norwak, CT) calibrated with standard acetanilide.

Results and Discussion Biofiltration Experiments Biofilter start-up and adaptation to gaseous n-hexane was studied using different carbon sources. Glycerol was selected because it is an easily degradable substrate, 1-hexanol, being the first product of n-hexane oxidation, has shown to have similar response than n-hexane (Vergara-Fernańdez et al., 2006). Finally, wheat bran has been often used for the solid cultivation of fungi and promotes abundant growth and the formation of diverse enzymes. Figure 2 shows the EC, the CO2 production rate, and pressure drop (Dp) evolution in the four experiments tested in this study. In the control experiment (B1; Fig. 2A), F. solani spores germinated and grew in mycelia form with gaseous n-hexane as the only carbon and energy source. Three different periods were observed, the first period (Period A), corresponds to the spores’ germination and the biofilter start-up. A second period (Period B) between days 6 and 35, corresponding to growth phase where n-hexane degradation was associated with CO2 production and the occupation of the void space by mycelia promotes an increase in pressure drop (25 mmH2O). Finally, the steady state (Period C) was attained between days 36 and 50, with a maximum and stable EC (225 g m3reactor h1). After the total operation time of 50 days, Table I, it was found that 5,280 g m3reactor

of n-hexane were consumed and 11,571 g CO2 m3reactor produced; corresponding to 71.25% mineralization. As shown in Table II, the average produced biomass was 101 mgbiomass g1dry perlite. In B2 (Fig. 2B), glycerol (10 g L1), was utilized for biofilter start-up in the first 11 days of biofilter operation (Period A). In this time, the experimentally measured total CO2 produced was around 6.3 g which corresponds to 81% of the theoretical value assuming complete oxidation. Thereafter, n-hexane was fed (Period B), and the biofilter was operated for 38 days, consuming 4,202 g m3reactor of nhexane with a 72.3% CO2 recovery. The steady state (Period C), was attained 24 days after the onset of n-hexane addition with an average EC of 200 g m3reactor h1 (Table I). This period was maintained for 14 days and showed a slow increase in EC. After 50 days the operation the final average biomass obtained was around 60 mgbiomass g1dry perlite (Table II). In experiment B3 (Fig. 2C), 1-hexanol initially at 10 g L1, was consumed in 22 days with relevant CO2 production due to its reduced state (2.6 g CO2 g1 1-hexanol as compared to 1.4 g CO2 g1 glycerol considering complete oxidation). After n-hexane feed was initiated on the 23rd day, 3,281 g m3reactor were consumed with 70.21% CO2 as seen in Table I. Figure 2 also shows that despite that initial growth on the auxiliary substrate was slower than glycerol as seen by CO2 production, a shorter adaptation to n-hexane biodegradation was observed and steady uptake was reached 14 days after n-hexane addition was initiated. This result can be related to the fact that 1-hexanol is the first product of the n-hexane degradation pathway, reducing the time required for the enzymatic induction. Although biofilters B1, B2, and B3 presented differences in adaptation to n-hexane degradation, the steady state of each biofilter was obtained at around of 35 days of operation. The adaptation time and the average ECs of 220 (30.9) g m3reactor h1 in different biofilters were similar to reported by Arriaga and Revah (2005a) and Spigno et al. (2003), both using malt extract for accelerate the start-up in a fungal biofilters for n-hexane elimination. In B4, wheat bran was mixed with the packing material, which resulted in an important CO2 production on the 5th day (590 greactor m3 h1; Fig. 2D). Gaseous n-hexane addition was started on the 10th day and steady consumption was attained 7 days later at about the same time that the maximum pressure drop. During the 16 days that the biofilter operated only with n-hexane as a carbon source, period B, 1,981 g m3reactor of n-hexane were transformed to CO2 with a 71.4% yield. In period C, an average EC of 150 g m3reactor h1 was maintained but CO2 production continued to increase probably due to the consumption of more recalcitrant bran components such as cellulose, hemicelluloses, etc. Experiment B4, presented the faster start-up of all experiments, but eventually attained the lowest EC of the four biofilters (150 g m3reactor h1). Despite the lower EC value in B4, it was similar to that reported by Kibazohi et al. (2004), using a peat–perlite


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Figure 2. Evolution of the EC of n-hexane (&), CO2 production (*), and pressure drop (~) over time, in four biofilters inoculated with Fusarium solani and an n-hexane inlet load of 325 (g m3reactor h1). Biofilter started with: (A) n-hexane (Biofilter 1); (B) glycerol and n-hexane (Biofilter 2); (C) 1-hexanol and n-hexane (Biofilter 3); (D) wheat bran and n-hexane (Biofilter 4).

mixture for the n-hexane elimination and sludge of a wastewater treatment as biological agent. Pressure drop, DP, is related to the reduction in the bed permeability associated to the fungal growth (Auria et al., 1995). DP increase in the initial period seems to be related to

the growth rate, represented by CO2 production, which was glycerol > 1-hexanol > n-hexane. In B4, CO2 production was higher from the complex substrates found in bran, which include easily assimilated carbohydrate substrates, but the DP evolution may be influenced by factors that

Table I. Biofilters operated only with n-hexane as carbon source and energy, operation time, total n-hexane consumption, total CO2 produced, EC average in the steady sate, and % mineralization reported as % n-hexane recovered as CO2.

Exp

Time to attain steady hexane uptake (days)a

Operation (days)b

Total consumed n-hexane (g m3reactor)

Total produced CO2 (g m3reactor)

Avg. EC in steady state (g m3reactor h1)

n-Hexane recovered as CO2 (%)c

B1 B2 B3 B4

36 24 14 7

49 38 23 15

5,280 4,202 3,281 1,981

1,1571 9,327 7,594 4,344

230 200 225 150

71.25 72.30 70.21 71.42

a

Days after gaseous hexane addition were started. Only n-hexane as carbon and energy source. c Considering theoretical CO2 production. b

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Table II. Biomass, n-hexane partition coefficient (HPC), maximum specific n-hexane consumption rate (Vmax), from biomass samples taken at the end of the experiment in the four biofilters. Parameters a

Biomass

HPCb Specific Vmaxc

Module

Biofilter B1

Biofilter B2

Biofilter B3

Biofilter B4

S1 (inlet) S2 S3 Average

130 5.2 98 2.4 74 1.9 101 29 0.31 0.01 4.3 0.21

81 3.8 53 1.9 47 0.9 60.3 20 0.63 0.04 3.7 0.16

104 4.3 82 3.5 69 1.2 85 19 0.35 0.09 3.9 0.11

— — — — 0.56 0.08 —

Final biomass (mgbiomass g1dry perlite). Average in the biofilter (ghexane g1biomass). c 1 According to Gompertz model (mghexane g1 biomass h ). Biofilter B1 (50 days operation), biofilters B2 (50 days operation), biofilters B3 (47 days operation), and biofilters B4 (27 days operation). a

b

include variations in bed permeability by insoluble substrate uptake or by increased water retention and swelling by the bran polymers (cellulose, hemicelluloses, etc). The difference between B1 and B4, corresponding to 43 and 55 mmH2O m1 of bed respectively could be possibly explained by the greater water retention capacity and swelling of the wheat bran utilized in the biofilter B4. Finally, the final DP obtained in B2 and B3 were similar to B1. In general, the final DP obtained in all biofilters were higher than the 12 mmH2O m1 bed previously reported by Kibazohi et al. (2004) for n-hexane elimination, using a microbial consortium, for a similar superficial velocity. However, these DP were similar to the 50 mmH2O m1 bed values reported by Arriaga and Revah (2005a) for a fungal biofilter. These results verify that the DP in fungal biofilters are higher than those observed in bacterial biofilter. At the end of the biofiltration experiments, the reactors were dismounted and samples of the packing material were taken from each module and were utilized for further characterization. From these samples, it was found that the water content was homogeneous throughout all the biofilters, obtaining average values of 52 (3)% in all cases. These values have been also reported to be favorable for fungal growth according to Cox et al. (1996). Table II shows the biomass profiles in the biofilter B1, B2, and B3 throughout the reactors. B4 was not evaluated as residual organic material the wheat bran interferes with the method used. In all cases, a decreasing biomass density was found from the inlet module to the gas outlet, obtaining approximately 40% less at the outlet. These profiles can be related with the diminishing concentration due to n-hexane consumption which affects both transfer and reaction rates. By comparison, biofilter B2 showed 29% and 40% less biomass with respect to B3 and B1, respectively. These differences can be attributed to the carbon source utilized for the start-up in each biofilter and the cellular yield (YX/S) for each carbon source. For 1-hexanol (C6H14O; 70.6% C and YX/S ¼ 0.82) the results were similar to the n-hexane, but 40% higher than with glycerol (C3H8O3; 34.6% C and YX/ S ¼ 0.49). The results were comparable to similar experiments reported previously by Arriaga and Revah (2005b). Hexane mineralization, once the initial substrate was used, was similar in B1, B2, and B3 but higher in B4 probably due

to the extra CO2 produced by bran components that were more slowly used. The HPC, results obtained with the final biomass, Table II showed that it diminished when the fungus was initially grown with a more hydrophobic carbon source. Lower HPC indicates improved n-hexane solubility in the biomass and thus its availability for both growth and mineralization. The values were consistent to those reported earlier by VergaraFernańdez et al. (2006), with in vitro systems. Furthermore, according to the report by Vergara-Fernańdez et al. (2006), the HPC obtained for F. solani grown on glycerol and 1-hexanol decreased after 1-week exposure to n-hexane vapors. When comparing the results of HPC (Table II) and those reported by Vergara-Fernańdez et al. (2006), it is possible to establish that important changes in the HPC do not occur when the fungus was exposed to n-hexane vapors for periods longer than 1 week. These results suggest that the better adaptation to n-hexane biodegradation observed in B3 agreed with the final HPC obtained (0.35 ghexane g1biomass), which was similar for B1, whereas in B2 was around 50% higher. The results indicate that the final EC in the biofilters was related to the corresponding HPC, however, it did not have a direct influence in the startup where alternative substrates may be used. The HPC values obtained in the steady state in the biofilters, with the fungus grown in glycerol and 1-hexanol and later adapted to n-hexane were similar to those previously obtained by Vergara-Fernańdez et al. (2006) in in vitro systems.

Microcosms Experiments Table II shows that the specific Vmax values for B1, B2, and B3 are within a 10% difference reflecting that the biomass adapts to the incoming n-hexane despite the initial substrate used. These values are in the range reported by Arriaga and Revah (2005b) for similar conditions. The results from the microcosm experiments using humid samples from B4 were consistently lower (around 40%) than the results with the other biofilters despite the fact that abundant biomass was observed, actual Vmax values were not calculated as residual biomass from the bran interferes with the protein determination. Possibly the initial bran, which has been


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used extensively in solid-state fermentations, promoted the colonization of other non n-hexane users fungi that competed in space and nutrients with F. solani reducing the volumetric uptake rates. Influence of Inlet Load on the EC Figure 3 shows the influence of n-hexane inlet load on the performance of the different biofilters. These experiments were performed after that steady state in the biofilters was attained. In the control biofilter, B1, 100% RE was obtained up to a n-hexane critical load of around 100 g m3reactor h1, with a maximum EC of 225 g m3reactor h1. The maximum EC and critical load obtained in B1 were approximately 38% and 26% greater than the reports by Arriaga and Revah (2005a) using F. solani and Spigno et al. (2003) using A. niger, respectively. On the other hand, lower critical loads are observed in the other reactors. These results may be attributed to the lower biomass content and possibly to

heterogeneous distribution within the reactor which may cause channeling effects.

Conclusion It was feasible to reduce the start-up time of a fungal biofilter for the elimination of hydrophobic VOC by using more available carbon sources as germination and growth promoters. This strategy would allow increasing the fungal biomass before the biofilter is connected to the polluted stream thus reducing the time the biofilter is operated with very low efficiencies as shown with the control experiment B1 where only gaseous n-hexane was fed. The results showed that the adaptation period could be reduced from 35 days in B1 to 24 days in B2, to 14 in B3, and to 8 in B4. On the other hand, these carbon sources have an effect on the HPC and the final EC. This occurs when the carbon source used for start-up was less hydrophobic than n-hexane. When wheat bran was used as carbon source, the start-up was diminished

Figure 3. Load effect on n-hexane EC in the four biofilters in the stationary state. Flow rate is 1.2 L min1 and HRT of 1.3 min. A: n-hexane, after 50 days operation (Biofilter 1); B: glycerol and n-hexane after, 50 days operation (Biofilter 2); C: 1-hexanol and n-hexane, after 47 days operation (Biofilter 3); D: wheat bran and n-hexane, after 27 days operation (Biofilter 4).

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20 days, with regard to the other biofilters using glycerol, 1hexanol, and n-hexane (control). However, this decrease in the time of start-up has to be evaluated considering that lower EC were obtained. The authors would like to thank Direccioń de Investigacioń de la Universidad Cato´lica de Temuco, Chile the Ph.D. scholarship of A. Vergara-Fernańdez and to Conacyt (Mexican Council for Science and Technology) Project SEMARNAT-00120.

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