- Email: [email protected]
Biodegradation of toluene at high initial concentration in an organic–aqueous phase bioprocess with nitrate respiration
Process Biochemistry 45 (2010) 1758–1762
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Biodegradation of toluene at high initial concentration in an organic–aqueous phase bioprocess with nitrate respiration Mehrdad Farhadian a,b, David Duchez a, Genevie`ve Gaudet c, Christian Larroche a,* a
Laboratoire de Ge´nie Chimique et Biochimique, Polytech’ Clermont-Ferrand, Universite´ Blaise Pascal, 24, avenue des Landais, BP 206, 63174 Aubiere Cedex, France Isfahan High Education and Research Institute, Isfahan, Iran c De´partement Ge´nie Biologique, Polytech’ Clermont-Ferrand, Universite´ Blaise Pascal, France b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 June 2009 Received in revised form 8 November 2009 Accepted 7 January 2010
An aerobic organic–aqueous system with forced aeration was shown to be inefficient in preventing significant volatile aromatic compounds loss in gassed systems since air sparging in n-hexadecane under abiotic conditions could reduce the toluene concentration from 2.1 g/L to about 0.5 g/L in 3 days with a gassing rate of 1VVM at 20 8C. However, the presence of such an organic phase was found to significantly reduce substrate loss in aerobic conditions in comparison to pure aqueous systems. It was thus decided to develop a new bioprocess based on an anaerobic microbial system operated in an organic–aqueous phase with nitrate respiration. The denitrifying bacterium used, Thauera aromatica K172, was produced by cultivation on sodium benzoate as carbon source under anaerobic conditions. This cultivated biomass (1.5 g/L) was shown to retain its ability to efficiently metabolize toluene in a biphasic medium without any significant loss of organic compound in the gas phase. Toluene biodegradation was thus performed in a biphasic system using a fed-batch technique involving sequential adding of both toluene and nitrate. The reaction rate with an initial concentration of toluene close to 14.5 g/L in hexadecane was found to be close to 0.5 g/L day and the molar stoichiometry of solute metabolization to nitrate reduction was close to 1:6. This work demonstrated that the denitrifying bacteria could efficiently degrade toluene in hexadecane– aqueous phase systems in which toxic compound release in the environment was prevented. ß 2010 Elsevier Ltd. All rights reserved.
Keywords: Monoaromatic hydrocarbon Biphasic system Partition coefficient Air sparging Anaerobic bioreactor Thauera aromatica
1. Introduction Remediation of monoaromatic compounds such as toluene is considered to be an environmental challenge because of their migration abilities and hazards for human health [1,2]. They enter the environment firstly through processes associated with evaporation of gasolin and chemical solvents, leakage of underground petroleum fuel storage tanks, industrial wastewater (effluents from manufacturing of paints, detergents, pesticides, etc.), and secondly through improper disposal and accidents during transportation in oil industries (oil refinery, petrochemical companies) [1,2]. Recently an aerobic system incorporating a two-phase partitioning bioreactor was applied for the treatment of monoaromatic hydrocarbon contaminants [3–9]. In a biphasic system, aromatic compounds can be solubilized in the water-immiscible organic phase and allowed to transfer into the aqueous phase [10–12]. The substrate delivery from organic to the aqueous phase is controlled by interfacial gradients of solute concentrations in a ternary system (air, water and solvent) (Fig. 1) [10,12]. Although some
* Corresponding author. Tel.: +33 4 73 40 74 29; fax: +33 4 73 40 78 29. E-mail address: [email protected] (C. Larroche). 1359-5113/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2010.01.006
bacteria are reported to be able to uptake toluene directly from an organic phase [13], the biomass generally degrades or transforms the substrate at the aqueous–organic interface and/or in the aqueous phase. In this system, monoaromatic concentration in the aqueous phase can be maintained below the inhibitory level (usually less than 50 mg/L toluene) for microorganisms. The partition process itself is also controlled to some extent by the metabolic activity of microorganisms [10–12]. The main drawback of aerobic biphasic bioprocess is that phenomena such as solute stripping are likely to contribute in a non-negligible amount to the pollutant removal from the liquid phase [14,23]. An anaerobic approach, able to strongly reduce these compound losses in a gas [1], has thus been considered. It is now well accepted that anaerobic microbial pathways are able to fully decompose monoaromatic hydrocarbons. The feasibility of toluene biodegradation by the denitrifying bacterium Thauera aromatica K172, in aqueous systems has been well documented in the last decades [15–21]. This strain is known to readily oxidize toluene to carbon dioxide in an anaerobic environment [22,23] and has been selected as a model to evaluate the true solute biodegradation in an anaerobic water–organic solvent, two-phase system. Hexadecane was selected as the organic solvent because it is now well
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2.4. Stripping experiments Studies on the stripping of monoaromatic hydrocarbons from artificially polluted water were carried out using a mechanically stirred bioreactor of 2 L working volume (Biostat MD, B. Braun, Melsungen, Germany) fitted with a mass flow controller. 2.5. Bacterial cultures 2.5.1. Inocula T. aromatica K172 (DSM 6984) was obtained from the German Resource Centre for Biological Material (Braunschweig, Germany). 2.5.2. Preculture The medium was prepared according to DSMZ protocol (http://www.dsmz.de). The strain was cultivated in a mineral-based medium with 0.72 g/L of sodium benzoate acting as a source of carbon and energy and 2 g/L of KNO3 serving as a terminal electron acceptor.
Fig. 1. Schematic diagram for a two liquid phase partitioning system. VG, VA, VS, volume of gas, aqueous and organic phases, respectively; C, mole concentrations in each phase (CGG mole concentration of gas in the gas phase (P/RT for an ideal gas)); y, mole fraction of the solute in the gas phase. Partition coefficients are defined as: KGS = (CG/CS), KSA = (CA/CS), KSG = (CS/CG).
established that it exhibits a very good biocompatibility with microorganisms [11]. 2. Materials and methods 2.1. Toluene analysis Identification and quantification of toluene in n-hexadecane (organic phase) were done by GC (6890, Agilent Technologies) equipped with a MS detector (5973, Agilent Technologies) and separated on a polar capillary column (HP-5MS, 30 m 0.1 mm i.d., 0.25 mm coating thickness). The carrier gas was ultra-purified helium (99.999%). The oven temperature was kept at 80 8C for 3 min, then raised with a constant rate of 30 8C/min to 300 8C, value held for 1 min. The injector and detector temperatures were both 250 8C; the split ratio for injection was 1:5. The injection volume was 1 mL. Mass spectrometry conditions involved an electronimpact ionization energy of 70 eV, an accelerating voltage of 1.1 kV, an emission current of 35 mA, and quadrupole, ion source and interface (line transfer) temperatures of 150, 230 and 280 8C, respectively. Data were analyzed using MSD ChemStation software (G1701CA, Agilent Technologies). Identification of monoaromatic compound was confirmed by comparison of collected mass spectra with those of authenticated standards and spectra of the National Institute for Standards and Technology (NIST) mass spectral library (version 1.7a, 2000). The monoaromatic hydrocarbon in aqueous phase was analyzed through HPLC (Waters system, USA) equipped with a Supelco Discovery C8 (4.6 mm 150 mm, 5 mm, Intact) silica-based reverse-phase column. The column temperature was kept at 35 8C by a column heater module. The mobile phase, containing a 60/40 volume percent mixture of methanol and water, was added at a flow rate of 1 mL/min. Compounds were detected using an UV/vis detector (Waters 2487 dual l absorbance detector). The volume of the injection loop was 50 mL (RHEODYNE, USA). The system also consisted of a Waters 515 high pressure pump, a degasser for the solvent (Waters In line model IDL) and a HPLC guard cartridge system (Security Guard Phenomenex, Analytical KJO-4282). The typical HPLC operating pressure was approximately 105 bar at the oven temperature used. 2.2. Nitrate and nitrite analysis Nitrate and nitrite concentrations were determined using a colorimetric assay (Hach DR/890 colorimeter, Hach Company, Loveland, USA) that required a sample volume of 10 mL. Nitrate was quantified using the cadmium reduction method (Hach Method 8039) which exhibited a linear detection range up to 30 mg/L NO3-N. Nitrite was determined using the ferrous sulfate method (Hach Method 8153) with a linear detection range up to 150 mg/L NO2-N. More detailed information on these two methods can be found at http://www.hach.com. 2.3. Carbon dioxide measurement The effluent gas was bubbled into a 800 mL, magnetically stirred, 0.5 M KOH solution. Its carbonate content was periodically determined by titration using HCl (0.127 N) [24].
2.5.3. Biomass production in the bioreactor Biomass production in the anaerobic bioreactor was carried out in a Biostat MD (B.Braun, Melsungen, Germany) equipped with pH, temperature and dissolved oxygen sensors. T. aromatica medium (4 L) was added to the bioreactor and after degassing of the medium with nitrogen (200 mL/min, 15 min), the bioreactor was inoculated with 400 mL of preculture (10%, v/v). The bioreactor was repeatedly fed with Na-benzoate (100 g/L) and KNO3 (100 g/L) at time intervals given in the Section 3. The temperature of the bioreactor was maintained at 30 8C. The solution was not agitated during the first 24 h, then the stirring rate was maintained at 50 rpm. The pH was maintained at 7.2 using NaOH (1 M) and H2SO4 (1 M) solutions. The medium was flushed with nitrogen (200 mL/min for 15 min) after each sampling to assay the carbon dioxide produced (see Section 2.3). 2.5.4. Two-phase partitioning bioreactor Hexadecane (800 mL) was added to the Biostat MD bioreactor after biomass production and finally pure toluene was injected into the organic phase using a HPLC micro-pump (100 mL/min, Waters 501, USA). The stirring rate was here also maintained at 50 rpm.
3. Results and discussion 3.1. Preliminary considerations on the behavior of toluene in an aerated water–organic solvent system The partition coefficient between the two liquid phases was defined as the ratio of concentrations of the aromatic compound between them at equilibrium. The solvent/aqueous phase partition coefficient was thus defined according to Eq. (1): K SA ¼
CS CA
(1)
where CS and CA were the equilibrium concentrations of a solute in the solvent and aqueous phase, respectively. This partition coefficient was measured using Teflon capped glass bottles containing 480 mL of water, 25 mL of hexadecane, 217.5 mL of toluene and 620 mL of air, the mixture being magnetically stirred at 600 rpm for 7 days. The average value was obtained from a linear regression approach as depicted in Fig. 2. The value obtained from the slope of the straight line in Fig. 2 gave 2.75 at 30 8C for the decimal logarithm of KSA, in good agreement with the literature value of 2.74 [25–27]. For example, in the aqueous/organic phase system at equilibrium at 30 8C, a toluene concentration of 40 g/L in hexadecane corresponded to a solute concentration in water close to 72 mg/L, a value significantly lower than the solubility in water. The preceding result allowed to consider that the experimental setup used in this work allowed to obtain results consistent with literature data. It was thus decided to use partition coefficients reported by Li and Carr [27] for the system toluene–water–air to predict solute loss in an aerated liquid– liquid system.
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Fig. 2. Solute transfer in aqueous/organic phase (temperature: 30 8C, hexadecane: 2 mL; water: 38 mL; shaker mixing: 250 rpm; glass bottles with Teflon caps: 40 mL; after 7 days).
Fig. 3. Toluene stripping from hexadecane by air sparging at 20 8C. Organic solvent volume, VS: 1 L, gas volume, VG: 1.9 L, air flow rate: 1 L/min (6.69 104 mol/s), stirring rate: 600 rpm. The slope of the straight line corresponded to the term KGSGin/CGGVS in Eq. (4) which took the predicted value of 0.024 h1.
The mass balance of a solute in such a system (Fig. 2) could be written as:
These equations clearly demonstrated that the solute stripping rate depended on the gas flow rate and on its activity. This last parameter took a value of 1 with a saturated aqueous solution and was always lower in a biphasic medium. Hence, the activity coefficient of toluene in hexadecane is close to unity [27] and, by definition, the mole fraction x is always less than 1. The stripping rate in a saturated aqueous solution could be estimated from Eqs. (5) and (6), using data available at http:// www.syrres.com/esc/physdemo.htm and a solute activity equal to 1 (Eq. (7)):
yGout ¼
dðV G C G Þ dðV A C A Þ dðV S C S Þ þ þ dt dt dt
(2)
It has already been demonstrated [28] that if (i) the mole fraction y in the gas is small, (ii) the solvents are poorly volatile, (iii) liquid phases are dilute, and (iv) the thermodynamic equilibrium is reached, this equation can be integrated to Eq. (3): Ln
C S0 K GS Gin ¼ t CS C GG ððV G K GS þ V A Þ=K SA Þ þ V S
(3)
where partition coefficients were defined in the legend of Fig. 2 and CS0 was the initial concentration of toluene in hexadecane. In the case of hydrophobic compounds, KSA was usually high while partition coefficients between a liquid and a gas, as defined here, were always small. This trend was true for toluene in a water–hexadecane system, since KGS and KSA were reported to be close to 4.07 104 and 5.5 102, respectively [27]. Eq. (3) could then be reduced with a reasonable approximation to Eq. (4), valid if VG/VS KGS: Ln
C S0 K GS Gin ¼ t CS C GG V S
(4)
This equation provided a very convenient way to predict the residual solute concentration in hexadecane as a function of the aeration rate. This equation remained true in a pure organic solvent medium (Fig. 3) because partition coefficients could be considered as independent on the presence of water due to the very low mutual solubilities (90.6 mg/L at 25 8C for the solubility of hexadecane in water, 36 mg/L for the solubility of water in hexadecane [26]). It was also possible to predict the solute stripping rate rst using Eq. (5): r st ¼ Gin y ¼
Gin C S K GS C GG
(5)
where Gin was the gas flow rate entering the system (Fig. 1). It should be noticed that, at thermodynamic equilibrium, y = (gx)S(P0/P) = (gx)A(P0/P) and thus C S K GS P0 ¼ ðg xÞ C GG P
(6)
where (gx)S and (gx)A were the activities of the solute (toluene) in the solvent and water phases, respectively. These two values were equal.
r st ¼ 3:74 102 Gin
(7)
Eq. (7) gave the maximal rate of pollutant loss in the gas, which was achieved with a pure aqueous system. A biphasic liquid–liquid system could allow a strong decrease in pollutant loss, especially at low concentrations, but this loss could remain significant if the system is subjected to forced aeration. It should be noticed that some researchers have recommended that the gas flow rate could be further reduced by using pure oxygen to decrease the loss of monoaromatics [5], but this approach could be considered as still unsatisfactory. The most efficient way to avoid pollutant loss in the atmosphere was in fact obviously the use of a process allowing Gin to take a zero value. 3.2. Anaerobic system 3.2.1. Biomass production Production of T. aromatica K172 biomass at the bioreactor scale has never, at the best of our knowledge, been reported. The use of the protocol described in Section 2, which involved fed-batch feedings on both carbon (total feedings 3.6 g/L) and nitrogen sources (total potassium nitrate feedings 10 g/L, see Section 2.5.3) allowed to obtain a significant cell growth from sodium benzoate and potassium nitrate as carbon and nitrogen sources, respectively, since about 1.5 g/L biomass was recovered after 40 h cultivation (Fig. 4) using 4 L of growth medium. It should be noticed that a quite long lag phase, close to 15 h, took place before the cell growth started. Analysis of curves in Fig. 4 allowed to estimate the biomass yield from sodium benzoate at a value close to 0.42 g/g, while that from nitrate was near 3.5 g/g. It was also shown that the carbon consumed in sodium benzoate (1.9 g/L) was fully recovered in the biomass (0.7 g/L) and carbon dioxide (1.2 g/L), which was consistent with the fact that no other metabolite was evidenced
M. Farhadian et al. / Process Biochemistry 45 (2010) 1758–1762
Fig. 4. Cultivation parameters observed during the anaerobic growth of Thauera aromatica K172 on sodium benzoate and potassium nitrate. (&) Biomass (OD600 0.33); (&) CO2 evolved; (~) NO3 consumed. Each arrow represents a feeding corresponding to 2.88 g sodium benzoate and 8 g nitrate. The 5 feedings led to the addition of 14.4 g (3.6 g/L benzoate) and 40 g (10 g/L nitrate). Biostat MD, medium volume 4 L, temperature 30 8C, pH 7.2.
in the medium. This feature also suggested that hexadecane was not metabolized, which was confirmed by the fact that the bacterium did not grow when this solvent was the sole carbon source present in the medium (results not shown). 3.2.2. Toluene biodegradation The biodegradation itself was initiated by adding hexadecane and toluene to the broth obtained after biomass production. The partition coefficient (Section 3.1) allowed interfacial transport phenomena and toluene could become available, at low concentration, to the biomass present in the aqueous layer. The denitrifying bacteria were shown to be able to metabolize toluene with an initial concentration in the organic phase as high as 14.5 g/L (Fig. 5). The molar stoichiometry of the toluene degradation and nitrate reduction was calculated from experimental data and was found to be close to 1:6 (from Fig. 6). This value was in good agreement with the theoretical stoichiometric ratio, as given in Eq. (8) [29], which showed that the toluene biodegradation was actually the main metabolism taking place in the present biphasic conditions: C7 H8 þ 6NO3 ! 7CO2 þ 4H2 O þ 3N2 þ 6e
(8)
This result was also consistent with the fact that no organic metabolite could be detected in the medium.
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Fig. 6. Toluene biodegradation in aqueous phase (&) and nitrate reduction () in the two-phase partitioning bioreactor (Biostat MD, 30 8C, pH 7.2, biomass concentration: 1325 mg dry mass/L, stirring rate: 50 rpm).
It should be pointed out that, in an independent experiment, the residual concentration of toluene in the abiotic two-phase partitioning reactor completely filled with liquid (VG = 0) was monitored for any potential losses due to possible adsorption phenomena on vessel glass or plugs. After 5 days, no change in the toluene level was detected in neither the aqueous nor the organic phase. It should also be emphasized that curves in Fig. 5 led to estimate that the average concentration of toluene in the gas phase was close to 0.037 mol/L (using data in Section 3.1 and assuming that the average concentration in hexadecane was 10 g/L), which corresponded to about 8% of the total aromatic hydrocarbon present in the system. This gaseous carbon source could be displaced towards the liquid phases at the end of an experiment, making it available to the biomass. These features again demonstrated the suitability of the anaerobic setup used for this work and allowed to consider that toluene disappearance observed in biodegradation experiments actually corresponded to the bacterial metabolism only. 4. Conclusion The efficiency of a biphasic anaerobic system to prevent loss of toluene by stripping while conducting the degradation of toluene to carbon dioxide was evaluated. It involved a nitrate-respiring bacterium which was shown to posses a great potential for volatile aromatic biodegradation without pollutant dispersal in the atmosphere. This anaerobic microbial process was reported for the first time to be feasible in a biphasic system operated in a bioreactor. These results demonstrated the feasibility of an approach where polluted water could be cleaned by pollutant extraction using a solvent highly insoluble in water and exhibiting a low vapor pressure, such as hexadecane. The resulting organic phase would be treated in a second step by the biological process shown in this work. Acknowledgments M. Farhadian would like to express his gratitude to Prof. Mehdi BORGHEI (BBRC, Sharif University of Technology, Iran) and Mr. Julien TROQUET (Director of Biobasic Environment Company) for supports and guides at this project.
Fig. 5. Toluene monitoring in the two-phase partitioning anaerobic bioreactor. Biostat MD (total volume 5 L), 30 8C, pH 7.2, biomass concentration: 1.3 g/L, mixing rate: 50 rpm; (&) toluene concentration in the aqueous phase; (&) toluene concentration in the organic phase. Volume of aqueous phase 4 L and volume of organic layer 0.8 L.
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Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Biodegradation of toluene at high initial concentration in an organic–aqueous phase bioprocess with nitrate respiration Mehrdad Farhadian a,b, David Duchez a, Genevie`ve Gaudet c, Christian Larroche a,* a
Laboratoire de Ge´nie Chimique et Biochimique, Polytech’ Clermont-Ferrand, Universite´ Blaise Pascal, 24, avenue des Landais, BP 206, 63174 Aubiere Cedex, France Isfahan High Education and Research Institute, Isfahan, Iran c De´partement Ge´nie Biologique, Polytech’ Clermont-Ferrand, Universite´ Blaise Pascal, France b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 June 2009 Received in revised form 8 November 2009 Accepted 7 January 2010
An aerobic organic–aqueous system with forced aeration was shown to be inefficient in preventing significant volatile aromatic compounds loss in gassed systems since air sparging in n-hexadecane under abiotic conditions could reduce the toluene concentration from 2.1 g/L to about 0.5 g/L in 3 days with a gassing rate of 1VVM at 20 8C. However, the presence of such an organic phase was found to significantly reduce substrate loss in aerobic conditions in comparison to pure aqueous systems. It was thus decided to develop a new bioprocess based on an anaerobic microbial system operated in an organic–aqueous phase with nitrate respiration. The denitrifying bacterium used, Thauera aromatica K172, was produced by cultivation on sodium benzoate as carbon source under anaerobic conditions. This cultivated biomass (1.5 g/L) was shown to retain its ability to efficiently metabolize toluene in a biphasic medium without any significant loss of organic compound in the gas phase. Toluene biodegradation was thus performed in a biphasic system using a fed-batch technique involving sequential adding of both toluene and nitrate. The reaction rate with an initial concentration of toluene close to 14.5 g/L in hexadecane was found to be close to 0.5 g/L day and the molar stoichiometry of solute metabolization to nitrate reduction was close to 1:6. This work demonstrated that the denitrifying bacteria could efficiently degrade toluene in hexadecane– aqueous phase systems in which toxic compound release in the environment was prevented. ß 2010 Elsevier Ltd. All rights reserved.
Keywords: Monoaromatic hydrocarbon Biphasic system Partition coefficient Air sparging Anaerobic bioreactor Thauera aromatica
1. Introduction Remediation of monoaromatic compounds such as toluene is considered to be an environmental challenge because of their migration abilities and hazards for human health [1,2]. They enter the environment firstly through processes associated with evaporation of gasolin and chemical solvents, leakage of underground petroleum fuel storage tanks, industrial wastewater (effluents from manufacturing of paints, detergents, pesticides, etc.), and secondly through improper disposal and accidents during transportation in oil industries (oil refinery, petrochemical companies) [1,2]. Recently an aerobic system incorporating a two-phase partitioning bioreactor was applied for the treatment of monoaromatic hydrocarbon contaminants [3–9]. In a biphasic system, aromatic compounds can be solubilized in the water-immiscible organic phase and allowed to transfer into the aqueous phase [10–12]. The substrate delivery from organic to the aqueous phase is controlled by interfacial gradients of solute concentrations in a ternary system (air, water and solvent) (Fig. 1) [10,12]. Although some
* Corresponding author. Tel.: +33 4 73 40 74 29; fax: +33 4 73 40 78 29. E-mail address: [email protected] (C. Larroche). 1359-5113/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2010.01.006
bacteria are reported to be able to uptake toluene directly from an organic phase [13], the biomass generally degrades or transforms the substrate at the aqueous–organic interface and/or in the aqueous phase. In this system, monoaromatic concentration in the aqueous phase can be maintained below the inhibitory level (usually less than 50 mg/L toluene) for microorganisms. The partition process itself is also controlled to some extent by the metabolic activity of microorganisms [10–12]. The main drawback of aerobic biphasic bioprocess is that phenomena such as solute stripping are likely to contribute in a non-negligible amount to the pollutant removal from the liquid phase [14,23]. An anaerobic approach, able to strongly reduce these compound losses in a gas [1], has thus been considered. It is now well accepted that anaerobic microbial pathways are able to fully decompose monoaromatic hydrocarbons. The feasibility of toluene biodegradation by the denitrifying bacterium Thauera aromatica K172, in aqueous systems has been well documented in the last decades [15–21]. This strain is known to readily oxidize toluene to carbon dioxide in an anaerobic environment [22,23] and has been selected as a model to evaluate the true solute biodegradation in an anaerobic water–organic solvent, two-phase system. Hexadecane was selected as the organic solvent because it is now well
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2.4. Stripping experiments Studies on the stripping of monoaromatic hydrocarbons from artificially polluted water were carried out using a mechanically stirred bioreactor of 2 L working volume (Biostat MD, B. Braun, Melsungen, Germany) fitted with a mass flow controller. 2.5. Bacterial cultures 2.5.1. Inocula T. aromatica K172 (DSM 6984) was obtained from the German Resource Centre for Biological Material (Braunschweig, Germany). 2.5.2. Preculture The medium was prepared according to DSMZ protocol (http://www.dsmz.de). The strain was cultivated in a mineral-based medium with 0.72 g/L of sodium benzoate acting as a source of carbon and energy and 2 g/L of KNO3 serving as a terminal electron acceptor.
Fig. 1. Schematic diagram for a two liquid phase partitioning system. VG, VA, VS, volume of gas, aqueous and organic phases, respectively; C, mole concentrations in each phase (CGG mole concentration of gas in the gas phase (P/RT for an ideal gas)); y, mole fraction of the solute in the gas phase. Partition coefficients are defined as: KGS = (CG/CS), KSA = (CA/CS), KSG = (CS/CG).
established that it exhibits a very good biocompatibility with microorganisms [11]. 2. Materials and methods 2.1. Toluene analysis Identification and quantification of toluene in n-hexadecane (organic phase) were done by GC (6890, Agilent Technologies) equipped with a MS detector (5973, Agilent Technologies) and separated on a polar capillary column (HP-5MS, 30 m 0.1 mm i.d., 0.25 mm coating thickness). The carrier gas was ultra-purified helium (99.999%). The oven temperature was kept at 80 8C for 3 min, then raised with a constant rate of 30 8C/min to 300 8C, value held for 1 min. The injector and detector temperatures were both 250 8C; the split ratio for injection was 1:5. The injection volume was 1 mL. Mass spectrometry conditions involved an electronimpact ionization energy of 70 eV, an accelerating voltage of 1.1 kV, an emission current of 35 mA, and quadrupole, ion source and interface (line transfer) temperatures of 150, 230 and 280 8C, respectively. Data were analyzed using MSD ChemStation software (G1701CA, Agilent Technologies). Identification of monoaromatic compound was confirmed by comparison of collected mass spectra with those of authenticated standards and spectra of the National Institute for Standards and Technology (NIST) mass spectral library (version 1.7a, 2000). The monoaromatic hydrocarbon in aqueous phase was analyzed through HPLC (Waters system, USA) equipped with a Supelco Discovery C8 (4.6 mm 150 mm, 5 mm, Intact) silica-based reverse-phase column. The column temperature was kept at 35 8C by a column heater module. The mobile phase, containing a 60/40 volume percent mixture of methanol and water, was added at a flow rate of 1 mL/min. Compounds were detected using an UV/vis detector (Waters 2487 dual l absorbance detector). The volume of the injection loop was 50 mL (RHEODYNE, USA). The system also consisted of a Waters 515 high pressure pump, a degasser for the solvent (Waters In line model IDL) and a HPLC guard cartridge system (Security Guard Phenomenex, Analytical KJO-4282). The typical HPLC operating pressure was approximately 105 bar at the oven temperature used. 2.2. Nitrate and nitrite analysis Nitrate and nitrite concentrations were determined using a colorimetric assay (Hach DR/890 colorimeter, Hach Company, Loveland, USA) that required a sample volume of 10 mL. Nitrate was quantified using the cadmium reduction method (Hach Method 8039) which exhibited a linear detection range up to 30 mg/L NO3-N. Nitrite was determined using the ferrous sulfate method (Hach Method 8153) with a linear detection range up to 150 mg/L NO2-N. More detailed information on these two methods can be found at http://www.hach.com. 2.3. Carbon dioxide measurement The effluent gas was bubbled into a 800 mL, magnetically stirred, 0.5 M KOH solution. Its carbonate content was periodically determined by titration using HCl (0.127 N) [24].
2.5.3. Biomass production in the bioreactor Biomass production in the anaerobic bioreactor was carried out in a Biostat MD (B.Braun, Melsungen, Germany) equipped with pH, temperature and dissolved oxygen sensors. T. aromatica medium (4 L) was added to the bioreactor and after degassing of the medium with nitrogen (200 mL/min, 15 min), the bioreactor was inoculated with 400 mL of preculture (10%, v/v). The bioreactor was repeatedly fed with Na-benzoate (100 g/L) and KNO3 (100 g/L) at time intervals given in the Section 3. The temperature of the bioreactor was maintained at 30 8C. The solution was not agitated during the first 24 h, then the stirring rate was maintained at 50 rpm. The pH was maintained at 7.2 using NaOH (1 M) and H2SO4 (1 M) solutions. The medium was flushed with nitrogen (200 mL/min for 15 min) after each sampling to assay the carbon dioxide produced (see Section 2.3). 2.5.4. Two-phase partitioning bioreactor Hexadecane (800 mL) was added to the Biostat MD bioreactor after biomass production and finally pure toluene was injected into the organic phase using a HPLC micro-pump (100 mL/min, Waters 501, USA). The stirring rate was here also maintained at 50 rpm.
3. Results and discussion 3.1. Preliminary considerations on the behavior of toluene in an aerated water–organic solvent system The partition coefficient between the two liquid phases was defined as the ratio of concentrations of the aromatic compound between them at equilibrium. The solvent/aqueous phase partition coefficient was thus defined according to Eq. (1): K SA ¼
CS CA
(1)
where CS and CA were the equilibrium concentrations of a solute in the solvent and aqueous phase, respectively. This partition coefficient was measured using Teflon capped glass bottles containing 480 mL of water, 25 mL of hexadecane, 217.5 mL of toluene and 620 mL of air, the mixture being magnetically stirred at 600 rpm for 7 days. The average value was obtained from a linear regression approach as depicted in Fig. 2. The value obtained from the slope of the straight line in Fig. 2 gave 2.75 at 30 8C for the decimal logarithm of KSA, in good agreement with the literature value of 2.74 [25–27]. For example, in the aqueous/organic phase system at equilibrium at 30 8C, a toluene concentration of 40 g/L in hexadecane corresponded to a solute concentration in water close to 72 mg/L, a value significantly lower than the solubility in water. The preceding result allowed to consider that the experimental setup used in this work allowed to obtain results consistent with literature data. It was thus decided to use partition coefficients reported by Li and Carr [27] for the system toluene–water–air to predict solute loss in an aerated liquid– liquid system.
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Fig. 2. Solute transfer in aqueous/organic phase (temperature: 30 8C, hexadecane: 2 mL; water: 38 mL; shaker mixing: 250 rpm; glass bottles with Teflon caps: 40 mL; after 7 days).
Fig. 3. Toluene stripping from hexadecane by air sparging at 20 8C. Organic solvent volume, VS: 1 L, gas volume, VG: 1.9 L, air flow rate: 1 L/min (6.69 104 mol/s), stirring rate: 600 rpm. The slope of the straight line corresponded to the term KGSGin/CGGVS in Eq. (4) which took the predicted value of 0.024 h1.
The mass balance of a solute in such a system (Fig. 2) could be written as:
These equations clearly demonstrated that the solute stripping rate depended on the gas flow rate and on its activity. This last parameter took a value of 1 with a saturated aqueous solution and was always lower in a biphasic medium. Hence, the activity coefficient of toluene in hexadecane is close to unity [27] and, by definition, the mole fraction x is always less than 1. The stripping rate in a saturated aqueous solution could be estimated from Eqs. (5) and (6), using data available at http:// www.syrres.com/esc/physdemo.htm and a solute activity equal to 1 (Eq. (7)):
yGout ¼
dðV G C G Þ dðV A C A Þ dðV S C S Þ þ þ dt dt dt
(2)
It has already been demonstrated [28] that if (i) the mole fraction y in the gas is small, (ii) the solvents are poorly volatile, (iii) liquid phases are dilute, and (iv) the thermodynamic equilibrium is reached, this equation can be integrated to Eq. (3): Ln
C S0 K GS Gin ¼ t CS C GG ððV G K GS þ V A Þ=K SA Þ þ V S
(3)
where partition coefficients were defined in the legend of Fig. 2 and CS0 was the initial concentration of toluene in hexadecane. In the case of hydrophobic compounds, KSA was usually high while partition coefficients between a liquid and a gas, as defined here, were always small. This trend was true for toluene in a water–hexadecane system, since KGS and KSA were reported to be close to 4.07 104 and 5.5 102, respectively [27]. Eq. (3) could then be reduced with a reasonable approximation to Eq. (4), valid if VG/VS KGS: Ln
C S0 K GS Gin ¼ t CS C GG V S
(4)
This equation provided a very convenient way to predict the residual solute concentration in hexadecane as a function of the aeration rate. This equation remained true in a pure organic solvent medium (Fig. 3) because partition coefficients could be considered as independent on the presence of water due to the very low mutual solubilities (90.6 mg/L at 25 8C for the solubility of hexadecane in water, 36 mg/L for the solubility of water in hexadecane [26]). It was also possible to predict the solute stripping rate rst using Eq. (5): r st ¼ Gin y ¼
Gin C S K GS C GG
(5)
where Gin was the gas flow rate entering the system (Fig. 1). It should be noticed that, at thermodynamic equilibrium, y = (gx)S(P0/P) = (gx)A(P0/P) and thus C S K GS P0 ¼ ðg xÞ C GG P
(6)
where (gx)S and (gx)A were the activities of the solute (toluene) in the solvent and water phases, respectively. These two values were equal.
r st ¼ 3:74 102 Gin
(7)
Eq. (7) gave the maximal rate of pollutant loss in the gas, which was achieved with a pure aqueous system. A biphasic liquid–liquid system could allow a strong decrease in pollutant loss, especially at low concentrations, but this loss could remain significant if the system is subjected to forced aeration. It should be noticed that some researchers have recommended that the gas flow rate could be further reduced by using pure oxygen to decrease the loss of monoaromatics [5], but this approach could be considered as still unsatisfactory. The most efficient way to avoid pollutant loss in the atmosphere was in fact obviously the use of a process allowing Gin to take a zero value. 3.2. Anaerobic system 3.2.1. Biomass production Production of T. aromatica K172 biomass at the bioreactor scale has never, at the best of our knowledge, been reported. The use of the protocol described in Section 2, which involved fed-batch feedings on both carbon (total feedings 3.6 g/L) and nitrogen sources (total potassium nitrate feedings 10 g/L, see Section 2.5.3) allowed to obtain a significant cell growth from sodium benzoate and potassium nitrate as carbon and nitrogen sources, respectively, since about 1.5 g/L biomass was recovered after 40 h cultivation (Fig. 4) using 4 L of growth medium. It should be noticed that a quite long lag phase, close to 15 h, took place before the cell growth started. Analysis of curves in Fig. 4 allowed to estimate the biomass yield from sodium benzoate at a value close to 0.42 g/g, while that from nitrate was near 3.5 g/g. It was also shown that the carbon consumed in sodium benzoate (1.9 g/L) was fully recovered in the biomass (0.7 g/L) and carbon dioxide (1.2 g/L), which was consistent with the fact that no other metabolite was evidenced
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Fig. 4. Cultivation parameters observed during the anaerobic growth of Thauera aromatica K172 on sodium benzoate and potassium nitrate. (&) Biomass (OD600 0.33); (&) CO2 evolved; (~) NO3 consumed. Each arrow represents a feeding corresponding to 2.88 g sodium benzoate and 8 g nitrate. The 5 feedings led to the addition of 14.4 g (3.6 g/L benzoate) and 40 g (10 g/L nitrate). Biostat MD, medium volume 4 L, temperature 30 8C, pH 7.2.
in the medium. This feature also suggested that hexadecane was not metabolized, which was confirmed by the fact that the bacterium did not grow when this solvent was the sole carbon source present in the medium (results not shown). 3.2.2. Toluene biodegradation The biodegradation itself was initiated by adding hexadecane and toluene to the broth obtained after biomass production. The partition coefficient (Section 3.1) allowed interfacial transport phenomena and toluene could become available, at low concentration, to the biomass present in the aqueous layer. The denitrifying bacteria were shown to be able to metabolize toluene with an initial concentration in the organic phase as high as 14.5 g/L (Fig. 5). The molar stoichiometry of the toluene degradation and nitrate reduction was calculated from experimental data and was found to be close to 1:6 (from Fig. 6). This value was in good agreement with the theoretical stoichiometric ratio, as given in Eq. (8) [29], which showed that the toluene biodegradation was actually the main metabolism taking place in the present biphasic conditions: C7 H8 þ 6NO3 ! 7CO2 þ 4H2 O þ 3N2 þ 6e
(8)
This result was also consistent with the fact that no organic metabolite could be detected in the medium.
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Fig. 6. Toluene biodegradation in aqueous phase (&) and nitrate reduction () in the two-phase partitioning bioreactor (Biostat MD, 30 8C, pH 7.2, biomass concentration: 1325 mg dry mass/L, stirring rate: 50 rpm).
It should be pointed out that, in an independent experiment, the residual concentration of toluene in the abiotic two-phase partitioning reactor completely filled with liquid (VG = 0) was monitored for any potential losses due to possible adsorption phenomena on vessel glass or plugs. After 5 days, no change in the toluene level was detected in neither the aqueous nor the organic phase. It should also be emphasized that curves in Fig. 5 led to estimate that the average concentration of toluene in the gas phase was close to 0.037 mol/L (using data in Section 3.1 and assuming that the average concentration in hexadecane was 10 g/L), which corresponded to about 8% of the total aromatic hydrocarbon present in the system. This gaseous carbon source could be displaced towards the liquid phases at the end of an experiment, making it available to the biomass. These features again demonstrated the suitability of the anaerobic setup used for this work and allowed to consider that toluene disappearance observed in biodegradation experiments actually corresponded to the bacterial metabolism only. 4. Conclusion The efficiency of a biphasic anaerobic system to prevent loss of toluene by stripping while conducting the degradation of toluene to carbon dioxide was evaluated. It involved a nitrate-respiring bacterium which was shown to posses a great potential for volatile aromatic biodegradation without pollutant dispersal in the atmosphere. This anaerobic microbial process was reported for the first time to be feasible in a biphasic system operated in a bioreactor. These results demonstrated the feasibility of an approach where polluted water could be cleaned by pollutant extraction using a solvent highly insoluble in water and exhibiting a low vapor pressure, such as hexadecane. The resulting organic phase would be treated in a second step by the biological process shown in this work. Acknowledgments M. Farhadian would like to express his gratitude to Prof. Mehdi BORGHEI (BBRC, Sharif University of Technology, Iran) and Mr. Julien TROQUET (Director of Biobasic Environment Company) for supports and guides at this project.
Fig. 5. Toluene monitoring in the two-phase partitioning anaerobic bioreactor. Biostat MD (total volume 5 L), 30 8C, pH 7.2, biomass concentration: 1.3 g/L, mixing rate: 50 rpm; (&) toluene concentration in the aqueous phase; (&) toluene concentration in the organic phase. Volume of aqueous phase 4 L and volume of organic layer 0.8 L.
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