Enhancement of cometabolic biodegradation of trichloroethylene (TCE) gas in biofiltration

JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 100, No. 6, 657–661. 2005 DOI: 10.1263/jbb.100.657

© 2005, The Society for Biotechnology, Japan

Enhancement of Cometabolic Biodegradation of Trichloroethylene (TCE) Gas in Biofiltration In-Gyung Jung1 and Ok-Hyun Park1* Department of Environmental Engineering, Pusan National University, Gumjung-gu, Busan 609-735, Korea1 Received 4 July 2005/Accepted 31 August 2005

A biofilter column inoculated with Pseudomonas putida F1 was operated to study cometabolic biodegradation of trichloroethylene (TCE) gas using toluene as a primary substrate. Variations in the efficiency and capacity of TCE elimination with different inlet concentrations of toluene and TCE were investigated in order to understand the competitive inhibition between toluene and TCE. Two toluene feeding methods, stage feeding along the column and cyclic feeding, were examined as strategies to enhance TCE cometabolic biodegradation by avoiding the toluene inhibition of TCE biodegradation and the toxic effect of TCE on cells and toluene dioxygenase enzymes. It was concluded that both methods are promising and that the determination of a suitable feeding frequency, recovery period, and inlet toluene concentration was required to optimize cyclic feeding in the cometabolic biodegradation of TCE. [Key words: trichloroethylene, Pseudomonas putida F1, biofiltration, cometabolic biodegradation]

ene, cresol, or phenol to induce the expression of enzymes with TCE degradation functions. P. mendocina KR-1 initially oxidizes toluene in the para-position to form paracresol, which has been observed to oxidize TCE (6). N. europaea is an ammonia-oxidizing bacterium of which the ammonia monooxygenase (AMO) has the ability to oxidize TCE (12). A number of studies concerning the biodegradation of TCE have been performed using conventional biofilters to understand the basic biokinetics of TCE cometabolic oxidation for various primary substrates (2). However, those studies were not able to present sustainable high degradation of TCE (2). Some studies tried to improve the TCE biodegradation by varying the inlet concentration of the primary substrate so that the competitive inhibition between TCE and the primary substrate could be avoided. Even though many positive results have been reported for pure cultures, this process has rarely been applied to large-volume TCE gas emitted from soil remediation areas (13). The purpose of the present research was to develop an economical and effective biofiltration technology for the treatment of gaseous chlorinated organic compounds. To achieve the maximum biodegradation efficiency of TCE, the competitive inhibition between the primary substrate and TCE should be minimized by introducing minimum amounts of the primary substrate. Two toluene feeding methods, stage feeding and cyclic feeding, were examined as strategies for enhancing TCE cometabolic biodegradation.

Remediation technologies that involve gas transport (e.g., soil vapor extraction and air sparging of groundwater) cause the emission of gases contaminated with chlorinated solvent. As trichloroethylene (TCE) mostly affects the nervous system, exposure to very high levels of TCE, even for short times, has caused unconsciousness and death. People who inhale moderate levels of TCE might suffer from headaches, dizziness, or an impaired ability to perform, and higher levels of exposure can cause liver and kidney damage (1). Although chlorinated organic compounds are not biodegraded easily, nonspecific oxygenase systems have the potential ability to catalyze the oxidation of a number of chlorinated aliphatic chemicals including TCE. For the cometabolic biodegradation of TCE to occur, one or more substrates must be supplied as both an inducer and as an exogenous electron donor capable of supplying NADH to the oxygenase system. Therefore, the biodegradation of TCE through cometabolic oxidation is intrinsically associated with the competitive inhibition between target substrates and exogenous electron donors, NADH limitation, and the toxicity of intermediates (2–4). Laboratory studies have shown that TCE can be oxidized by oxygenase-producing bacteria such as Methylosinus trichosporium OB3b, Burkholderia cepacia G4, Pseudomonas mendocina KR-1, Nitrosomonas europaea, and Pseudomonas putida F1 (2, 5–8). Methanotrophs, such as M. trichosporium OB3b, grow on methane as an energy and carbon source and their methane monooxygenase (MMO) enzyme degrades TCE (9–11). B. cepacia G4 requires the addition of exogenous substrates such as tolu-

MATERIALS AND METHODS Preparation of inoculums and biofilter packing material P. putida F1 obtained from David T. Gibson (University of Iowa) was routinely cultivated at 25°C in a 300-ml flask containing 50 ml

* Corresponding author. e-mail: [email protected] phone: +82-51-510-2415 fax: +82-51-514-9574 657

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TABLE 1. Composition of culture medium Compound Solution A KH2PO4 K2HPO4 Solution B Nitrilotriacetic acid MgSO4 or MgSO4 ⋅ 7H2O CaCl2 ⋅ 2H2O (NH4)6Mo7O24 ⋅ 4H2O FeSO4 ⋅7H2O Metals 44 Solution C (NH4)2SO4

Concentration (g l–1) 2.15 5.30 10 14.45 3.33 0.00925 0.099 50 ml 60 g/300 ml H2O

TABLE 2. Composition of 44 metals mineral solution Compound EDTA ZnSO4 ⋅7H2O (250 mg Zn) MnSO4 ⋅ H2O (50 mg Mn) FeSO4 ⋅7H2O (100 mg Fe) CuSO4 ⋅5H2O (10 mg Cu) Co(NO3)2 ⋅6H2O (5 mg Co) Na2B4O7 ⋅ 10H2O (2 mg B)

Concentration (mg ml–1) 250 109.5 154 500 39.20 24.80 17.10

of modified Hunters medium (16). The medium consisted of three solutions; the inorganic constituents for each solution are shown in Tables 1 and 2. Phosphate buffer mixed with KH2PO4 (2.15 g l–1) and K2HPO4 (5.3 g l–1) was added to the medium, and the final pH was adjusted to 7.0. Toluene was supplied using a toluene-filled glass bulb. Toluene vapor from the toluene-filled glass bulb was dissolved in the medium. The culture was incubated in a shaker (VS-5500N; Vision, Seoul, Korea), and cells in exponential phase (OD600 =0.8–1.0) were inoculated into 500-ml flasks containing 100 ml of fresh medium and spherical ceramic particles (200 ml) of 1 cm diameter and 0.04 m2 m–3 specific surface area (SH VOC-01; Sam Whan, Busan, Korea). After 3 d of cell growth in a shaker at 100 rpm, the particles in the flasks were put in an aseptic column. Air, containing 950 µg l–1 of toluene, was supplied through the top of the column at a rate of 800 ml min–1 for 4 d. Biofilter system configuration and operating conditions Figure 1 shows the experimental apparatus. The biofilter consisted of a gas mixture generation system, a nutrient supply system, and a cylindrical three-plate glass column (11 cm inside diameter×95 cm height; Fig. 1). In each part of the column, spherical ceramic particles were packed up to an 18 cm height. The whole system was placed in a 25°C temperature-controlled room. The column temperature was maintained at 25°C using a water jacket. Mass flow controllers (5850E; Brooks, Hatfield, PA, USA) were used to control the concentrations of toluene and TCE as well as the flow rate of the air-toluene-TCE mixture. The empty bed residence time (EBRT) of the column was 6 min 24 s, air containing 950 µg l–1 of toluene was supplied through the top of the column at 500 ml min–1 for approximately 3 weeks before the start of the experiments. The medium was also supplied through the top of the column, at 1 ml min–1. Analytical methods Concentrations of toluene and TCE in the gas sample were analyzed by using a gas chromatograph (Autosystem XL; Perkin-Elmer, Wellesley, MA, USA) equipped with a flame ionization detector (FID) and a capillary column (DB-WAX; J&W Scientific, Köln, Germany). The temperatures of the oven, the detector, and the injector were controlled at 100°C, 200°C and 150°C, respectively. Gas samples (400 µl each) were

FIG. 1. Schematic diagram of biofilter system purifying TCE with stage feeding of toluene. A–E, Sampling ports.

taken in duplicate at the sampling ports using a 5-ml gas-tight syringe. The OD of the culture was measured at 600 nm wavelength by using a spectrophotometer (UV-1601; Shimadzu, Kyoto). To determine the amount of the attached biomass, two particles taken from each stage of the column were immersed for 1 h in a 20 ml solution of NaP2O7 (0.1%), and sonicated for 3 s three times at 30 s intervals. The volatile suspended solid (VSS) in the obtained solution was analyzed as described in APHA (16).

RESULTS AND DISCUSSION TCE elimination efficiency with TCE inflow concentration at a constant loading rate of toluene Toluene at 1800 µg l–1 as the primary substrate was fed through a biofilter at a flow rate of 500 ml min–1. The TCE elimination efficiency and the elimination rate were determined while the TCE inflow concentration increased gradually from 6 to 1150 µg l–1. Samples were collected after 200 min operations. Figure 2 shows the variations in the efficiency and rate of TCE elimination with various inflow concentrations of TCE at a fixed loading rate of toluene. At the lower range of TCE inflow concentration, the elimination efficiency was over 95%; however, it gradually decreased with TCE inlet concentration. An elimination efficiency of 15% was observed at 1153 µg l–1 of TCE and 0.45 load ratio of TCE to toluene. It was found that the reduction in the elimination rate resulted from the inactivation of enzyme activity at TCE concentrations above 700 µg l–1. This appears to be attributed to the toxic effect of TCE on cells and enzymes. As shown in Fig. 2, the elimination rate of TCE vapor decreased with an increase in the TCE/toluene loading ratio above 0.3 due to the toxic effect of TCE. When the TCE/toluene load ratio was below 0.3, the TCE biodegradation rate increased with TCE inflow concentrations. This suggests that the load ratio of TCE/toluene should be maintained below 0.3 in order to maximize TCE elimination capacity at a feeding toluene concentration of 1800 g l–1 and a flow rate of 500 ml min–1.

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FIG. 3. Variation of TCE elimination rate with inflow toluene concentration for both TCE inflow concentrations of 500 µg l–1 and 40 µg l–1. Symbols: triangles, 500 µg l–1 of TCE; circles, 40 µg l–1 of TCE. FIG. 2. Variations in efficiency and rate of TCE elimination with its inlet concentrations for consistent feeding of 1800 µg l–1 toluene at 500 ml min–1 of gas flow rate. Symbols: triangles, elimination rate; circles, elimination efficiency.

Toluene inhibition from TCE biodegradation The effect of toluene input on TCE biodegradation was examined to understand the inhibitory effect of toluene on TCE biodegradation, which was caused by the preference of the enzyme (2). Figure 3 shows the TCE elimination rates for various inflow toluene concentrations at constant TCE inflow concentrations of 500 µg l–1 and 40 µg l–1. TCE outflow concentrations were measured after 8 h operation once TCE outflow concentrations became stabilized. At a TCE inflow concentration of 500 µg l–1, the TCE elimination rate was observed to decrease with operating time at inflow toluene concentrations of less than 2800 µg l–1 as indicated by the vertical line in Fig. 3, an effect which was probably due to the inactivation of the toluene dioxygenase enzyme. For the cases of inflow toluene concentrations of less than 2800 µg l–1 and an inflow TCE concentration of 500 µg l–1, the TCE elimination rate appeared to increase with the toluene concentration, suggesting that newly fed toluene could compensate for toluene dioxygenase inactivated by TCE degradation. At inflow toluene concentrations higher than 2800 µg l–1, the TCE elimination rate per unit biomass decreased exponentially with toluene concentration, an effect which was attributed to the toluene inhibition from TCE biodegradation. The inhibitory effect of toluene on TCE biodegradation occurred in a similar pattern for both inflow TCE concentrations of 500 µg l–1 and 40 µg l–1. This suggests that once toluene induces enough toluene dioxygenase, only the minimum amount of toluene sufficient to maintain the activity of the enzyme should be fed through the biofilter in order to biologically degrade TCE efficiently. Enhancement of TCE elimination performance by modifying feeding method of toluene Toluene vapor should be continuously fed to the microorganisms to sustain enzyme production and to support biomass growth. Therefore, the appropriate amount of toluene vapor had to be determined that would recover enzyme activity while avoid-

ing the effect of toluene inhibition caused by the enzyme’s preference for toluene rather than TCE. In the case of gas feeding from the top to the bottom of the biofilter, the biofilm was observed to be thickest near the inflow inlet of the biofilter column because the major fraction of the fed toluene was degraded directly after the inflow into the column. As a result, lesser TCE degradation occurred at the lower part of the column compared with the upper part, because of the lack of biomass and the toluene dioxygenase enzyme. To make bacterial cells grow evenly throughout the whole column, it is necessary to maintain an appropriate inlet toluene concentration so as to supply enough growth substrate. However, this caused an increase in the inhibition of TCE biodegradation. Therefore, it was expected that TCE elimination capacity could be improved if toluene was fed individually to each stage along the column. The experiment using the stage feeding method was conducted to verify the enhancement of TCE biodegradation capacity. The gas mixture was introduced at a flow rate of 500 ml min–1 through the top of the column with a stepwise increase in TCE concentrations, while a total toluene loading of 112 µg l–3 min–1 was divided into three inflow lines and fed to three different stages along the column. Figure 4 shows the TCE elimination efficiency and rates for both the stage feeding and the single feeding of toluene. The stage feeding of toluene enhanced TCE elimination efficiency by 10–30% compared with the single feeding. In particular, at TCE inflow concentrations over 800 µg l–1, there was a significant difference in the TCE elimination rate between the methods. The biofilter with the stage feeding was operated for as long as 4 d to observe the variations in TCE elimination capacity under the inlet conditions of 110 µg l–1 TCE, 220 µg l–1 toluene, and a gas flow rate of 500 ml min–1, conditions that were designed to maintain high TCE removal efficiency. Figure 5 shows the results of the above experiment on the TCE elimination efficiency for the stage feeding of toluene during 4 d of early stage operation. The efficiency was maintained at over 80% during the period. The cyclic feeding of toluene was also considered as a strategy to reduce the toluene inhibition of TCE cometabolic biodegradation. Table 3 shows the operating condi-

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FIG. 5. TCE elimination efficiency for stage feeding of toluene during 4 d operation at input TCE concentration of 110 µg l–1 gas flow rate of 500 ml min–1, and stage feeding toluene concentration of 220 µg l–1.

FIG. 4. TCE elimination efficiency (a) and rate (b) with different feeding methods at toluene concentration of 1800 µg l–1 and gas flow rate of 500 ml min–1. Symbols: closed triangles, stage feeding; open triangles, single feeding.

tions and results for the TCE removal experiment using cyclic feeding of toluene. Each set of experiments was conducted for 24 h with various combinations of recovery periods, feeding frequencies, and toluene inlet concentrations. The time variation of TCE elimination efficiency during our set 1 experiment is shown in Fig. 6. Although Segar et al. (17), who studied the method of feeding phenol as a primary substrate to enhance TCE elimination efficiency, concluded that TCE elimination efficiency for the feeding frequency of 1/12 (h/h) was higher than that for the frequency of 1/24 (h/h), in our set 1 and set 2 experiments, there was not a significant difference in the average TCE elimination efficiency depending on the feeding frequency (Table 3). The set 1 experiment had double the recovery period of the set 2 experiment, resulting in a slightly higher average TCE elimination efficiency. For the set 3 experiment, which involved identical conditions of feeding frequency and recovery period of TCE elimination efficiency, the efficiency was less

FIG. 6. Variation of TCE elimination efficiency for cyclic feeding of toluene during set 1 experiment at TCE concentration of 110 µg l–1 and gas flow rate of 800 ml min–1. Symbols: closed triangles, TCE elimination for cyclic feeding of toluene; open triangles, TCE elimination for consistent feeding of toluene; closed circles, inflow toluene concentration; open bars, data set for cyclic toluene feeding duration.

than 60% because of the high inflow toluene input. It was concluded that adequate determinations of the toluene feeding frequency and recovery period were required to enhance TCE elimination efficiency in biofiltration; moreover, the inflow toluene concentration was a more important factor for enhancement than either of the other factors.

TABLE 3. Summary of operating conditions and results for TCE removal experiments using cyclic feeding of toluene Toluene inlet Recovery Average TCE Feeding concentration period elimination frequency (µg l–1) (h) efficiency, η (%) (number/h) 1 3/24 2 470 70 2 6/24 1 470 67 3 3/24 1 940 59 a ∆η is difference of TCE elimination efficiency between, before and after toluene cyclic feeding. Set

Average, ∆ηa (%) 9 6 8

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