Biodegradation of fluorinated environmental pollutants under aerobic conditions Hasan, Syed Adnan
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2010
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Hasan, S. A. (2010). Biodegradation of fluorinated environmental pollutants under aerobic conditions. s.n.
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3
Degradation kinetics of 4-fluorocinnamic acid by a
consortium of Arthrobacter sp. strain G1 and Ralstonia sp. strain H1
Syed A. Hasan, Maria I. M. Ferreira, and Dick B. Janssen
Under review
ABSTRACT
Arthrobacter sp. strain G1 was isolated based on its ability to grow on 4-fluorocinnamic acid (4-FCA) as sole carbon source. The organism converts 4-FCA into 4-fluorobenzoic acid (4-FBA) and utilizes the two-carbon side-chain for growth with some formation of 4-fluoroacetophenone (4-FAP) as a dead-end side product. We also have isolated Ralstonia sp. strain H1, an organism that degrades 4-FBA. A consortium of strains G1 and H1 degraded 4-FCA with Monod kinetics during growth in batch and continuous cultures. Specific growth rates of strain G1 and specific degradation rates of 4-FCA were observed to follow substrate inhibition kinetics, which could be modeled using the kinetic models of Haldane-Andrew and Luong-Levenspiel. The mixed culture showed complete mineralization of 4-fluorocinnamic acid with quantitative release of fluoride, both in batch and continuous cultures. Steady-state chemostat cultures that were exposed to shock loadings of substrate responded with rapid degradation and returned to steady-state in 10-15 h, indicating that the mixed culture provided a robust system for 4-fluorocinnamic acid degradation.
INTRODUCTION
Fluorinated chemicals are prominent xenobiotics that are used in pharmaceutical, agricultural and other applications because of their chemical stability, high lipophilicity and ability to resist metabolic conversion, thus increasing application lifetime (29, 34). Fluoro- substitution can cause significant biological effects, such as enzyme inhibition, changes in cell-to-cell communication, disruption of transport over the membrane and inhibition of energy generation (19). Even though the synthesis and use of fluoroorganics is growing and they often inevitably end up in the environment, knowledge about the biodegradation of fluorinated compounds is scarce. Biodegradation studies with halogenated chemicals have mostly been focused on brominated and chlorinated chemicals (10, 14, 16). Fluorinated compounds have so far received less attention because they are considered very stable which is sometimes supposed to indicate little impact on human and environmental health (28).
The use of organofluorides in open applications and improper waste disposal has led to their occurrence as ubiquitous environmental contaminants (19). Polymers of 4-FCA are used in electronic industries (12) and can cause environmental contamination if the industrial effluent is discharged untreated. Treatment of 4-FCA containing waste streams is necessary
to preserve the environmental quality of soil and water. Among the various techniques available for removing xenobiotic chemicals, biological treatment often is the most economical and versatile approach, as it leads to complete mineralization (31). For the design of processes for treatment of toxic waste streams, an understanding of the degradation kinetics is very important. Mathematical models that include bacterial growth and substrate utilization have been developed to describe the biodegradation kinetics of contaminants. The microbial growth rate is most often described using Monod kinetics (27). However, high substrate concentrations may become inhibitory to growth. Several mathematical models have been developed to quantify inhibitory effects of toxic substrates on microbial growth kinetics. The Haldane-Andrew (1, 13) and Luong-Levenspiel (23, 24) models are the most commonly used ways to describe microbial metabolism that includes growth inhibition kinetics.
Recently, we have demonstrated the possibility of partial degradation of 4-FCA by Arthrobacter sp. strain G1 and complete degradation by a mixed culture of strain G1 and Ralstonia sp. strain H1 (15). Several strains of the genus Arthrobacter are known to utilize similar aromatic compounds which include p-hydroxybenzoic acid (17), gentisic acid (12), 4-chlorobenzoate (25, 28, 37), mono- and dichlorinated biphenyls (11), 3-aminophenol (21), phenol (18), 4-fluorophenol (9) and a mixture of phenols (35). Members of the genus Ralstonia are also known for their potential to mineralize aromatic compounds (2, 3, 4, 5, 22, 32).
The aerobic degradation of 4-FCA was reported earlier to proceed via 4-fluoro- acetophenone (4-FAP) to form 4-fluorobenzoic acid (4-FBA) as the end product (6, 8, 30).
These studies were performed with activated sludge that was used for the treatment of a pharmaceutical waste. We could propose a degradation pathway of 4-FCA by studying a consortium of the newly isolated Arthrobacter sp. strain G1 and Ralstonia sp. strain H1 (15).
We discovered that strain G1 utilizes 4-FCA for growth by cleaving off the two-carbon side- chain with release of 4-FBA as the major product. Traces of 4-FAP were also formed. In the current study, we further have evaluated the possibility of the partial and complete mineralization of 4-FCA by a pure and mixed cultures under batch and continuous cultivation conditions.
This study focuses on the determination of the kinetic properties of the pure culture of Arthrobacter sp. strain G1 and the mixed culture of strain G1 and Ralstonia sp. strain H1 by conducting batch tests at various initial 4-FCA concentrations. We also demonstrate the
chemostat culture, and provide information about the degradation kinetics. The consortium established by the symbiotic relationship of these strains may be useful to understand and improve bioremediation of toxic organofluorine waste in the environment.
MATERIALS AND METHODS
Growth conditions. Cells of strains G1 and H1 were grown aerobically at 30°C in flasks under rotary shaking or in a fermentor. Growth medium (MMY) contained per liter:
5.37 g of Na2HPO4.12H2O, 1.36 g of KH2PO4, 0.5 g of (NH4)2SO4, 0.2 g of MgSO4.7H2O.
The media was supplemented with a trace elements solution (5 ml l-1) that contained: 780 mg of Ca(NO3)2.4H2O, 200 mg of FeSO4.7H2O, 20 mg of Na2SeO4.10H2O, 10 mg of ZnSO4.7H2O, 10 mg of H3BO3, 10 mg of CoCl2.6H2O, 10 mg of CuSO4.5H2O, 4 mg of MnSO4.H2O, 3 mg of Na2MoO4.2H2O, 2 mg of NiCl2.6H2O and 2 mg of Na2WO4.2H2O, and 10 mg yeast extract (Difco Laboratories).
In batch culture experiments, cells of strain G1 and H1 were grown separately on 4-FCA and 4-FBA, respectively, as sole carbon source. For biodegradation experiments with cell suspensions, cells were harvested (4,000 × g for 10 min) at mid log phase at an optical density at 450 nm of approximately 0.5. Cells were washed twice with 100 mM potassium phosphate buffer (pH 6.8) and resuspended in the same buffer. To follow biodegradation of 4-FCA, cell suspensions were added to 250 ml flasks containing 100 ml of MMY medium supplemented with 4-FCA at a concentration ranging from 0.2 mM to 15 mM. To study biodegradation of 4-FBA, cells were incubated similarly in MMY medium supplemented with 2 mM 4-FBA. Cells were incubated in a rotary shaker at 30°C and 150 rpm. Samples were taken with suitable time intervals, centrifuged at 16,000 × g, and analyzed immediately by HPLC, LC-MS and ion chromatography. To study the kinetics of complete mineralization of 4-FCA, mixed cultures of strains G1 and H1 were inoculated into MMY medium containing 0.1-30 mM 4-FCA.
Optical densities were monitored using a spectrophotometer at a wavelength of 450 nm. The OD450 values were then converted to dry cell mass (OD450 = 0.16 corresponds to 1 g/l dry weight).
Mathematical models. For studying growth kinetics of strain G1 with 4-FCA as a limiting substrate, we used the Monod equation (27):
S K
S
s +
= µmax
µ (1)
Where µ is the specific growth rate (h-1), µmax the maximum specific growth rate (h-1), S the substrate concentration (mM) at time t, and Ks is the half saturation coefficient (mM).
For describing inhibitory growth of microorganisms at high substrate concentrations, the Haldane-Andrew model (1, 13) was used, which is given by the following equation:
i
s K
S S K
S
2 max
+ +
= µ
µ (2)
Where Ki is the substrate inhibition constant (mM). Luong (24) applied the equation of Levenspiel (23), which is an extended Monod type model that can also be applied to describe growth inhibition at high substrate concentrations:
n
m
s S
S S K
S
−
= µmax+ 1
µ (3)
Where Sm is the critical inhibitor concentration above which growth stops and n is an empirical constant.
In order to establish the effect of the 4-FCA concentration on growth, specific growth rates at different 4-FCA concentrations were calculated with the following equation:
) (
) / ln(
1 2
1 2
t t
X X
= −
µ (4)
Where X is biomass concentration (mg l-1) at time t (h).
The specific substrate degradation rate q (h-1) was calculated using the following equation:
dt dS
q X1
−
= (5)
The model equations were solved by using non-linear regression method of Microcal Origin 7.
Continuous culture. For growth of strains in continuous culture, we used a 3 l fermentor filled with 2.5 l of MMY medium. The pH was maintained at 7.0 with a sterile solution of 2 M NaOH added by a pump connected to a pH controller. The reactor was kept at 30°C by a temperature sensor and controller, and the agitator speed was adjusted to 250 rpm. Culture medium was supplied to the reactor vessel with a peristaltic pump. Sterile air was supplied to the fermentor by passing it through a 0.45 µm pore size filter. The airflow
rate was controlled with a mass flow controller. The purity of the cultures during operation of the fermentor was regularly checked by plating culture samples on NB and LB plates, which were incubated at 30°C. When a culture attained steady state (after 5 volume changes), samples were collected for estimation of dry cell mass, fluoride released and residual 4-FCA.
The values of maximum specific growth rate (µmax) were determined by the method of washout i.e., when D > µmax. The biomass decreases by the following expression (26).
Xo
t D
X ( ) ln
ln = µmax − + (6)
or D
X X
t o +
= ln1 µmax
where Xo is the initial biomass concentration and X is the biomass concentration at time t (h).
The Monod half-saturation constant (Ks) was determined by analysis of substrate concentrations at different dilution rates using the Monod equation (27).
Chemostat pulse experiments. The effect of a pulse addition of 4-FCA on the growth of the consortium was studied with a supply of 10 mM 4-FCA to a continuous culture that was operated at a dilution rate of 0.033 h-1. Steady-state conditions were disturbed by injecting 4-FCA through a septum directly into the reactor vessel to a final concentration of 10-16 mM. One sample was taken immediately after 4-FCA injection, centrifuged, and the concentrations of 4-FCA, 4-FBA and fluoride were measured. Subsequent samples were taken with suitable intervals until the entire amount of 4-FCA added was removed due to dilution and conversion by the cells, which can be described with a standard differential equation.
dt =
dS supply to the culture – washout by dilution – biodegradation
S X K
S S Y
S dt D
dS
s
o − − +
= 1 max
)
( µ
(7)
The rate of change of biomass concentration of the bacterial consortium in continuous culture may be described by:
DX S X
K S dt
dX
s
+ −
= µmax
(8)
The rate of fluoride formation during a pulse experiment in continuous culture is described by the equation:
DF S X
K S Y dt dF
s
+ −
= 1 µmax
(9)
In equations (7), (8), and (9), So is the 4-FCA concentration in the chemostat reservoir (mM), S is the concentration of 4-FCA in the chemostat culture at time t (mM), F is the fluoride concentration in the effluent (mM), µmax is the maximum specific rate of 4-FCA conversion (mmoles 4-FCA transformed per g biomass in 1 h, h-1), Ks is the Monod constant for the conversion of 4-FCA and formation of fluoride (mM), D is the dilution rate (h-1), X is the biomass concentration at time t (g l-1), and Y is the yield coefficient (g mmol-1, biomass produced from one mmol 4-FCA).
Parameter estimation for equations (7), (8) and (9) was carried out using the program ScientistTM (MicroMath Inc., Salt Lake City, UT).
Analytical methods. Concentrations of 4-FCA, 4-FBA and their metabolites in culture supernatants were determined by reverse phase HPLC (Jasco PU-2086 pump and Jasco AS-2051 autosampler), using a Lichrosorb C18 column (250 × 4.6 mm, 5 µm particle size). The mobile phase was 0.02 M ammonium acetate adjusted to pH 4.5 with 70/30 (v/v) acetic acid/methanol. The injection volume was 10 µl, the flow rate was 0.8 ml min-1, and detection was at 254 nm with a variable UV-absorbance detector (Jasco UV-2075).
LC-MS was carried out using a Micromass ZMD equipped with a 996 Waters photodiode array detector and an Alliance 2690 separation module. HPLC conditions were as described in the previous paragraph. The mass spectrometer scan range was from m/z 50 to 600 and detection was in the negative ion mode. The source and desolvation temperatures were set to 125 and 150°C, respectively. The cone and capillary voltages were set to 30 and 2.25 V, respectively.
Fluoride measurements were performed by ion chromatography (IC) using a DX 120 ion chromatograph (Dionex, Sunnyvale, CA, USA) connected to an autosampler. This was equipped with an Altech A-2 anion (100 × 4.6 mm, 7 µm) column and an Altech guard (50 × 4 mm) column. The injection volume was 50 µl. The column temperature was set to 30°C.
The eluent used was a mixture of NaHCO3 and Na2CO3 in deionized water at a flow rate of 1.2 ml min–1.
RESULTS AND DISCUSSION
Kinetics of 4-FCA degradation. In order to investigate the kinetic properties of Arthrobacter sp. strain G1 and Ralstonia sp. strain H1, we performed a number of batch experiments. In MMY medium, strain G1 grew exponentially between 3 to 16 h with specific
growth rate of 0.11 h-1 (Fig. 1) when 2 mM 4-FCA was supplied as the sole source of carbon and energy. Strain H1 degraded 4-FBA, forming 4-fluorocatechol (4-FC) which led to ring fission with fluoride release (Fig. 2). Traces of 4-FC remained in the culture medium for a prolonged period.
0 5 10 15 20
0.0 0.4 0.8 1.2 1.6 2.0
OD450
Time (h)
Concentration (mM)
0.00 0.05 0.10 0.15 0.20
0 10 20 30 40 50
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
OD 450
Time (h)
Concentration (mM)
0.00 0.05 0.10 0.15 0.20 0.25 0.30
In order to examine the toxic effects of 4-FCA and its degradation products on growth, cells of strain G1 were incubated in MMY medium containing increasing concentrations (0.2 to 15 mM) of 4-FCA. For comparison, mixed cultures of strains G1 and H1 were incubated in separate flasks containing MMY medium supplemented with 0.4 to 30 mM 4-FCA. For each batch culture with a certain 4-FCA concentration, cell growth and substrate degradation were measured at a function of time (Fig. 3A and Fig. 3B for pure cultures and Fig. 4A-4D for the mixed cultures). The specific growth and degradation rates of pure and mixed cultures were calculated by using equation (4) and (5), respectively. The
FIG. 1. Growth of strain G1 in MMY medium supplemented with 2 mM 4-FCA. Symbols: ■, 4-FCA concen- tration; ●, 4-FBA concentration;
○, optical density at 450 nm; and
□, 4-FAP concentration.
FIG. 2. Growth of Ralstonia sp. H1 in MMY supplemented with 2 mM 4-FBA.
Symbols: ●, 4-FBA concen-tration;
○, optical density at 450 nm; ▲, F– concentration; ▼, 4-fluorocatechol concentration.
experimental data of specific growth rates are plotted in Fig. 5A and Fig. 6A for pure and mixed cultures and the degradation data are plotted in Fig. 5B and Fig. 6B for pure and mixed cultures, respectively.
0 20 40 60 80 100 120 140 160 180 0.0
0.2 0.4 0.6 0.8 1.0
OD 450
Time (h)
0.3 mM 0.5 mM 0.8 mM 1.0 mM 2.0 mM 3.0 mM 5.0 mM 8.0 mM 10.0 mM 15.0 mM
A
0 20 40 60 80 100 120 140 160 180 200 220 0
2 4 6 8 10 12 14 16 18
Concentration 4-FCA (mM)
Time (h)
0.3 mM 0.5 mM 0.8 mM 1.0 mM 2.0 mM 3.0 mM 5.0 mM 8.0 mM 10.0 mM 15.0 mM
B
FIG. 3. Time-course profiles of cell growth (A) and 4-FCA degradation (B) of Arthrobacter sp. G1 in MMY medium supplemented with different 4-FCA concentrations.
From the results, it is quite clear that both the specific growth rate and degradation rate increase with an increase in substrate concentration until a maximum value is reached.
However, above 2 mM in pure culture and above 3.5 mM in case of the mixed culture, both the specific growth rate and the degradation rate started to decrease, indicating substrate inhibition of both growth and degradation. This may be due to cell damage or disruption of membrane integrity at higher 4-FCA concentrations (33, 36).
The kinetics of partial degradation of 4-FCA by strain G1 and of the complete degradation by the mixed culture of strain G1 and H1 were studied by using growth and substrate inhibition models. The Monod model was used to determine the growth parameters and the Haldane-Andrew and Luong-Levenspiel models were used to quantify the effects of substrate inhibition on growth and degradation rates. To fit the data and estimate the values of the biokinetic constants of these models, non-linear regression was done with Microcal Origin 7. The parameters estimated by using these models are summarized in Table 1.
0 10 20 30 40 50 60 70 80 90 0.0
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
OD450
Time (h)
0.4 mM 0.6 mM 1.0 mM 1.4 mM 1.8 mM 2.2 mM 2.6 mM 3.0 mM 3.5 mM 4.0 mM 5.0 mM 6.0 mM
A
0 100 200 300 400 500 600
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
OD450
Time (h)
10.0 mM 15.0 mM 20.0 mM 30.0 mM
B
0 20 40 60 80 100
0 1 2 3 4 5 6
Concentration 4-FCA (mM)
Time (h)
0.4 mM 0.6 mM 1.0 mM 1.4 mM 1.8 mM 2.2 mM 2.6 mM 3.0 mM 3.5 mM 4.0 mM 5.0 mM 6.0 mM
C
0 100 200 300 400 500 600
0 4 8 12 16 20 24 28 32
Concentration 4-FCA (mM)
Time (h)
10.0 mM 15.0 mM 20.0 mM 30.0 mM
D
FIG. 4. Time-course profiles of cell growth (A and B) and 4-FCA degradation (C and D) of a mixed culture of Arthrobacter sp. G1 and Ralstonia sp. H1 in MMY medium supplemented with different 4-FCA concentrations.
The Monod model was used to fit specific growth rate data from low-concentration region and those from high concentration region were fitted to Haldane-Andrew and Luong- Levenspiel inhibition models. Values of Ks indicate the ability of microbes to grow at low substrate levels. The Haldane-Andrew model predicts the Ki values that indicate the sensitivity of the culture to substrate inhibition above which the specific growth and substrate degradation rates decline. A higher Ki value physically means that the culture is less sensitive to substrate inhibition. A higher Ki (18 mM) of mixed culture compared to the lower value of
pure culture (1.5 mM) indicated a high tolerance level in growth of mixed culture. The mixed culture can also degrade up to 8 mM without any significant inhibition, whereas the Ki of pure culture was calculated as 2.6 mM.
0 2 4 6 8 10 12 14 16
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14
Specific growth rate (h-1 )
Concentration (mM)
A
0 2 4 6 8 10 12 14 16
0 10 20 30 40
Specific degradation rate (µmol/min/g cells)
Concentration (mM)
B
FIG. 5. Specific growth rates (A) and degradation rates (B) of Arthrobacter sp. G1 at different 4-FCA concentrations and fitting with the kinetic models. Symbols: ■, experimental data; —, Haldane-Andrew regression curve; and ----, Luong-Levenspiel regression curve. Growth and degradation rates were calculated from the time-course profiles shown in Fig. 3.
0 5 10 15 20 25 30
0.00 0.02 0.04 0.06 0.08 0.10 0.12
Specific growth rate (h-1 )
Concentration (mM)
A
0 5 10 15 20 25 30
0 10 20 30 40
Specific degradation rate (µmol/min/g cells)
Concentration (mM)
B
FIG. 6. Specific growth rates (A) and degradation rates (B) of a mixed culture of Arthrobacter sp. G1 and Ralstonia sp. H1 at different 4-FCA concentrations and fitting of kinetic models. Symbols: ■, experimental data; —, Haldane-Andrew regression curve; and ----, Luong-Levenspiel regression curve. Growth and degradation rates were calculated from the time-course profiles shown in Fig. 4.
TABLE 1. Kinetic parameters with Monod, Haldane-Andrew, and Luong-Levenspiel models in batch conditions with (a) pure culture of Arthrobacter sp. strain G1 and (b) mixed culture of strains G1 and Ralstonia sp. strain H1, growing in MMY supplemented with 4-FCA.
(a)
Kinetic
parameter 4-FCA growth kinetics 4-FCA-limited degradation kinetics Monod
model
Haldane-Andrew model
Luong- Levenspiel model
Haldane-Andrew model
Luong- Levenspiel model
µmax (h-1) 0.12 0.28 0.12 55 32
Ks (mM) 0.15 0.72 0.14 0.27 0.08
Ki (mM) - 1.5 - 2.6 -
Sm (mM) - - 15 - 17
n - - 1 - 1
m - - - - -
R2 0.90 0.94 0.85 0.79 0.69
(b)
Kinetic
parameter 4-FCA growth kinetics 4-FCA-limited degradation kinetics Monod
model
Haldane-Andrew model
Luong- Levenspiel model
Haldane-Andrew model
Luong- Levenspiel model
µmax (h-1) 0.10 0.11 0.10 47 31
Ks (mM) 0.16 0.21 0.15 0.84 0.30
Ki (mM) - 18 - 8 -
Sm (mM) - - 44 - 39
n - - 1 - 1
m - - - - -
R2 0.90 0.88 0.88 0.84 0.77
The Luong-Levenspiel model uses a critical substrate concentration (Sm) value, at which the growth rate falls to zero. A high Sm was found for the mixed culture, indicating that the mixed culture can survive and grow up to 44 mM 4-FCA and degrade up to 39 mM. In case of the pure culture, a two-fold lower Sm was found. Of the inhibition models, the Haldane-Andrew model gave a somewhat better fit to our data than the Luong-Levenspiel model. The latter suggests a critical inhibitor concentration (Sm) above which growth is blocked, but our data suggest a more general effect that slows down growth.
Kinetics of 4-FCA degradation by strain G1 in chemostat culture. In order to evaluate the effect of the growth rate on the 4-FCA removal efficiency of strain G1 in continuous culture the bioreactor was inoculated to an OD450 of 0.013 with a batch culture of G1 pre-grown on 5 mM 4-FCA. Growth of strain G1 with 5 mM 4-FCA was followed in batch mode to an OD450 of 0.81. As soon as the stationary growth phase was reached, the culture was switched to continuous mode. Growth kinetics was subsequently studied at different dilution rates, starting with 0.007 h-1 and increasing until the critical dilution rate was reached (Fig. 7A). The µmax of strain G1 on 4-FCA determined under washout conditions (using equation 6) was 0.042 h-1. Steady states were established at four dilution rates between 0.007 h-1 and 0.038 h-1. The growth of strain G1 followed Monod kinetics. The Ks was 41 µM as calculated using the Monod equation. Cell mass was almost constant up to a dilution rate of 0.038 h-1, but at higher dilution rates it decreased sharply. The growth yield was almost constant during steady-states and it was determined as 18.8 g of cell dry-mass per mole of substrate. No wall growth of cells was observed and no formation of fluoride occurred during the course of the experiment, but the concentration of 4-FBA was 4.8 ± 0.1 mM up to the maximum dilution rate of 0.06 h-1. The concentration of 4-FBA in the fermentor decreased sharply beyond 0.06 h-1 and reduced to almost zero at 0.072 h-1.
To study the complete biodegradation of 4-FCA, a consortium of strains G1 and H1 was inoculated in a chemostat with a continuous supply of 10 mM 4-FCA. Cell mass and concentrations of 4-FCA, 4-FBA and F– were measured at different dilutions rates. The culture density was almost constant up to a dilution rate of 0.15 h-1, but decreased sharply with higher dilution rates (Fig. 7B). The residual concentration of 4-FCA was below 0.05 mM from 0.008 to 0.15 h-1, but increased sharply at higher dilution rates. The apparent Ks value of the consortium was 47 µM and the µmax obtained was 0.11 h-1.
Irrespective of the main product of 4-FCA degradation, the low values of Ks indicate that under both pure and mixed culture conditions the bacterial cultures are capable of efficient degradation of 4-FCA. A low cell yield was noted during operation of the reactor when a pure culture of strain G1 was employed to degrade 4-FCA at dilution rates 0.007 to 0.038 h-1. In contrast, the cell yield estimated in case of the consortium of strains G1 and H1 was 34.5 g of cell dry mass per mole of substrate consumed at dilution rates of 0.033 to 0.12 h-1.
The µmax determined (using equation 6) for strain G1 (Fig. 7A) and for the consortium of strains G1 and H1 (Fig. 7B) was lower than the chemostat dilution rate at which wash-out
started. In contrast, an early wash-out at D < µmax was reported for a multi-species culture growing aerobically on 6-aminoaphthalene-2-sulphonic acid (7) and another one growing anaerobically on phenol (20). The authors have not clearly explained this observation.
However, a possible reason could be the early wash-out of some stabilizing escorting species which is not directly involved in the degradation process. In our system, both strains of the mixed culture are involved in the degradation of substrate and metabolites. The fact that the chemostat dilution rate could be increased to values exceeding the µmax determined by the wash-out experiment may be due to adaptation of the cells to higher flow rates if they are kept in the chemostat at increasing dilution rates.
0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0
1 2 3 4 5
Cell mass (g l-1 )
D (h-1)
Concentration (mM)
0.00 0.05 0.10 0.15 0.20
A
0.00 0.03 0.06 0.09 0.12 0.15 0.18 0.21 0
2 4 6 8 10
Cell mass (g l-1 )
D (h-1)
Concentration (mM)
0.00 0.20 0.40 0.60 0.80
B
FIG. 7. Utilization of (A) 5 mM 4-FCA in a continuous culture of Arthrobacter sp. G1 and (B) 10 mM 4-FCA in a continuous culture of a consortium of Arthrobacter sp. G1 and Ralstonia sp. H1 at different dilution rates. Symbols: ■, 4-FBA concentration; ●, biomass; ▲, 4-FCA concentration; □, F– concentration.
In the mixed culture a two-fold higher concentration (10 mM) of 4-FCA was supplied to the mixed culture as compared to the pure culture (5 mM). A constant cell mass was obtained up to D = 0.15 h-1, which is four times higher than the cell mass obtained with single strain G1 (up to D = 0.038 h-1). The higher substrate concentration also produced a five times higher cell yield in mixed culture. This confirms that the mixed culture is much more efficient than the pure culture, and this will contribute to stability when the consortium is used in a bioremediation process,
Effect of shock loadings of 4-FCA to the chemostat culture. The capability of the consortium of strains G1 and H1 to degrade shock loadings of 4-FCA in continuous mode was tested by pulse additions of 10 mM and 16 mM of 4-FCA to a steady-state culture growing on 10 mM 4-FCA at a dilution rate of 0.033 h-1 (Fig. 8). Before the pulse, the steady-
state concentration of 4-FCA was below detection limit (<0.02 mM) and the fluoride concentration was 9.5 mM. After a pulse, the consortium completely removed the 10 mM and 16 mM pulse additions of 4-FCA in 10 h and 15 h, respectively. During consumption of pulsed 4-FCA, cell growth resumed and the same steady-state conditions as before the pulse addition of 4-FCA were obtained.
A model based on Monod kinetics (equations 7-9) was appropriate for describing the conversion of 4-FCA during the pulse experiments (Fig. 8), because no inhibition of growth or degradation was observed. Only 53% and 60% of the expected liberated fluorine was detected as free fluoride after the 10 mM and 16 mM pulses of 4-FCA, respectively. This might be due to the formation of some intermediate organofluorines that were washed out and remained undetected in the effluent.
0 2 4 6 8 10
0 2 4 6 8 10 12
Concentration (mM)
Time (h)
A
0 2 4 6 8 10 12 14 16
0 3 6 9 12 15 18
Concentration (mM)
Time (h)
B
FIG. 8. Effect of 4-FCA shock loadings on substrate depletion and fluoride formation by a consortium of Arthrobacter sp. G1 and Ralstonia sp. H1 growing in a bioreactor that is continuously supplied with 10 mM 4-FCA at the rate of 0.033 h-1. (A) A pulse of 10 mM 4-FCA was injected directly into chemostat vessel. 4-FCA (■) was depleted (µmax = 2.80 mmoles h-1 g-1 cells) and fluoride (▲) (µmax = 1.23 mmoles h-1 g-1 cells) was released; (B) in a pulse of 16 mM the rate of 4-FCA (■) depletion was (µmax = 3.0 mmoles h-1 g-1 cells) and fluoride (▲) was formed at the rate of (µmax = 1.22 mmoles h-1 g-1 cells). The residual concentration of 4-FBA (●) in the reactor was remained < 0.01 mM. The solid lines show the fit with Monod’s equations (7 and 9) for the removal of 4-FCA and release of fluoride.
The maximum 4-FCA conversion rates (µmax) in the case of 10 mM and 16 mM pulses of 4-FCA were estimated as 2.8 mmoles h-1 g-1 cells and 3.0 mmoles h-1 g-1 cells, respectively. The maximum fluoride formation rate for 10 mM and 16 mM of 4-FCA could
The removal of a high 4-FCA concentration and the final return to the initial conditions indicated that the consortium of strains G1 and H1 is highly resistant to shock loadings and can withstand a temporary exposure to a high concentration of 4-FCA in continuous culture. The increasing biomass concentration and the quick depletion of 4-FCA from the bioreactor indicates that the metabolites formed by side-chain cleavage of 4-FCA, especially 4-FBA, are not toxic for strain H1, and the consortium readily consumes these metabolites.
CONCLUSION
The data reported here show that Arthrobacter sp. strain G1 which can degrade 4-FCA by removing the side-chain of two-carbon atoms, can form a consortium with Ralstonia sp. strain H1. The mixed culture has a high tolerance to 4-FCA toxicity and is capable to degrade up to 16 mM 4-FCA without any significant inhibition, both in batch mode and continuous culture. It can also absorb shock loadings of higher concentrations of 4-FCA. This feature potentially enables the consortium to be used for the treatment of industrial wastewater or in situ bioremediation of contaminated soils.
ACKNOWLEDGEMENT
We thank Pieter Wietzes for technical support and Theodora Tiemersma for help in LC-MS analysis. S. A. Hasan gratefully acknowledges the HEC (Higher Education Commission), Government of Pakistan for financial support under the HEC-NUFFIC overseas scholarship program.
REFERENCES
1. Andrew, J. F. 1968. A mathematical model for continuous culture of microorganisms utilizing inhibitory substrates. Biotechnol. Bioeng. 10:707-723.
2. Baggi, G., L. Cavalca, P. Francia, and M. Zangrossi. 2004. Chlorophenol removal from soil suspensions: effects of a specialized microbial inoculum and a degradable analogue.
Biodegradation 15:153-160.
3. Bailey, J. E., and D. F. Ollis. 1986. Biochemical engineering fundamentals, 2nd edition.
McGraw-Hill, New York.
4. Chen, B. –Y., W. –M. Chen, and J. H. Chang. 2007. Optimal biostimulation strategy for phenol degradation with indigenous rhizobium Ralstonia taiwanensis. J. Haz. Mat. B1 39:232- 237.
5. Chen, W. –M., J. –S. Chang, C. –H. Wu, and S. –C. Chang. 2004. Characterization of phenol and trichloroethene degradation by the rhizobium Ralstonia taiwanensis. Res. Microbiol.
155:672-680.
6. Creaser, C., L. F. dos Santos, D. G. Lamarca, A. New, and J. C. Wolff. 2002.
Biodegradation studies of 4-fluorobenzoic acid and 4-fluorocinnamic acid: An evaluation of membrane inlet mass spectrometry as an alternative to high performance liquid chromatography and ion chromatography. Anal. Chim. Act. 454:137-145.
7. Diekmann, R. B., B. Nortemann, D. C. Hempel, and H. –J. Knackmuss. 1988. Degradation of 6-aminoaphthalene-2-sulfonic acid by mixed cultures: kinetic analysis. Appl. Microbiol.
Biotechnol. 29:85-88.
8. dos Santos, L. M. F., A. Spicq, A. P. New, G. L. Biundo, J. C. Wolff, and A. Edwards. 2001.
Aerobic biotransformation of 4-fluorocinnamic acid to 4-fluorobenzoic acid. Biodegradation 12:23-29.
9. Ferreira, M. I. M., J. R. Marchesi, and D. B. Janssen. 2008. Degradation of 4-fluorophenol by Arthrobacter sp. strain IF1. Appl. Microbiol. Biotechnol. 78:709-717.
10. Fetzner, S. 1998. Bacterial dehalogenation. Appl. Microbiol. Biotechnol. 50:633-657.
11. Furukawa, K., and A. M. Chakrabarty. 1982. Involvement of plasmids in total degradation of chlorinated biphenyls. Appl. Environ. Microbiol. 44:619-626.
12. Gerus, I., A. Glushchenko, Y. Kurioz, Y. Reznikov, and O. Tereshchenko. 2004. Sticking of liquid crystal on photosensitive polymer layers. Opto. Elect. Rev. 12:281-284.
13. Haldane, J. S. B. 1930. Enzymes, longmans, green, UK. Republished by MIT press, Cambridge, MA (1965).
14. Hardman, D. J. 1991. Biotransformation of halogenated compounds. Crit. Rev. Biotechnol.
11:1-40.
15. Hasan, S. A., M. I. M. Ferreira, M. J. Koetsier, M. I. Arif, and D. B. Janssen. 2010.
Complete biodegradation of 4-fluorocinnamic acid by a consortium of Arthrobacter sp. strain G1 and Ralstonia sp. strain H1. Submitted for publication.
16. Janssen, D. B., J. E. Oppentocht, and G. J. Poelarends. 2001. Microbial dehalogenation.
Curr. Opin. Biotechnol. 12:254-258.
17. Johnson, T. A., G. K. Sims, T. R. Ellsworth, and A. R. Balance. 1999. Effects of moisture and sorption on bioavailability of p-hydroxybenzoic acid to Arthrobacter sp. in soil. Microbiol.
Res. 153:349-353.
18. Karigar, C., A. Mahesh, M. Nagenahalli, and D. J. Yun. 2006. Phenol degradation by immobilized cells of Arthrobacter citreus. Biodegradation 17:47-55.
19. Key, B. D., R. D. Howell, and C. S. Criddle. 1997. Fluorinated organics in the biosphere.
Environ. Sci. Technol. 31:2445-2454.
20. Khoury, N., D. Wolfgang, and P. Kampfer. 1992. Anaerobic degradation of phenol in batch and continuous cultures by a denitrifying bacterial consortium. Appl. Microbiol. Biotechnol.
37:524-528.
21. Lechner, U., and G. Straube. 1988. Degradation of 3-aminophenol by Arthrobacter sp. mA3.
J. Basic Microbiol. 28:629-637.
22. Leonard, D., and N. A. Lindly. 1999. Growth of Ralstonia eutropha on inhibitory concentration of phenol: Diminished growth can be attributed to hydrophilic perturbation of phenol hydroxylase activity. Enz. Microbial. Tech. 25:271-277.
23. Levenspiel, O. 1980. The Monod equation: A revisit and a generalization to product inhibition situations. Biotechnol. Bioeng. 22:1671-1687.
24. Luong, J. H. T. 1987. Generalization of Monod kinetics for analysis of growth data with substrate inhibition. Biotechnol. Bioeng. 29:242-248.
25. Marks, T. S., R. Wait, and A. R. Smith. 1984. The origin of the oxygen incorporated during the dehalogenation/hydroxylation of 4-chlorobenzoate by an Arthrobacter sp. Biochem.
Biophys. Res. Commun. 124:669-674.
26. Molin, G. 1983. Measurement of the maximum specific growth-rate in chemostat of Pseudomonas sp. with different abilities for biofilm formation. Eur. J. Appl. Microbiol.
Biotechnol. 18:303-307.
28. Muller, R., R. H. Oltmanns, and F. Lingens. 1988. Enzymatic dehalogenation of 4- chlorobenzoate by extracts from Arthrobacter sp. SU DSM 20407. Biol. Chem. Hoppe-Seyler 369:567-571.
29. Natarajan, R., R. Azerad, B. Badet, and E. Copin. 2005. Microbial cleavage of C-F bond. J.
Fluor. Chem. 126:425-436.
30. New, A. P., L. M. F. dos Santos, G. L. Biundo, and A. Spicq. 2000. Analytical techniques used for monitoring the biodegradation of fluorinated compounds in waste streams from pharmaceutical production. J. Chrom. A 889:177-184.
31. Parales, R. E., N. C. Bruce, A. Schmid, and L. P. Wackett. 2002. Biodegradation, biotransformation and biocatalysis (B3). Appl. Env. Microbiol. 68:4699-4709.
32. Salehi, Z., M. Sohrabi, F. Vahabzadeh, S. Fatemi, and Y. Kawase. 2010. Modeling of p- nitrophenol biodegradation by Ralstonia eutropha via application of the substrate inhibition concept. J. Haz. Mat. 177:582-585.
33. Sikkema, J., J. A. de Bont, and B. Poolman. 1995. Mechanisms of membrane toxicity of hydrocarbons. Microbiol Rev. 59:201-222.
34. Tavener, S. J., and J. H. Clark. 2003. Can fluorine chemistry be green chemistry? J. Fluor.
Chem. 123:31-36.
35. Unell, M., K. Nordin, C. Jernberg, J. Stenstrom, and J. K. Jansson. 2008. Degradation of mixtures of phenolic compounds by Arthrobacter chlorophenolicus A6. Biodegradation 19:495- 505.
36. Weber, F. J., and J. A. de Bont. 1996. Adaptation mechanisms of microorganisms to the toxic effects of organic solvents on membranes. Biochim. Biophys. Acta. 1286:225-245.
37. Zaitsev, G. M., T. V. Tsoi, V. G. Grishenkov, E. G. Plotnikova, and A. M. Boronin. 1991.
Genetic control of degradation of chlorinated benzoic acids in Arthrobacter globiformis, Corynebacterium sepedonicum and Pseudomonas cepacia strains. FEMS Microbiol. Lett.
65:171-176.
4
Genes and proteins involved in the degradation of 4-fluorophenol by Arthrobacter sp. strain IF1
Most of the results described in this chapter have been published: Ferreira, M.I.M., T. Iida, S.A. Hasan, K. Nakamura, M.W. Fraaije, D.B. Janssen, and T Kudo (2009) Analysis of two gene clusters involved in the degradation of 4-fluorophenol by Arthrobacter sp. strain IF1.
Appl. Environ. Microbiol. 75:7767–7773.
The cloning and sequencing was done by M.I.M.Ferreira during a research stay at the Environmental Molecular Biology Laboratory, RIKEN, Wako, Japan.
The mass spectrometry analysis was done by H.P. Permentier, University of Groningen.
ABSTRACT
Arthrobacter sp. strain IF1 is able to grow on 4-fluorophenol (4-FP) as a sole source of carbon and energy. We report the cloning and sequencing of two gene clusters that each harbor a monooxygenase with high sequence similarity to the oxygenase component of 4-nitrophenol-, 4-chlorophenol- and 4-hydroxyphenyl acetate monooxygenase systems. One cluster also contained a gene for a flavin reductase. The monooxygenase (FpdA2) and reductase (FpdB) were purified from E. coli cells expressing the corresponding genes. The expression of the FpdA2 was very good but the (purified) protein was unstable. We characterized the inactivated enzyme by MALDI-MS/MS and the results suggested that FpdA2 underwent truncation by the activity of a protease. FpdA2 and FpdB together catalyzed NADH-dependent hydroxylation and dehalogenation of para-substituted phenols.
The results indicate that strain IF1 transforms 4-FP to hydroquinone by a two-component monooxygenase system of which one component provides reduced FAD at the expense of NADH and the other catalyzes para-hydroxylation of 4-FP and other 4-substituted phenols.
INTRODUCTION
Halogenated phenols are used as building blocks in the synthesis of pharmaceuticals, agrochemicals, and performance materials. As a result of improper waste disposal or open applications, they frequently occur as pollutants in water and soil. Some microorganisms have evolved pathways that allow biodegradation of these compounds (10, 20, 28, 52). Most studies on such pathways have been done with bacteria that degrade and dehalogenate chlorinated phenols. Since the 1990s, the industrial use of fluorinated compounds has been growing (24, 49).
The cleavage of the carbon-fluorine bond in organofluorine compounds is especially interesting in view of its kinetic stability and high bond energy. However, pathways for the biodegradation of fluorinated compounds and the enzymes catalyzing defluorination have been scarcely examined, although some routes are known (37).
Defluorination of fluoroaromatics can occur prior to ring cleavage, e.g. via oxygenases that defluorinate 2-fluorobenzoate (12, 39) or fluorobenzene (9). In other cases, defluorination occurs after ring cleavage via the formation of fluorinated muconolactones
(45), which can be produced from 4-fluorobenzoate (21) and fluorobenzene (9) via 4-fluorocatechol. Bacterial and fungal phenol hydroxylases can convert fluorophenols to
fluorocatechols or fluoropyrogallols, which are metabolized to fluoromuconic acids by ring- cleavage dioxygenases (6, 7). Defluorination of 4-fluorophenol prior to ring cleavage has recently been described by us in a strain of Arthrobacter, but the enzymatic basis of defluorination was not solved in detail (14).
For chlorinated phenols, two main metabolic routes have been described. Pathways in which the chlorophenol is oxidized to a substituted catechol, in some cases with partial dehalogenation, followed by ortho-cleavage of the aromatic ring and post ring-cleavage dehalogenation, occur in bacteria that degrade mono- and dichlorophenols (20, 23, 52, 53).
On the other hand, routes in which the substituted phenol is converted via hydroquinone (or a substituted hydroquinone) to maleylacetate are also known, mainly in organisms that grow on polyhalophenols (31, 33, 34, 41, 48, 55). The further aerobic metabolism of hydroquinone may proceed via direct ring fission (8, 35) or via hydroxylation to hydroxyhydroquinone (1,2,4-trihydroxybenzene) (13), which can undergo ring fission by an intradiol dioxygenase (27, 32, 36, 38). Genes for this latter hydroquinone degradation route have been cloned from Cupriavidus necator (formerly Ralstonia eutropha) strain JMP134 and Ralstonia pickettii DTP0602 which both grow on 2,4,6-trichlorophenol (22, 33, 34) and from a strain of Sphingobium chlorophenolicum that can grow on pentachlorophenol (10, 40).
Here, we report the characterization of two 4-fluorophenol (4-FP) catabolic gene clusters from Arthrobacter sp. strain IF1, an organism that was isolated on basis of its capability to utilize 4-fluorophenol as carbon source for growth (14). We describe the two- component flavin monooxygenase genes that are involved in the initial steps of 4-FP degradation. The activities of the encoded monooxygenase and its associated reductase are also described.
MATERIALS AND METHODS
Bacterial strains and culture conditions. Arthrobacter sp. strain IF1 was grown in Luria-Bertani (LB) medium or in a synthetic medium (14) at 30°C. Escherichia coli BL21(DE3) (Stratagene) was grown in LB medium and when necessary 1 mM of IPTG and 100 µg/ml of ampicillin were added.
Cloning and sequencing of monooxygenase genes. The cloning and sequencing of the fluorophenol catabolic gene clusters was done by M. I. M. Ferreira in the laboratory of T. Kudo. DNA isolation and cloning were done as described by Sambrook et al. (44).
with the Large-Construct Isolation Kit from Qiagen. The degenerate primer set and the PCR procedures that were used for the cloning have been described by Ferreira et al. (15).
Genomic DNA was separately digested by ApaI and BamHI, and putative monooxygenase sequences were detected by Southern blotting with a DIG-hybridization system as described (15, 25), using a probe obtained by labeling of PCR products that were obtained by amplification from genomic DNA as mentioned above. A band of 5 kb was detected with genomic DNA that was treated with ApaI and fragments of this size were cloned into pBluescriptII KS+ (Stratagene) to give library A. Fragments of 9 kb were detected with BamHI-restricted DNA and cloned into pHSG397 (Takara) to give library B.
For screening, the libraries were transformed into E. coli cells and transformants were inoculated in several Falcon tubes containing 2 ml of LB and chloramphenicol (pHSG397) or ampicillin (pBluescriptII KS+). After overnight growth at 30°C, DNA was isolated and screened for the presence of the 4-FP monooxygenase gene by PCR with the primers used earlier for preparation of the probe. Positive cultures were plated and colonies were screened again by PCR. DNA was isolated from the positive clones, subcloned into pUC19 (Takara), and used for sequencing.
Dideoxy sequencing was done using an ABI PRISM BigDye Ready Reaction kit and ABI Model 3700 sequencer and sequences were analyzed as described (25).
Sequence analysis comparison and structural model. The amino acid sequence of FpdA2 was initially compared to those in the databases using the BLASTp program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Amino acid sequences, which showed high similarity, were aligned using the CLUSTAL W program (www.ebi.ac.uk/clustalW).
A predicted structure was obtained using the Swiss-Model server (http://swissmodel.expasy.org) (3), with 4-chlorophenol monooxygenase (CPMO) of Burkholderia cepacia AC1100 (PDB 3HWC, unpublished) as the template (61% sequence identity).
Expression of fpd genes in E. coli. The nucleotide sequences of fpdA1, fpdA2 and fpdB were amplified with PCR primers (sequences mentioned above) and cloned in pET17b (Novagen) as translational fusions in the NdeI restriction site of the vector. E. coli BL21(DE3) was used for expression.
Purification of 4-fluorophenol monooxygenase (FpdA2). The 4-FP monooxygenase (FpdA2) was purified from E. coli BL21(DE3)(pETfpdA2). Cells were grown in LB medium containing ampicillin until the OD600 reached 0.5. IPTG was then added (0.5 mM) and the culture was incubated overnight at 20-22°C with shaking. Cells were harvested by
centrifugation, washed twice with TEMG buffer (50 mM Tris-SO4, pH 7.5, 0.5 mM EDTA, 1 mM β-mercaptoethanol, 5% glycerol), resuspended in the same buffer, and disrupted by sonication. After centrifugation (40,000 × g, 60 min), the extract was loaded on a DEAE Sepharose column (60 ml bed volume) pre-equilibrated with TEMG buffer. FpdA2 was eluted with a linear gradient of 0–0.5 M (NH4)2SO4 in TEMG, concentrated by ultrafiltration (Amicon YM-30 membrane), and separated on a hydroxyapatite column (50 ml) using 10- 400 mM potassium phosphate buffer (pH 7.0) containing 1 mM β-mercaptoethanol and 5%
glycerol. FpdA2 was concentrated by ultrafiltration and stored at -20°C.
Purification of flavin reductase (FpdB). Flavin reductase was purified from E. coli BL21(DE3)(pETfpdB), cultivated, induced and lysed as described above for FpdA2. Cell- free extract was fractionated on a DEAE Sepharose column, after which FpdB protein was concentrated and dialyzed against 1.5 M (NH4)2SO4 in TEMG buffer, which caused precipitation. The protein pellet was dissolved in 4 ml TEMG buffer (pH 7.5) and fractionated on a Superdex 200 column (320 ml bed volume) using TEMG buffer containing 0.15 M NaCl. FpdB was concentrated by ultrafiltration and stored at -20°C.
Analysis of protein by mass spectrometry. Selected protein bands (A, B and C) from SDS-PAGE (Fig. 4A, lane 1) were excised, destained and digested with trypsin (Progema, Madison, WI, USA). After washing twice with 25 mM ammonium bicarbonate and 50% acetonitrile, gel pieces were dried in a Speed-Vac. For tryptic digestion, dried gel pieces were swollen in 10 ng/µl trypsin solution that was prepared in 100 mM NH4HCO3 and incubated at 37°C for 12 to 15 h. Peptides were recovered by adding a mixture of 75%
acetonitrile and 25% of 5% formic acid in water. Samples from digested proteins were prepared for MS by mixing 0.5 µl of the sample with 0.5 µl matrix solution (5 mg/ml α-cyano-4-hydroxycinnamic acid in 50% acetonitrile containing 0.1% trifluoroacetate) and spotted on a stainless steel 192-well target plate. They were allowed to air dry at room temperature, and analyzed on a 4700 Proteomics Analyzer (Applied Biosystems, Foster City, CA, USA) MALDI-TOF/TOF mass spectrometer. For MS spectra, 1500 laser shots were acquired, and subsequently precursors from the resulting peptide spectra in the m/z range 840-4000 with a signal-to-noise threshold of 50 were automatically selected for analysis by MS/MS, with a maximum of 25 precursors per spot, excluding the most commonly observed peptide peaks of trypsin and keratin.
Enzyme assays. 4-Fluorophenol monooxygenase was measured at 25°C in incubations containing 50 mM phosphate buffer (pH 7.0), a suitable amount of
