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J. Phys. Chem. B 2006, 110, 8100-8104
Biofilm Effects on the Peroxidase-Oxidase Reaction Deyana D. Lewis,† Michael L. Ruane, and Alexander Scheeline* Department of Chemistry, UniVersity of Illinois at Urbana-Champaign, 600 South Mathews AVenue, Urbana, Illinois 61801 ReceiVed: NoVember 13, 2005; In Final Form: January 20, 2006
In experiments on the kinetics of the peroxidase-oxidase oscillatory reaction in pH 5.l acetate buffer, biofilms form in less than 48 h on the quartz reactor surface. The nominally homogeneous peroxidase system shows dynamical changes in response to this biofilm growth, partially explaining subtle differences among dynamics observed over time and between laboratories. Kinetics data and model computations are correlated with micrographs of biofilm formation. It is evident that bare quartz also interacts with reaction species, so that the surface area-to-volume ratio is an important parameter on which observed dynamics depend.
Introduction The peroxidase-oxidase (PO) reaction has been one of the most heavily studied biochemical oscillators.1-35 The mechanism has become reasonably well understood; the Bronnikova, Fed’kina, Schaffer, and Olson (BFSO) model is the current consensus as dynamically and chemically the most descriptive and predictive.36-38 We thus were perplexed when attempts to reproduce our earlier work, particularly that of Olson,16 proved to be difficult. The observations corresponded to some early problems we had in reproducing Olsen’s results6,8,39-42 and that had led Olsen to simplify the system by excluding 2,4dichlorophenol from the usual PO reaction mixture. Here we report a source of the irreproducibility and its consequences for interpreting most if not all of the PO experiments to date. Olson noted in his thesis11 that the damping of the PO reaction depended on the composition of the reactor, so that Pyrex, poly(methyl methacrylate), and polyethylene all gave rise to lesser numbers of oscillations than quartz prior to achievement of a low-oxygen steady state. Use of bovine serum albumin to passivate the quartz and stirrer surfaces also changed the damping. Nevertheless, data interpretation proceeded on the assumption that the reaction vessel surface had no effect on kinetics. To our best knowledge, this assumption has been used for all analyses of the PO system with one exception: We noted that different redox states of horseradish peroxidase (HRP) adsorb differentially on quartz.31 Protein adsorption on mesoporous silicates (similar chemistry to quartz and glass) has been shown to be related to protein charge.43 We later devised a model that showed changes in simulations of the system when adsorption/desorption dynamics were added to the BFSO schema. We did not publish at that time, as the model had arbitrary rate parameters and no experimental verification. SciFinder searches for other citations of the effect of the reactor surface on oscillatory peroxidase kinetics proved fruitless, although porphines adsorbed on silica catalyze NADH reduction better than the same unsupported catalysts.44,45 The rate of HRP adsorption on glass at pH 6 is similar to the rate at which Olson’s * Author to whom correspondence should be addressed. E-mail: scheelin@ scs.uiuc.edu. † Current address: Genome Sequencing Center, Washington University School of Medicine, Campus Box 8501, 4444 Forest Park, St. Louis, MO 63108.
oscillatory reactions damped46 but fails to explain Lvovich’s result that adding silica surface area to the PO reaction actually accelerates oxygen consumption.47 It has long been known that one of the cofactors in the PO reaction, methylene blue (MB), adsorbs on quartz.48 Olson’s anomalous result that adding MB to the PO system slowed the consumption of oxygen14 contrasts with Hauser’s conclusion26 that MB suppresses enzyme degradation and either prevents formation of ferroperoxidase or accelerates its consumption. Nowhere in Hauser’s work are surface processes implicated. Biofilms have been known for some time,49 but since the mid-1980s have garnered more attention in the medical and processing industries. Bacteria in biofilms on medical implants and catheters are more resistant to antibiotics than exogenous bacteria, and biofilms in industrial pipelines decrease flow rates due to increased skin friction. The presence of a biofilm in our system is, in hindsight, not unexpected. The reactor environment is very conducive to biofilm formation, with turbulent, highshear surroundings containing reactive oxygen species (ROS). The presence of one or more ROS provides a stress to planktonic (free floating) bacteria, one which is better dealt with by sessile (anchored) bacteria.50 Turbulent flow helps biofilm growth, in part by impinging bacteria onto a surface.51 Biofilms are primarily composed of exopolysaccharides. Thus, as they grow, heterogeneous reactions in vessels coated with such films cease occurring on quartz (with SiO moieties) and instead occur with polysaccharides. The role of the reactor surface in the PO reaction has been minimally characterized, with the influence only hinted at. The current paper shows that the reactor surface is in fact critical to the observed reaction dynamics, that biofilm formation is a central feature unless the reactor is aggressively cleaned before each experiment, and that all previous data on the PO system that has assumed homogeneous solution-phase kinetics has overlooked a critical aspect of the reaction mechanism. It appears that biofilm coating of quartz is helpful in passivating the reactor. Experimental Section Materials. β-Nicotinamide adenine dinucleotide (NADH, 98%, reduced disodium salt) was purchased from Sigma and kept in a dark 0 °C desiccator. Horseradish peroxidase (HRP,
10.1021/jp0565608 CCC: $30.25 © 2006 American Chemical Society Published on Web 03/30/2006
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Figure 1. Sketch of cuvette including slides and Teflon retainer ring. Gray shading indicates approximate solution filling.
EIA grade) was purchased from Boehringer Mannheim and kept in darkness at -20 °C. Stock solutions consisted of 10 mM 2,4-dichlorophenol (2,4-DCP) (Aldrich) in ethanol and 20 µM methylene blue (MB) (Sigma) in water. The 2,4-DCP solution was stored at room temperature, exposed to light, as was the MB solution. The HRP and NADH solutions were made up immediately before each run. The HRP solution contained 0.5 mg of HRP (2.3 µM after dilution), 3.00 µL of 2,4-DCP (6 µM), and 3.00 µL of MB (12 nM) in 5.0 mL of pH 5.1 acetate buffer. The NADH solution contained 22 mg of NADH (6.2 mM) in 5.0 mL of pH 7.0 0.1 M acetate buffer, from which particulates were removed with a 0.45-µm filter prior to use. The HRP solution was placed into a 5-cm-tall, cylindrical quartz cuvette with a 2-cm inside diameter, which in turn was placed in a thermostating jacket. The system was operated as a continuously stirred fill reactor (CSFR). The CSFR was maintained at 26 ( 1 °C and stirred at 1100 rpm with a Teflon impellor. The apparatus was completely covered, shielding the cuvette from ambient light. NADH was added to the HRP solution via a 75-µm-innerdiameter capillary. The NADH vial was pressurized with N2, creating a constant flow into the CSFR. The NADH flow rate could be controlled by varying the pressure in the vial with an MKS type 146 pressure controller and 248A solenoid valve. The NADH vial is kept in an ice bath to slow NADH degradation.52 Oxygen and nitrogen were supplied to the reactor headspace via Sierra model 840 mass flow controllers. The reaction was monitored with a Clark oxygen electrode (Microelectrodes, Inc.).14 To more easily study the formation of a biofilm, a quartz microscope slide (1-mm-thick) was cut into smaller pieces, each roughly 2.0 cm × 0.75 cm, and placed on the cuvette wall, secured by a thin Teflon ring. Three or four pieces were placed in the cuvette, covering approximately half the circumference. These slides acted as a virtual reactor wall and were easily removed one at a time to study the growth of the biofilm after each experiment. The geometry is sketched in Figure 1. Procedure. The cuvette was cleaned using one of two methods. At the start of each series of runs, the cuvette and slides were submerged in a NoChromix solution and rinsed with
distilled water. This ensured that the inner surface exposed to the reaction was organic-free quartz. Thereafter, the cuvette was only rinsed with (and stored in) distilled water. Once each solution was in place, the O2/N2 feed tube was inserted in the Teflon cuvette cover, together with the stirrer, NADH feed capillary, and O2 electrode. Nitrogen was purged through the reactor headspace, removing nearly all dissolved O2 from the HRP solution. After the O2 levels were adequately low, O2 (S. J. Smith Welding Co., 2.00% O2 in N2) replaced the N2 flow to the headspace, allowing O2 to dissolve in solution, approaching equilibrium at ∼17 µM O2.14 Once equilibrium was reached, NADH was fed into the reactor with an N2 pressure of 1810 Torr, corresponding to 636 µL h-1. After approximately one-half hour, the pressure was reduced to 1350 Torr, corresponding to 474 µL h-1. This contrasts to Olson’s procedure of reducing flow at the second oxygen minimum and was adopted when we found it difficult to initiate oscillations. Note that evaporation approximately balances fluid influx, maintaining overall reaction volume. After each run, one quartz slide piece was removed and placed in a desiccator until it could be examined with an environmental scanning electron microscope (ESEM, FEI/Philips XL30 ESEMFEG). Biofilm growth rate and properties are strong functions of the shear forces present during formation,51 so care was taken to study the side of the slide facing solution not the cuvette. Results and Discussion Difficulty reproducing the work of Olson11 first led us to investigate surface conditions more closely. Given seemingly identical starting conditions, the oscillator would behave differently from one experiment to the next, frequently going to a nonoscillatory steady state. After an extensive search through parameter space, sustained oscillations were found under conditions outlined in Table 1. After cleaning with dilute HNO3, oscillations again disappeared, only to return under the conditions shown after several experiments. Thus, a correlation between cuvette cleaning procedures and oscillator behavior was established. Evidence of biofilm formation was found after just one experimental run. Reaction dynamics also changed with the growth of a biofilm as the appearance of the film correlated with the appearance of periodic oscillations. Figures 2 and 3 each show a series of three runs each (2 months of experiments separate the data in Figure 2 from that in Figure 3), with the number of oscillations achieved in each run growing along with the biofilm. In both figures, t ) 0 corresponds to the time when purging of oxygen from buffer equilibrated with air began; the exponential decay to low [O2] is omitted from the figures. In Figure 2, the growth of [O2] after an N2 purge was completed begins the series, while in Figure 3 [O2] growth is nearly complete at the start of each plotted series. Admission of NADH starts oxygen consumption. (A glitch in the first trace in Figure 3 shows the time at which NADH addition commenced.) In all cases, there is an initial, rapid consumption of O2, almost assuredly from a free radical chain reaction. The following time series shows differences depending on the reactor’s surface
TABLE 1: Comparison of Conditions under Which Stable Oscillations Were Obtained variable
current work
Olson’s work
buffer NADH maintenance flow rate [HRP] [2,4-DCP] [MB]
pH 5.1, 0.1 M sodium acetate 2.94 µmol/h 2.5 µM 60 µM 12 nM
pH 5.1, 0.1 M sodium acetate 1.97 µmol/h 7.9 µM 10 µM 200 nM
8102 J. Phys. Chem. B, Vol. 110, No. 15, 2006
Figure 2. Three consecutive runs. The cuvette had been cleaned with dilute HNO3 prior to run A, then cleaned with distilled water prior to runs B and C.
Figure 3. Three consecutive runs, under conditions similar to Figure 1, with quartz slides in the reactor for convenient transfer to the electron microscope.
conditions. In Figure 2, trace A, the system goes to nearly steady state for several hours before the reaction accelerates to consume oxygen fast enough to reach a low-oxygen steady state. In trace B, an oscillatory reaction occurs early, evolving to a low-oxygen steady state in approximately 5 h. Trace C shows sustained oscillations. The key observations are: (1) Oxygen consumption accelerates during the course of an experiment, and (2) the rate of oxygen consumption is lowest for the runs with the cleanest quartz surface. It has been puzzling for many years why the reaction appeared under many circumstances to accelerate through the course of the reaction.11,14,15,23,31 Unlike Olsen,6,8,18,39,53 we maintained a constant flow rate feed of NADH (after an initial surge to initiate reaction, as described in the Experimental Section) rather than servo-controlling the concentration of NADH. Evolution to a low-oxygen steady state would thus require either decelerated uptake of oxygen (for which we have no evidence and no plausible mechanism), increasing levels of NADH (no evidence, no plausible mechanism), accelerated catalysis (under conditions where enzyme will eventually react to noncatalytic forms such as P470, compound X, or compound Y), or decreased inhibition. The latter is the most plausible explanation: Radical scavenging at the walls appears to be effective on bare silica.23,31 Here, it would seem that radical scavenging or surface recombination decreases as biofilm coverage increases. In fact, because the biofilm appears to passivate the reactor, a dirty, biofilm-covered reactor may result in fewer measurement artifacts than a clean
Lewis et al.
Figure 4. ESEM image of the quartz slide exposed to experimental conditions for one run, showing rapid formation of the biofilm matrix.
surface. Additionally, if enzyme adsorbs on bare quartz, then a condition similar to that of Iwado et al. with silanol groups cocatalyzing NADH reduction and H2O2 formation may pertain.44 Furthermore, Olson’s observation that MB initially decreased the rate of oxygen consumption is consistent with the known adsorption of MB on quartz, passivating silanol groups.14,48 In the case of Figure 3, four 2 cm × 0.75 cm quartz slides as described in the Experimental Section and shown in Figure 1 were inserted into the reactor, with one slide withdrawn for SEM imaging after each experiment, with the last slide allowed to age an additional 2 days in distilled water before microscopy. Thus, the surface area was higher (and varying) for the runs in Figure 3 compared to those in Figure 2. Consistent with the explanation that surface reactions damp oscillations, sustained oscillations are not seen in this higher surface area experiment. Complicating the experiment is the change in mixing due to the faceted slides and stagnant fluid regions between the slides and the cylindrical reactor walls. The first, clean surface experiment shows behavior similar to that attributed to a homoclinic orbit.8,17 The later experiments show more early oscillations and decreased damping than the earlier experiment, consistent with the data shown in Figure 2. An inconsistency is that a burst of oscillations does appear at later times in trace A in Figure 3. Because the reaction slowed to the point that a high oxygen steady state was obtained, the oscillations starting at ∼14 800 s in effect are initiating an entirely fresh reaction sequence, but with much higher [NADH] than the series starting near t ) 2000 s. Thus, the reaction conditions at this late time are not directly comparable to others shown. It is conceivable that, by this time, the clean quartz surfaces are so fouled that they cease scavenging radicals. Figures 4 and 5 show biofilm formation following one and three experiments, respectively, for the experimental time series shown in Figure 3. (Figure 5 is of the slide aged an additional 2 days following experiment 3, trace C.) Correlation between the film growth and the change in dynamics is evident. As noted for the trace A in Figure 3, the rapid biofilm formation (less than 48 h) raises concerns that reaction dynamics change not only between runs but also within experiments. The deposit on the quartz slide in Figure 4, obtained after a single ∼6 h experiment and the ∼6 h period over which Olson’s oscillations damped,16 match to a degree, which is suggestive that biofilms have formed rapidly in our system and, by implication, in many others.
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Figure 5. ESEM image of the quartz slide exposed to experimental conditions three times, while sitting in distilled water between runs. The three-dimensional structure and bacteria are visible, as is exopolysaccharide matrix material.
Figure 7. Simulation of the effects of reactor walls on peroxidaseoxidase oscillations: (A) assuming no biofilm coverage, (B) 15% biofilm coverage, (C) 20% biofilm coverage, (D) 100% coverage, so no surface reaction occurring. Rate constants are as described in the text and listed in Table 2.
Simulation
Figure 6. ESEM image of the quartz reactor cuvette, only rinsed with distilled water after each run and air-dried between an indeterminate number of runs over a period of several months. Multiple species of bacteria as well as the exopolysaccharide matrix material are visible.
The type of bacteria that make up the biofilm is still unknown, but we do know that at least two different species are present, because two different bacterial shapes (spherical and cylindrical) can be seen in Figure 6. The presence and effect of the biofilm suggest that surface chemistry involving both quartz and polysaccharides is an important factor in the PO reaction and is not included in any current models. The closest approximation to a surface/bulk interaction model is the coupled reactor approach reported by Kirkor and Scheeline.54 That model looked only at adsorption/ desorption of two redox states of HRP, ignoring reaction other than oxidation of adsorbed ferriperoxidase to peroxidase compound I on the surface (followed by rapid desorption). Radical scavenging by the surface was not considered.
To demonstrate how prior models of the PO system have been compromised by wall catalysis, we modified our 1995 “Urbanalator” model,16 with ad hoc embellishments to take wall effects into account. These changes should not be thought of as a definitive mechanism for at least the following reasons: (a) The model uses ordinary differential equations under circumstances where spatiotemporal coupling is obviously occurring. (b) No surface catalysis rate constants for the hypothesized reactions were measured, nor are they available to our knowledge in the literature. (The closest work being with nicotinamde and protein but not oxygen radical systems.43-45) (c) To emphasize the qualitative nature of the present model, we chose not to use the BFSO model,36 generally believed (including by us) to be superior to the Urbanalator. (d) The chemistry of 2,4-DCP and MB does not explicitly appear. (e) The biofilm was presumed to be inert. The rate constants and reaction steps were those of the Urbanalator,16 augmented as shown in Table 2. In place of explicitly modeling heterogeneity, surface-bound species were accounted for as separate species. By specification of small formation rate constants, only a small fraction of the peroxidase enzyme (native ferriperoxidase which has been observed to adsorb on quartz31) is construed to be removed from bulk solution. Similarly, transport of NAD• and O2-• to the quartz surface is mimicked as a slow, first-order reaction to adsorbed species. Radical-radical annihilation on the surface is rapid compared to transport to the surface, though the assignment of the annihilation rate constant (last reaction in Table 2) is arbitrary. The simulated NADH feed mimicked that in the experiments: an initial feed rate of 570 µM h-1, followed (after 2000 s) by a reduced feed rate of 430 µM h-1. The effect on both the amplitude and the form of the oscillations is profound. Figure 7, trace A, uses the adsorption modification to the Urbanalator. Oxygen is consumed so rapidly
TABLE 2: Modifications to the Peroxidase-Oxidase Oscillator Model To Include Wall Effects added species Per3+
(adsorbed on quartz)
Per5+ (adsorbed on quartz) O2-• NAD•
added reactions 3+
3+
Per f Per (ads) Per3+(ads) + H2O2 f Per5+(ads) Per5+(ads) f Per5+ O2-• f O2-•(ads) 2 O2-•(ads) f H2O2 + O2 NAD• f NAD•(ads) NAD•(ads) + O2-•(ads) f NAD+ + H2O2
putative rate constants 0.01 s-1 50 M-1 s-1 0.01 s-1 0.02 s-1 100 M-1 s-1 0.001 s-1 1000 M-1 s-1
8104 J. Phys. Chem. B, Vol. 110, No. 15, 2006 that the system goes to a low-oxygen steady state. For surface coverage of 16%, 20%, and 100%, traces B-D show that covering the quartz, thus reducing adsorption, restores the expected oscillatory behavior. A bare surface suppresses oscillation, as shown in Figure 2, trace A. A partially covered surface may either damp oscillations (Figure 2, trace B; Figure 7, trace B) or alter the oscillation frequency (Figure 7, trace C). Trace C also shows that the change in NADH feed rate at 2000 s significantly perturbs the oscillation pattern just as, in Figure 3, trace C, the dynamics change as the NADH feed is reduced. While the simulation shown in Figure 7 does not replicate the behavior in either Figure 2 or 3 precisely, it shows some qualitative features consistent with the trends seen and explanations given. It is well-known that the PO system is capable of showing chaotic behavior. If surface reactions are significant, then this nonlinear domain could well be due in part to coupling between surface and homogeneous reactions in addition to the homogeneous reactions to which oscillation is usually ascribed. While we have shown that surface reactions slow in the presence of a biofilm, it is important to note that we have no proof that the saccharide film is passive. Considering that quartz cells are used for a large fraction of all biochemical rate measurements and that the substances used to passivate quartz surfaces (bovine serum albumin, poly(ethylene glycol)) can be reactive, our observations raise a general concern for the accuracy of many heretofore published kinetics results. Acknowledgment. We thank the U. S. National Science Foundation (Grant No. PHY-01-40179) and Research Corporation (Grant No. RA0333) for partial support of this work. The assistance of Rebekah Wilson in obtaining ESEM photographs is appreciated. Scott Robinson assisted in formatting these photographs. References and Notes (1) Yamazaki, I.; Yokota, K.; Nakajima, R. Biochem. Biophys. Res. Commun. 1965, 21, 582. (2) Degn, H. Nature 1968, 217, 1047. (3) Nakamura, S.; Yokota, K.; Yamazaki, I. Nature 1969, 222, 794. (4) Yamazaki, I.; Yokoto, K. A Siphon Model for Oscillatory Reactions in the Reduced Pyridine Nucleotide, Oxygen, and Peroxidase System. In Biological and Biochemical Oscillators; Chance, B., Ghosh, A. K., Hess, B., Eds., Academic Press: New York, 1973. (5) Olsen, L. F. Biochim. Biophys. Acta 1978, 527, 212. (6) Olsen, L. F.; Degn, H. Biochim. Biophys. Acta 1978, 523, 321. (7) Fed’kina, V. R.; Bronnikova, T. V.; Ataullakhanov, F. I. Stud. Biophys. 1981, 82, 159. (8) Aguda, B. D.; Hofmann-Frisch, L.-L.; Olsen, L. F. J. Am. Chem Soc. 1990, 112, 6652. (9) Aguda, B. D.; Larter, R. J. Am. Chem. Soc. 1990, 112, 2167. (10) Geest, T.; Olsen, L. F.; Steinmetz, C. G.; Larter, R.; Schaffer, W. M. J. Phys. Chem. 1993, 97, 8431. (11) Olson, D. L. Experimental and Theoretical Studies of the Peroxidase-NADH Biochemical Oscillator: An Enzyme-Mediated Chemical Switch. Ph.D. Dissertation, University of Illinois at Urbana-Champaign, Urbana, IL, 1994. (12) Hemkin, S.; Larter, R. M. J. Phys. Chem. 1995, 100, 18924. (13) Krylov, S. N.; Aguda, B. D.; Ljubimova, M. L. Biophys. Chem. 1995, 53, 213.
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