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Diffusion in Pseudomonas aeruginosa biofilms: a pulsed field gradient NMR study
Journal of Biotechnology 77 (2000) 137 – 146 www.elsevier.com/locate/jbiotec
Diffusion in Pseudomonas aeruginosa biofilms: a pulsed field gradient NMR study M. Vogt a, H.-C. Flemming b, W.S. Veeman a,* a
Institute of Physical and Theoretical Chemistry, Gerhard-Mercator-Uni6ersity Duisburg, Lotharstrasse 1, 47057 Duisburg, Germany b Faculty for Aquatic Microbiology, Gerhard-Mercator-Uni6ersity Duisburg, Lotharstrasse 1, 47057 Duisburg, Germany Received 8 February 1999; received in revised form 9 July 1999; accepted 23 July 1999
Abstract A Pseudomonas aeruginosa biofilm is studied with pulsed field gradient echo nuclear magnetic resonance. Although not all spectral components are assigned yet, the experimental results show that a biofilm consists of components with very different diffusion coefficients. The various biofilm components that give motionally narrowed 1H NMR signals, can be grouped into five classes with diffusion coefficients, ranging from 2 × 10 − 9 to 1 × 10 − 13 m2 s − 1. Investigation of the diffusion behavior of water in the biofilm shows three fractions with different diffusion coefficients. Besides the highly mobile bulk water at least two other fractions with much lower diffusion coefficients are detected. It is shown that one of the fractions with the low diffusion coefficient probably arises from intracellular water. Also for another component of the biofilm, glycerol, three fractions with diffusion coefficients that differ more than a factor ten are detected. Also a group of signals exists which result from practically immobile components. © 2000 Elsevier Science B.V. All rights reserved. Keywords: PFG-NMR; Diffusion; Water; Biofilm; Pseudomonas aeruginosa
1. Introduction The aim of many studies of biofilms is to investigate either the structure of the biofilms or the diffusion of small molecules in biofilms. Especially the last aspect can provide insight how to optimize bioreactors and sludge dewatering while * Corresponding author. Tel.: + 49-203-3793320; fax: +49203-3793522. E-mail address: [email protected] (W.S. Veeman)
it determines the transport of nutrients, oxygen, disinfectants and water in wide areas of the biofilm (de Beer et al., 1994a; Chen and Steward, 1996; Xu et al., 1996; Beyenal et al., 1997; Beuling et al., 1998). The diffusion of small molecules in a biofilm can be determined without knowing the detailed biofilm structure, but can of course only be understood when the microscopical structure of the biofilm is known. Reversely, knowing how the various components in a biofilm can diffuse, in principle yields information about the biofilm structure.
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Many publications deal with the general aspects of diffusion in biological systems (Moonen and van Zijl, 1990; van Zijl et al., 1991; Hinton and Johnson, 1993; Ohtsuka et al., 1994; Ohtsuka and Watanabe, 1996; Anisimov et al., 1998; van As et al., 1998), but only a few publications deal with the mobility of water in sludges or biofilms. Lens et al. (1997, 1999) have shown that there are several types of water in sludges, e.g. intracellular water and water in cavities. van den Heuvel et al. (1997) have examined the mobility of water in methanogenic granules and found convective flow during pressure oscillations. Other authors differentiate the water in sludges in free or bulk water and bound water (‘freezable’ and ‘non-freezable’ water, respectively). Whereas the free water can be easily removed from sludges, the bound water can only be removed at a high expense of energy (Katsiris and Kouzeli-Katsiri, 1987; Colin and Gazbat, 1995; Smith and Vesilind, 1995; La Heij and Kerkhoff, 1996; La Heij et al., 1996). The mobility of water in several artificial and natural biofilms was examined by Beuling et al. (1998) and by van Zijl et al. (1991). Four methods are commonly used to determine diffusion coefficients in biological systems. Two of these methods use fluorescent labelled tracers and a confocal laser microscope (Scanning Confocal Laser Microscopy (SCLM) and Fluorescence Recovery After Photobleaching (FRAP)). SCLM and FRAP are excellent for three-dimensional studies of the mobility (diffusion and/or convection) of fluorescent molecules or particles in biofilms (de Beer et al., 1997; Okabe et al., 1997). With these methods de Beer et al. (1994b) have shown that there is liquid flow in biofilms. A disadvantage of both methods is that they need fluorescent molecules or particles. Therefore it is not possible to observe water or other small molecules directly. The third method uses microelectrodes, which are sensitive for one type of molecule (de Beer et al., 1994a; Lewandowski et al., 1994; de Beer and Stoodley, 1995). The last method is nuclear magnetic resonance, which uses NMR-active atoms (e.g. 1H, 13C, 15N, 31P) as a tracer. NMR can be used to get spatial information about the structure of biofilms or sludges by NMR-imaging techniques (Lewandowski et al.,
1994; La Heij and Kerkhoff, 1996; van As and van Dusschoten, 1997), about the self-diffusion of molecules by pulsed field gradient NMR (PFGNMR; van Zijl et al., 1991; Volke et al., 1994; Lens et al., 1997; Manz et al., 1997; Beuling et al., 1998) and about the chemical structure of molecules and their interactions with other biofilm constituents (Casu, 1985; Skja˚k-Bræk et al., 1986; Colquhoun et al., 1995; Lemercinier and Jones, 1996). Although the PFG-NMR method is known since the 1960s (Stejskal and Tanner, 1965; Tanner, 1968), only in recent years the PFG-NMR method has become very popular, especially to study restricted diffusion in porous systems (von Meerwall, 1983; Callaghan, 1991; Ka¨rger and Ruthven, 1992; Latour et al., 1993; Bar et al., 1998; Zakhartchenko et al., 1998). PFG-NMR uses pulsed magnetic field gradients to gain information about the average displacement of the spins. With PFG-NMR it is in principle possible to measure the self-diffusion coefficients of all NMR-detectable molecules. The PFG-NMR method is reviewed by, e.g. Stilbs (1987). The most sensitive NMR nucleus is the proton and since in biological systems nearly all molecules contain protons, it is the most favorable NMR probe for diffusion studies in biofilms. A problem in such systems is the large water content (up to 98%) whose NMR resonance dominates the proton spectrum of the biofilm. In this paper we use the PFG-NMR method for a diffusion ordered examination of biofilms. That means that the proton spectra of molecular systems in biofilms with different mobility (different self-diffusion coefficients) can be separately obtained. Similar experiments on biological materials have been reported by van Zijl et al. (1991) and Hinton and Johnson (1993). In addition we use PFG-NMR to suppress the signal of free water (Stilbs, 1987; van Zijl et al., 1991), which is the component with the highest mobility in biofilms (Otting, 1997). 1H spectra of the biofilm can then be obtained without the necessity of D2O– H2O exchange to eliminate the dominating water resonance (Skja˚k-Bræk et al., 1986). When the dominating signal of fast diffusing bulk water is suppressed, the diffusion coefficients from the
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other biofilm resonances can be measured. It will be shown that the NMR signals of a biofilm result from components with very different mobility. Some signals represent molecular systems with extremely low diffusion coefficients. On the other hand it should be realized that the employed NMR method is a typical high-resolution ‘liquidtype’ experiment. Bacteria in biofilms, for instance, contain many protons, but it must be expected that for proton spins in the bacteria cell walls, the absence of fast motional averaging will broaden their signals beyond recognition. As biofilm, Pseudomonas aeruginosa was chosen because it produces a well known extracellular polymeric substance (EPS), i.e. alginate, and because a highly mucoid strain (Grobe et al., 1995) was available which allowed the harvest of sufficient quantities of defined biomass for the NMR investigations. It has to be acknowledged that the collected P. aeruginosa biomass in the NMR tube, of course, is not completely equivalent to a thin biofilm.
2. Materials and methods In this study we use P. aeruginosa biofilms. The biofilms were cultivated on Pseudomonas Isolation Agar (Difco) in petri dishes for 24 or 96 h at 30°C (Grobe et al., 1995; Mayer et al., 1999). The biofilm was carefully removed from the plate and was transferred into a NMR-sample tube with a diameter of 5 mm. The biofilm contained (93 9 1)% water and 6.9 × 1011 cells per g dry weight. With a density of 1.016 for the biofilm one finds 4.9 ×1010 cells per cm3. The NMR-spectra were recorded with a Bruker Avance DRX 500 spectrometer, equipped with a gradient amplifier BAFPA 40 and a Bruker Diff30 probehead with 5 mm 1H-coil.
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All PFG-NMR experiments were performed with a stimulated echo pulse sequence (Fig. 1; Tanner, 1970) with 11 ms 90° pulses and a 10 s recycle delay. The echo intensity I can be written as: I= I0 e − Db with:
(1)
1 b= g 2 d 2 g 2 D− d 3
(2)
where I0 is the echo intensity without field gradients; D is the apparent self-diffusion coefficient (m2 s − 1); g is the gyromagnetic ratio (T − 1 rad s − 1); g is the gradient strength (T m − 1); d is the gradient pulse length (s); D is the time between the pulsed gradients= diffusion time (s). For simplicity we will in the following use the expression ‘diffusion coefficient’ instead of ‘apparent self-diffusion coefficient’. In principle, the diffusion coefficient D can be determined by varying g, d or D. To eliminate the effect of spin relaxation we kept for most experiments d and D constant and varied g. The spectrum is obtained after Fourier transformation of the echo signal. The diffusion coefficients of the biofilm components were determined by deconvolution of the 1H spectrum with 20 separate lines (Bruker Winfit). After deconvolution the signal intensities were fitted mono-, and when necessary, biexponentially with the program Microcal Origin. The diffusion coefficients were determined by varying the gradient strength g in 32 steps, in two runs of each 16 steps, between 0.5 and 8 T m − 1 with a diffusion time D of 10 ms and a gradient pulse length d of 1 ms. The gradient system was calibrated using the known self-diffusion coefficients of pure water and pure glycerol, respectively, at room temperature (T= 295 K).
3. Results and discussion
Fig. 1. Pulse scheme of the PFG-NMR stimulated echo sequence.
Fig. 2 shows the logarithmic plot of the echo intensity of the water signal at 4.77 ppm of the 24 h biofilm as a function of g 2. Fitting the exponen-
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Fig. 2. Logarithmic plot of the signal attenuation of the water resonance at 4.77 ppm in a P. aeruginosa biofilm.
tial curve (not shown) with Eq. (1) up to a gradient strength of 3 T m − 1 yields a diffusion coefficient for the bulk biofilm water Dbulk of 1.8×10 − 9 m2 s − 1. The diffusion coefficient of water in the biofilm matrix corresponds to 85% of the value in pure water (2.1 ×10 − 9 m2 s − 1). When we simplify the biofilm structure to a liquid phase containing for water impermeable bacterial cells, we can apply the following relation between Dbulk and the diffusion coefficient of pure water Daq (Beuling et al., 1998): Dbulk =
1−f D 1 aq 1+ f 2
(3)
where f is the volume fraction of the impermeable phase. The resulting value for f is 10.5%, which seems too high in comparison to the biofilm water weight fraction of ca. 93% and a dry mass of ca. 7% (Mayer et al., 1999). The twophase model used here is certainly too simple while the signal we ascribe to bulk water results from water in the channels of the biofilm and in the extracellular polymeric substance (EPS), with unequal diffusion coefficients. Besides allowing the determination of diffusion coefficients PFG-NMR also makes it possible to
suppress the signal of mobile water in hydrogels and biofilms (van Zijl et al., 1991; Hinton and Johnson, 1993). Fig. 3 shows the calculated intensity of the echo signal as a function of the gradient strength g by constant D=10 ms and d=1 ms for different diffusion coefficients (D=1.8×10 − 9, D= 1.0× 10 − 10, D= 1.0× 10 − 11 m2 s − 1). It is clear that for larger gradients the signals from components with high diffusion coefficients will be suppressed. This technique can be employed to eliminate the dominating water peak, and other peaks from components with high mobility, from the 1H spectrum of the biofilm. In Fig. 4 the 1H spectrum of a P. aeruginosa biofilm (24 h) is shown. The mobile water signal at 4.77 ppm dominates the spectrum. Fig. 5 shows the spectrum when the signal from very mobile water is suppressed by using g= 3 T m − 1. This method allows to observe biofilm spectra without any further preparation like exchanging the free water with heavy water. Signals of fast diffusing components in the biofilm are of course also suppressed by this method. In the spectrum with water suppression there is still a signal at 4.77 ppm left. This signal results from water with a significantly lower mobility than the biofilm bulk water, discussed above.
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Fig. 3. Calculated intensity I of a NMR-echo-signal for different diffusion coefficients D as a function of gradient strength (diffusion time D = 10 ms, gradient pulse length d= 1 ms).
Fig. 4. (a) 1H spectrum of a P. aeruginosa biofilm (24 h); and (b) enlargement of spectrum a (× 270).
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Fig. 5. PFG-1H spectrum of a P. aeruginosa (24 h) biofilm with water suppression (g =3 T m − 1, D =10 ms, d =1 ms).
To determine the diffusion coefficients of the various components of the biofilm, including the slowly diffusing water, the echo signal intensities are plotted for 16 gradient values above 3 T m − 1. In the spectra many overlapping signals can be found. Instead of integrating the signals the complete spectrum was deconvoluted with 20 signals. For each of the 20 signals the diffusion coefficient can then be determined by fitting the intensity as a function of g with Eq. (1). The resulting diffusion coefficients can be sorted into five groups with different diffusion coefficients, see Table 1. Group one contains only the signal of the biofilm bulk water at 4.77 ppm, as discussed above. The second group contains three resonances, at 3.49, 3.58 and 3.71 ppm, which result from glycerol. Glycerol is added to the agar as nutrient (20 g l − 1). The effective diffusion coefficient of the glycerol in the biofilm (6.2 ×10 − 10 m2 s − 1) is significantly lower than that of diluted glycerol in water at 20°C (8×10 − 10 m2 s − 1); the diffusion of glycerol in the biofilm is similarly affected by the biofilm as the diffusion of water: Dglycerol in biofilm =0.34 Dwater in biofilm Dglycerol in water =0.38 Dwater in water
Since also much slower diffusing glycerol is detected (see below), the fast diffusing fraction (90%) is believed to be located in the water filled pores of the biofilm. Most of the detected biofilm resonances belong to the third (0.5× 10 − 10 B DB2.5× 10 − 10 m2 s − 1) and fourth group (0.6× 10 − 11 B DB2.5× 10 − 11 m2 s − 1). As yet, we cannot assign these resonances, with the exception of the signal at 2.1 ppm, which results from acetyl groups of the polysaccharides from P. aeruginosa. Between 3.5 and 4.5 ppm the C-H protons of polysaccharides can be expected (Skja˚k-Bræk et al., 1986). A direct comparison to the results of Skja˚k-Bræk is Table 1 The NMR signals can be grouped into five groups with respect to their different diffusion coefficient Group
D (m2 s−1)
Signals (ppm)
1 2
1.8×10−9 6.0×10−10 – 6.3×10−10 0.5×10−10 – 2.5×10−10
4.77 3.49–3.58–3.71
3
and 4 5
0.6×10−11 – 2.5×10−11 1–5×10−13
0.77–0.87–1.18–1.33–1.61–1.96 – 2.23–3.80–3.86–4.09–4.34–4.55 – 4.77–8.04 2.06–3.49–3.58–3.71–4.09–4.34 – 4.77 2.06–3.49–3.58–3.71
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Fig. 6. PFG-spectra of P. aeruginosa biofilm (96 h) with constant t2-time, constant b and increasing diffusion time D by increasing t1.
not possible due to different experimental conditions, especially the higher temperature in their experiments (92°C) and the pH- or pD-dependence of the chemical shifts. The signals at 3.49, 3.58 and 3.71 ppm in group 4 arise from 10% of the glycerol molecules, which have an order of magnitude lower mobility than the glycerol molecules of group 2. We tentatively assign these slowly diffusing glycerol molecules to glycerol in the EPS network. That would imply that the diffusion of glycerol in the EPS network is 50 times slower than in the pores of the biofilm. The water fraction in group 3 (volume fraction of the total amount of water = 0.004) might be intracellular water. Its diffusion coefficient of 0.2 ×10 − 9 m2 s − 1 agrees well with the results of van Zijl et al. (1991). With 4.9×1010 cells pro cm3 biofilm and an estimated average cell volume of 2× 10 − 13 cm3, we find a cell volume fraction of the biofilm of 0.01, which is of the same order of magnitude as the fraction of water. In group 4 another resonance at 4.77 ppm is found. This third water fraction (B 0.1%) has a very low diffusion coefficient, only 0.01 of that of
pure water. It might be due to water entrapped in the secondary structure of EPS molecules, so that the mobility of the water molecules is strongly hindered (Belton, 1997). Most signals of the group four display a second, very small diffusion coefficient in the order of 1–5× 10 − 13 m2 s − 1 (group 5). For the determination of these extremely low diffusion coefficients it was necessary to use longer gradient pulses (d= 2 ms) and longer diffusion times (D= 40 ms). The resonances in these spectra result mainly from glycerol and the acetyl group of the polysaccharides of the EPS. So far we discussed experiments where D and d are constant but g is varied. In PFG-NMR it is also possible to vary the diffusion time. We have performed some experiments with increasing diffusion times D and constant b (b= g 2d 2g 2(D−1/ 3d)). Keeping b constant has the advantage that D can be varied without any effect of diffusion on the signal intensity. The signal intensity is then only affected by T1/T2 relaxation and/or exchange processes during D. To keep b constant we decreased the gradient strength g and kept the gradi-
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Fig. 7. PFG-spectra of P. aeruginosa biofilm (24 h) with constant t1-time, constant b and increasing diffusion time D by increasing t2.
ent pulse duration d constant. We performed two sets of experiments with the stimulated echo sequence, with t2 constant (Fig. 6) and with t1 constant (Fig. 7). The spectrum of Fig. 6 is from a biofilm which was cultivated for 96 h. This biofilm shows broader lines and slight differences in the signal intensities compared to the sample cultivated for 24 h. All diffusion coefficients of the 96 h biofilm, however, are within the experimental error equal to those of the 24 h biofilm. By increasing D from 10 to 30 ms the signal intensity of only one signal is significantly effected: the resonance intensity at 4.77 ppm of the water molecules with the low diffusion coefficients (from groups 3 and 4) decreases very quickly with increasing D. When we assume the 4.77 ppm signals of groups 3 and 4 are due to entrapped water (van Zijl et al., 1991; Belton, 1997; Beuling et al., 1998), these water molecules have either intrinsic shorter T1 and T2 relaxation times or with longer diffusion time D the entrapped water molecules increasingly exchange with the mobile water of group 1. The much larger D of the
mobile water then decreases the echo intensity according to Eq. (1).
4. Conclusions The PFG-NMR is a powerful method for studying the mobility of biofilm components. The results of the experiments demonstrate the extreme heterogeneity of a biofilm. We ordered the diffusion coefficients of 20 proton NMR signals into five groups with different ranges of the diffusion coefficient, from 10 − 9 to 10 − 13 m2 s − 1. The most prominent signals are from water and glycerol, of which at least three significantly different diffusion coefficients can be detected.
Acknowledgements We thank Mr Za¨hres for his technical support and Dr Wingender for cultivating the biofilm and for helpful discussions.
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References Anisimov, A.V., Sorokina, N.Y., Dautova, N.R., 1998. Water diffusion in biological porous systems: a NMR approach. Magn. Res. Imag. 16 (5/6), 565–568. Bar, N.K., Ka¨rger, J., Pfeifer, H., Scha¨fer, H., Schmitz, W., 1998. Diffusion anisotropy in natural Chabazite. Microporous Mesoporous Mater. 22, 289–295. Belton, P.S., 1997. NMR and the mobility of water in polysaccharide gels. Int. J. Biol. Macromol. 21, 81–88. Beuling, E.E., van Dusschoten, D., Lens, P., van den Heuvel, J.C., van As, H., Ottengraf, S.P.P., 1998. Characterization of the diffusive properties of biofilms using pulsed field gradient-nuclear magnetic resonance. Biotechnol. Bioeng. 60 (3), 283 – 291. Beyenal, H., Seker, S., Tanyolac, A., Salih, B., 1997. Diffusion coefficients of phenol and oxygen in a biofilm of Pseudomonas putida. AIChE 43, 242–250. Callaghan, P.T., 1991. Principles of Nuclear Magnetic Resonance Microscopy. Oxford University Press, Oxford. Casu, B., 1985. Nuclear magnetic resonance studies of polysaccharide structure and interactions. In: Atkins, E.D.T. (Ed.), Polysaccharides: Topics in Structure and Morphology. VCH, Weinheim, pp. 1–39. Chen, X., Steward, P.S., 1996. Chlorine penetration into artificial biofilm is limited by a reaction-diffusion interaction. Environ. Sci. Technol. 30, 2078–2083. Colin, F., Gazbat, S., 1995. Distribution of water in sludges in relation to their mechanical dewatering. Water Res. 29, 2000 – 2005. Colquhoun, I.J., Defernez, M., Morris, V.J., 1995. NMR studies of acetan and the related bacterial polysaccharide, CR1/4, produced by a mutant strain of Acetobacter xylinum. Carbohydr. Res. 269, 319–331. de Beer, D., Stoodley, P., Roe, F., Lewandowski, Z., 1994a. Effects of biofilm structures on oxygen distribution and mass transport. Biotechnol. Bioeng. 43, 1131–1138. de Beer, D., Stoodley, P., Lewandowski, Z., 1994b. Liquid flow in heterogeneous biofilms. Biotechnol. Bioeng. 53, 151 – 158. de Beer, D., Stoodley, P., 1995. Relation between the structure of an aerobic biofilm and transport phenomena. Water Sci. Tech. 32, 11 – 18. de Beer, D., Stoodley, P., Lewandowski, Z., 1997. Measurement of local diffusion coefficients in biofilms by microinjection and confocal microscopy. Biotechnol. Bioeng. 53, 151 – 158. Grobe, S., Wingender, J., Tru¨per, H.-G., 1995. Characterisation of mucoid Pseudomonas aeruginosa strains isolated of technical water systems. J. Appl. Bacteriol. 79, 94–102. Hinton, D.P., Johnson, C.S. Jr, 1993. Diffusion ordered 2D NMR spectroscopy of phospholipid vesicles: determination of vesicle size distributions. J. Phys. Chem. 97, 9064–9072. Ka¨rger, J., Ruthven, D.M., 1992. Diffusion in Zeolites and Other Microporous Solids. John Wiley, New York. Katsiris, N., Kouzeli-Katsiri, A., 1987. Bound water content of biological sludges in relation to filtration and dewatering. Water Res. 21, 1319–1327.
145
La Heij, E.J., Kerkhoff, P.J.A.M., 1996. Determining porosity profiles during filtration and expression of sewage sludge by NMR imaging. AIChE 42, 959 – 959. La Heij, E.J., Kerkhoff, P.J.A.M., Herwijn, A.J.M., Coumans, W.J., 1996. Fundamental aspects of sludge filtration and expression. Water Res. 30, 697 – 703. Latour, L.L., Mitra, P.P., Kleinberg, R.L., Sotak, C.H., 1993. Time dependent diffusion coefficient of fluids in porous media as a probe of surface-to-volume ratio. J. Magn. Res. A 101, 342 – 346. Lemercinier, X., Jones, C., 1996. Full 1H-assignment and detailed O-acetylation patterns of capsular polysaccharides from Neisseria meningitidis use in vaccine production. Carbohydr. Res. 296, 83 – 96. Lens, P., Pol, L.H., Lettinga, G., van As, H., 1997. Use of 1H NMR to study transport processes in sulfidogenic granular sludge. Water Sci. Tech. 36, 157 – 163. Lens, P., Vergeldt, F., Lettinga, G., van As, H., 1999. 1H NMR characterisation of the diffusional properties of methanogenic granular sludge. Water Sci. Tech. 39, 187 – 194. Lewandowski, Z., Stoodley, P., Altobelli, S., Fukushima, E., 1994. Hydrodynamics and kinetics in biofilm systems-recent advances and new problems. Water Sci. Tech. 29, 223 – 229. Manz, B., Seymour, J.D., Callaghan, P.T., 1997. PGSE NMR measurement of convection in a capillary. J. Magn. Res. 125, 153 – 158. Mayer, C., Moritz, R., Kirschner, C., Borchard, W., Maibaum, R., Wingender, J., Flemming, H.-C., 1999. The role of intermolecular interactions: studies on model systems for bacterial biofilms. J. Biol. Macromol. 26, 3 – 16. Moonen, C.T.W., van Zijl, P.C.M., 1990. Highly effective water suppression for in vivo proton NMR spectroscopy. J. Magn. Res. 88, 28 – 41. Ohtsuka, A., Watanabe, T., 1996. The network structure of gellan gum hydrogels based on the structural parameters by the analysis of the restricted diffusion of water. Carbohydr. Polym. 30, 135 – 140. Ohtsuka, A., Watanabe, T., Suzuki, T., 1994. Gel structure and water diffusion phenomena in starch gels studied by pulsed field gradient stimulated echo NMR. Carbohydr. Polym. 25, 95 – 100. Okabe, S., Yasuda, T., Watanabe, Y., 1997. Uptake and release of inert fluorescent particles by mixed population biofilms. Biotechnol. Bioeng. 53, 459 – 469. Otting, G., 1997. NMR studies of water bound to biological molecules. Prog. Nucl. Magn. Reson. Spectrosc. 31, 259 – 285. Skja˚k-Bræk, G., Grasdalen, H., Larsen, B., 1986. Monomer sequences and acetylation patterns in some bacterial alginates. Carbohydr. Res. 154, 239 – 250. Smith, J.K., Vesilind, P.A., 1995. Dilatrometric measurement of bound water in waste water sludge. Water Res. 29, 2621 – 2626. Stejskal, E.O., Tanner, J.E., 1965. Spin diffusion measurements: spin echoes in the presence of a time-dependent field gradient. J. Chem. Phys. 42, 288 – 292.
146
M. Vogt et al. / Journal of Biotechnology 77 (2000) 137–146
Stilbs, P., 1987. Fourier transform pulsed-gradient spin-echo studies of molecular diffusion. Progr. NMR Spectr. 19, 1–45. Tanner, J.E., 1968. Transient diffusion in a system partitioned by permeable barriers. Applications to NMR measurements with a pulsed field gradient. J. Chem. Phys. 69, 1748 – 1754. Tanner, J.E., 1970. Use of stimulated echo in NMR diffusion studies. J. Chem. Phys. 52, 2523–2526. van As, H., van Dusschoten, D., 1997. NMR methods for imaging of transport processes in micro-porous systems. Geoderma 80, 389 – 403. van As, H., Palstra, W., Tallarek, U., van Duschoten, D., 1998. Flow and transport studies in (non)consolidated porous (bio)systems consisting of solid or porous beads by PFG NMR. Magn. Res. Imag. 16, 569–573. van den Heuvel, J.C., Beuling, E.E., van Dusschoten, D., Roosenschoon, O.L., Verschuren, P.G., 1997. Convective flow in methanogenic granules. Water Sci. Tech. 36, 311– 316.
.
van Zijl, P.C.M., Moonen, C.T.W., Faustino, P., Pekar, J., Kaplan, O., Cohen, J.S., 1991. Complete separation of intracellular and extracellular information in NMR spectra of perfused cells by diffusion-weighted spectroscopy. Proc. Natl. Acad. Sci. USA 80, 3228 – 3232. Volke, F., Eisenbla¨tter, S., Galle, J., Klose, G., 1994. Dynamic properties of water at phosphatidylcholine lipidbilayer surfaces as seen by deuterium and pulsed field gradient proton NMR. Chem. Phys. Lipids 70, 121 – 131. von Meerwall, E.D., 1983. Self-diffusion in Polymer Systems, Measured with Field-Gradient Spin Echo NMR Methods. Advances in Polymer Science, 54. Springer-Verlag, Berlin Heidelberg, p. 3. Xu, X., Stewart, P.S., Chen, X., 1996. Transport limitation of chlorine disinfection of Pseudomonas aeruginosa entrapped in alginate beads. Biotechnol. Bioeng. 49, 93 – 100. Zakhartchenko, N.L., Skirda, V.D., Valiullin, R., 1998. Selfdiffusion of water and oil in peanuts investigated by PFG NMR. Magn. Res. Imag. 16, 583 – 586.
Diffusion in Pseudomonas aeruginosa biofilms: a pulsed field gradient NMR study M. Vogt a, H.-C. Flemming b, W.S. Veeman a,* a
Institute of Physical and Theoretical Chemistry, Gerhard-Mercator-Uni6ersity Duisburg, Lotharstrasse 1, 47057 Duisburg, Germany b Faculty for Aquatic Microbiology, Gerhard-Mercator-Uni6ersity Duisburg, Lotharstrasse 1, 47057 Duisburg, Germany Received 8 February 1999; received in revised form 9 July 1999; accepted 23 July 1999
Abstract A Pseudomonas aeruginosa biofilm is studied with pulsed field gradient echo nuclear magnetic resonance. Although not all spectral components are assigned yet, the experimental results show that a biofilm consists of components with very different diffusion coefficients. The various biofilm components that give motionally narrowed 1H NMR signals, can be grouped into five classes with diffusion coefficients, ranging from 2 × 10 − 9 to 1 × 10 − 13 m2 s − 1. Investigation of the diffusion behavior of water in the biofilm shows three fractions with different diffusion coefficients. Besides the highly mobile bulk water at least two other fractions with much lower diffusion coefficients are detected. It is shown that one of the fractions with the low diffusion coefficient probably arises from intracellular water. Also for another component of the biofilm, glycerol, three fractions with diffusion coefficients that differ more than a factor ten are detected. Also a group of signals exists which result from practically immobile components. © 2000 Elsevier Science B.V. All rights reserved. Keywords: PFG-NMR; Diffusion; Water; Biofilm; Pseudomonas aeruginosa
1. Introduction The aim of many studies of biofilms is to investigate either the structure of the biofilms or the diffusion of small molecules in biofilms. Especially the last aspect can provide insight how to optimize bioreactors and sludge dewatering while * Corresponding author. Tel.: + 49-203-3793320; fax: +49203-3793522. E-mail address: [email protected] (W.S. Veeman)
it determines the transport of nutrients, oxygen, disinfectants and water in wide areas of the biofilm (de Beer et al., 1994a; Chen and Steward, 1996; Xu et al., 1996; Beyenal et al., 1997; Beuling et al., 1998). The diffusion of small molecules in a biofilm can be determined without knowing the detailed biofilm structure, but can of course only be understood when the microscopical structure of the biofilm is known. Reversely, knowing how the various components in a biofilm can diffuse, in principle yields information about the biofilm structure.
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Many publications deal with the general aspects of diffusion in biological systems (Moonen and van Zijl, 1990; van Zijl et al., 1991; Hinton and Johnson, 1993; Ohtsuka et al., 1994; Ohtsuka and Watanabe, 1996; Anisimov et al., 1998; van As et al., 1998), but only a few publications deal with the mobility of water in sludges or biofilms. Lens et al. (1997, 1999) have shown that there are several types of water in sludges, e.g. intracellular water and water in cavities. van den Heuvel et al. (1997) have examined the mobility of water in methanogenic granules and found convective flow during pressure oscillations. Other authors differentiate the water in sludges in free or bulk water and bound water (‘freezable’ and ‘non-freezable’ water, respectively). Whereas the free water can be easily removed from sludges, the bound water can only be removed at a high expense of energy (Katsiris and Kouzeli-Katsiri, 1987; Colin and Gazbat, 1995; Smith and Vesilind, 1995; La Heij and Kerkhoff, 1996; La Heij et al., 1996). The mobility of water in several artificial and natural biofilms was examined by Beuling et al. (1998) and by van Zijl et al. (1991). Four methods are commonly used to determine diffusion coefficients in biological systems. Two of these methods use fluorescent labelled tracers and a confocal laser microscope (Scanning Confocal Laser Microscopy (SCLM) and Fluorescence Recovery After Photobleaching (FRAP)). SCLM and FRAP are excellent for three-dimensional studies of the mobility (diffusion and/or convection) of fluorescent molecules or particles in biofilms (de Beer et al., 1997; Okabe et al., 1997). With these methods de Beer et al. (1994b) have shown that there is liquid flow in biofilms. A disadvantage of both methods is that they need fluorescent molecules or particles. Therefore it is not possible to observe water or other small molecules directly. The third method uses microelectrodes, which are sensitive for one type of molecule (de Beer et al., 1994a; Lewandowski et al., 1994; de Beer and Stoodley, 1995). The last method is nuclear magnetic resonance, which uses NMR-active atoms (e.g. 1H, 13C, 15N, 31P) as a tracer. NMR can be used to get spatial information about the structure of biofilms or sludges by NMR-imaging techniques (Lewandowski et al.,
1994; La Heij and Kerkhoff, 1996; van As and van Dusschoten, 1997), about the self-diffusion of molecules by pulsed field gradient NMR (PFGNMR; van Zijl et al., 1991; Volke et al., 1994; Lens et al., 1997; Manz et al., 1997; Beuling et al., 1998) and about the chemical structure of molecules and their interactions with other biofilm constituents (Casu, 1985; Skja˚k-Bræk et al., 1986; Colquhoun et al., 1995; Lemercinier and Jones, 1996). Although the PFG-NMR method is known since the 1960s (Stejskal and Tanner, 1965; Tanner, 1968), only in recent years the PFG-NMR method has become very popular, especially to study restricted diffusion in porous systems (von Meerwall, 1983; Callaghan, 1991; Ka¨rger and Ruthven, 1992; Latour et al., 1993; Bar et al., 1998; Zakhartchenko et al., 1998). PFG-NMR uses pulsed magnetic field gradients to gain information about the average displacement of the spins. With PFG-NMR it is in principle possible to measure the self-diffusion coefficients of all NMR-detectable molecules. The PFG-NMR method is reviewed by, e.g. Stilbs (1987). The most sensitive NMR nucleus is the proton and since in biological systems nearly all molecules contain protons, it is the most favorable NMR probe for diffusion studies in biofilms. A problem in such systems is the large water content (up to 98%) whose NMR resonance dominates the proton spectrum of the biofilm. In this paper we use the PFG-NMR method for a diffusion ordered examination of biofilms. That means that the proton spectra of molecular systems in biofilms with different mobility (different self-diffusion coefficients) can be separately obtained. Similar experiments on biological materials have been reported by van Zijl et al. (1991) and Hinton and Johnson (1993). In addition we use PFG-NMR to suppress the signal of free water (Stilbs, 1987; van Zijl et al., 1991), which is the component with the highest mobility in biofilms (Otting, 1997). 1H spectra of the biofilm can then be obtained without the necessity of D2O– H2O exchange to eliminate the dominating water resonance (Skja˚k-Bræk et al., 1986). When the dominating signal of fast diffusing bulk water is suppressed, the diffusion coefficients from the
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other biofilm resonances can be measured. It will be shown that the NMR signals of a biofilm result from components with very different mobility. Some signals represent molecular systems with extremely low diffusion coefficients. On the other hand it should be realized that the employed NMR method is a typical high-resolution ‘liquidtype’ experiment. Bacteria in biofilms, for instance, contain many protons, but it must be expected that for proton spins in the bacteria cell walls, the absence of fast motional averaging will broaden their signals beyond recognition. As biofilm, Pseudomonas aeruginosa was chosen because it produces a well known extracellular polymeric substance (EPS), i.e. alginate, and because a highly mucoid strain (Grobe et al., 1995) was available which allowed the harvest of sufficient quantities of defined biomass for the NMR investigations. It has to be acknowledged that the collected P. aeruginosa biomass in the NMR tube, of course, is not completely equivalent to a thin biofilm.
2. Materials and methods In this study we use P. aeruginosa biofilms. The biofilms were cultivated on Pseudomonas Isolation Agar (Difco) in petri dishes for 24 or 96 h at 30°C (Grobe et al., 1995; Mayer et al., 1999). The biofilm was carefully removed from the plate and was transferred into a NMR-sample tube with a diameter of 5 mm. The biofilm contained (93 9 1)% water and 6.9 × 1011 cells per g dry weight. With a density of 1.016 for the biofilm one finds 4.9 ×1010 cells per cm3. The NMR-spectra were recorded with a Bruker Avance DRX 500 spectrometer, equipped with a gradient amplifier BAFPA 40 and a Bruker Diff30 probehead with 5 mm 1H-coil.
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All PFG-NMR experiments were performed with a stimulated echo pulse sequence (Fig. 1; Tanner, 1970) with 11 ms 90° pulses and a 10 s recycle delay. The echo intensity I can be written as: I= I0 e − Db with:
(1)
1 b= g 2 d 2 g 2 D− d 3
(2)
where I0 is the echo intensity without field gradients; D is the apparent self-diffusion coefficient (m2 s − 1); g is the gyromagnetic ratio (T − 1 rad s − 1); g is the gradient strength (T m − 1); d is the gradient pulse length (s); D is the time between the pulsed gradients= diffusion time (s). For simplicity we will in the following use the expression ‘diffusion coefficient’ instead of ‘apparent self-diffusion coefficient’. In principle, the diffusion coefficient D can be determined by varying g, d or D. To eliminate the effect of spin relaxation we kept for most experiments d and D constant and varied g. The spectrum is obtained after Fourier transformation of the echo signal. The diffusion coefficients of the biofilm components were determined by deconvolution of the 1H spectrum with 20 separate lines (Bruker Winfit). After deconvolution the signal intensities were fitted mono-, and when necessary, biexponentially with the program Microcal Origin. The diffusion coefficients were determined by varying the gradient strength g in 32 steps, in two runs of each 16 steps, between 0.5 and 8 T m − 1 with a diffusion time D of 10 ms and a gradient pulse length d of 1 ms. The gradient system was calibrated using the known self-diffusion coefficients of pure water and pure glycerol, respectively, at room temperature (T= 295 K).
3. Results and discussion
Fig. 1. Pulse scheme of the PFG-NMR stimulated echo sequence.
Fig. 2 shows the logarithmic plot of the echo intensity of the water signal at 4.77 ppm of the 24 h biofilm as a function of g 2. Fitting the exponen-
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Fig. 2. Logarithmic plot of the signal attenuation of the water resonance at 4.77 ppm in a P. aeruginosa biofilm.
tial curve (not shown) with Eq. (1) up to a gradient strength of 3 T m − 1 yields a diffusion coefficient for the bulk biofilm water Dbulk of 1.8×10 − 9 m2 s − 1. The diffusion coefficient of water in the biofilm matrix corresponds to 85% of the value in pure water (2.1 ×10 − 9 m2 s − 1). When we simplify the biofilm structure to a liquid phase containing for water impermeable bacterial cells, we can apply the following relation between Dbulk and the diffusion coefficient of pure water Daq (Beuling et al., 1998): Dbulk =
1−f D 1 aq 1+ f 2
(3)
where f is the volume fraction of the impermeable phase. The resulting value for f is 10.5%, which seems too high in comparison to the biofilm water weight fraction of ca. 93% and a dry mass of ca. 7% (Mayer et al., 1999). The twophase model used here is certainly too simple while the signal we ascribe to bulk water results from water in the channels of the biofilm and in the extracellular polymeric substance (EPS), with unequal diffusion coefficients. Besides allowing the determination of diffusion coefficients PFG-NMR also makes it possible to
suppress the signal of mobile water in hydrogels and biofilms (van Zijl et al., 1991; Hinton and Johnson, 1993). Fig. 3 shows the calculated intensity of the echo signal as a function of the gradient strength g by constant D=10 ms and d=1 ms for different diffusion coefficients (D=1.8×10 − 9, D= 1.0× 10 − 10, D= 1.0× 10 − 11 m2 s − 1). It is clear that for larger gradients the signals from components with high diffusion coefficients will be suppressed. This technique can be employed to eliminate the dominating water peak, and other peaks from components with high mobility, from the 1H spectrum of the biofilm. In Fig. 4 the 1H spectrum of a P. aeruginosa biofilm (24 h) is shown. The mobile water signal at 4.77 ppm dominates the spectrum. Fig. 5 shows the spectrum when the signal from very mobile water is suppressed by using g= 3 T m − 1. This method allows to observe biofilm spectra without any further preparation like exchanging the free water with heavy water. Signals of fast diffusing components in the biofilm are of course also suppressed by this method. In the spectrum with water suppression there is still a signal at 4.77 ppm left. This signal results from water with a significantly lower mobility than the biofilm bulk water, discussed above.
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Fig. 3. Calculated intensity I of a NMR-echo-signal for different diffusion coefficients D as a function of gradient strength (diffusion time D = 10 ms, gradient pulse length d= 1 ms).
Fig. 4. (a) 1H spectrum of a P. aeruginosa biofilm (24 h); and (b) enlargement of spectrum a (× 270).
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Fig. 5. PFG-1H spectrum of a P. aeruginosa (24 h) biofilm with water suppression (g =3 T m − 1, D =10 ms, d =1 ms).
To determine the diffusion coefficients of the various components of the biofilm, including the slowly diffusing water, the echo signal intensities are plotted for 16 gradient values above 3 T m − 1. In the spectra many overlapping signals can be found. Instead of integrating the signals the complete spectrum was deconvoluted with 20 signals. For each of the 20 signals the diffusion coefficient can then be determined by fitting the intensity as a function of g with Eq. (1). The resulting diffusion coefficients can be sorted into five groups with different diffusion coefficients, see Table 1. Group one contains only the signal of the biofilm bulk water at 4.77 ppm, as discussed above. The second group contains three resonances, at 3.49, 3.58 and 3.71 ppm, which result from glycerol. Glycerol is added to the agar as nutrient (20 g l − 1). The effective diffusion coefficient of the glycerol in the biofilm (6.2 ×10 − 10 m2 s − 1) is significantly lower than that of diluted glycerol in water at 20°C (8×10 − 10 m2 s − 1); the diffusion of glycerol in the biofilm is similarly affected by the biofilm as the diffusion of water: Dglycerol in biofilm =0.34 Dwater in biofilm Dglycerol in water =0.38 Dwater in water
Since also much slower diffusing glycerol is detected (see below), the fast diffusing fraction (90%) is believed to be located in the water filled pores of the biofilm. Most of the detected biofilm resonances belong to the third (0.5× 10 − 10 B DB2.5× 10 − 10 m2 s − 1) and fourth group (0.6× 10 − 11 B DB2.5× 10 − 11 m2 s − 1). As yet, we cannot assign these resonances, with the exception of the signal at 2.1 ppm, which results from acetyl groups of the polysaccharides from P. aeruginosa. Between 3.5 and 4.5 ppm the C-H protons of polysaccharides can be expected (Skja˚k-Bræk et al., 1986). A direct comparison to the results of Skja˚k-Bræk is Table 1 The NMR signals can be grouped into five groups with respect to their different diffusion coefficient Group
D (m2 s−1)
Signals (ppm)
1 2
1.8×10−9 6.0×10−10 – 6.3×10−10 0.5×10−10 – 2.5×10−10
4.77 3.49–3.58–3.71
3
and 4 5
0.6×10−11 – 2.5×10−11 1–5×10−13
0.77–0.87–1.18–1.33–1.61–1.96 – 2.23–3.80–3.86–4.09–4.34–4.55 – 4.77–8.04 2.06–3.49–3.58–3.71–4.09–4.34 – 4.77 2.06–3.49–3.58–3.71
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Fig. 6. PFG-spectra of P. aeruginosa biofilm (96 h) with constant t2-time, constant b and increasing diffusion time D by increasing t1.
not possible due to different experimental conditions, especially the higher temperature in their experiments (92°C) and the pH- or pD-dependence of the chemical shifts. The signals at 3.49, 3.58 and 3.71 ppm in group 4 arise from 10% of the glycerol molecules, which have an order of magnitude lower mobility than the glycerol molecules of group 2. We tentatively assign these slowly diffusing glycerol molecules to glycerol in the EPS network. That would imply that the diffusion of glycerol in the EPS network is 50 times slower than in the pores of the biofilm. The water fraction in group 3 (volume fraction of the total amount of water = 0.004) might be intracellular water. Its diffusion coefficient of 0.2 ×10 − 9 m2 s − 1 agrees well with the results of van Zijl et al. (1991). With 4.9×1010 cells pro cm3 biofilm and an estimated average cell volume of 2× 10 − 13 cm3, we find a cell volume fraction of the biofilm of 0.01, which is of the same order of magnitude as the fraction of water. In group 4 another resonance at 4.77 ppm is found. This third water fraction (B 0.1%) has a very low diffusion coefficient, only 0.01 of that of
pure water. It might be due to water entrapped in the secondary structure of EPS molecules, so that the mobility of the water molecules is strongly hindered (Belton, 1997). Most signals of the group four display a second, very small diffusion coefficient in the order of 1–5× 10 − 13 m2 s − 1 (group 5). For the determination of these extremely low diffusion coefficients it was necessary to use longer gradient pulses (d= 2 ms) and longer diffusion times (D= 40 ms). The resonances in these spectra result mainly from glycerol and the acetyl group of the polysaccharides of the EPS. So far we discussed experiments where D and d are constant but g is varied. In PFG-NMR it is also possible to vary the diffusion time. We have performed some experiments with increasing diffusion times D and constant b (b= g 2d 2g 2(D−1/ 3d)). Keeping b constant has the advantage that D can be varied without any effect of diffusion on the signal intensity. The signal intensity is then only affected by T1/T2 relaxation and/or exchange processes during D. To keep b constant we decreased the gradient strength g and kept the gradi-
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Fig. 7. PFG-spectra of P. aeruginosa biofilm (24 h) with constant t1-time, constant b and increasing diffusion time D by increasing t2.
ent pulse duration d constant. We performed two sets of experiments with the stimulated echo sequence, with t2 constant (Fig. 6) and with t1 constant (Fig. 7). The spectrum of Fig. 6 is from a biofilm which was cultivated for 96 h. This biofilm shows broader lines and slight differences in the signal intensities compared to the sample cultivated for 24 h. All diffusion coefficients of the 96 h biofilm, however, are within the experimental error equal to those of the 24 h biofilm. By increasing D from 10 to 30 ms the signal intensity of only one signal is significantly effected: the resonance intensity at 4.77 ppm of the water molecules with the low diffusion coefficients (from groups 3 and 4) decreases very quickly with increasing D. When we assume the 4.77 ppm signals of groups 3 and 4 are due to entrapped water (van Zijl et al., 1991; Belton, 1997; Beuling et al., 1998), these water molecules have either intrinsic shorter T1 and T2 relaxation times or with longer diffusion time D the entrapped water molecules increasingly exchange with the mobile water of group 1. The much larger D of the
mobile water then decreases the echo intensity according to Eq. (1).
4. Conclusions The PFG-NMR is a powerful method for studying the mobility of biofilm components. The results of the experiments demonstrate the extreme heterogeneity of a biofilm. We ordered the diffusion coefficients of 20 proton NMR signals into five groups with different ranges of the diffusion coefficient, from 10 − 9 to 10 − 13 m2 s − 1. The most prominent signals are from water and glycerol, of which at least three significantly different diffusion coefficients can be detected.
Acknowledgements We thank Mr Za¨hres for his technical support and Dr Wingender for cultivating the biofilm and for helpful discussions.
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References Anisimov, A.V., Sorokina, N.Y., Dautova, N.R., 1998. Water diffusion in biological porous systems: a NMR approach. Magn. Res. Imag. 16 (5/6), 565–568. Bar, N.K., Ka¨rger, J., Pfeifer, H., Scha¨fer, H., Schmitz, W., 1998. Diffusion anisotropy in natural Chabazite. Microporous Mesoporous Mater. 22, 289–295. Belton, P.S., 1997. NMR and the mobility of water in polysaccharide gels. Int. J. Biol. Macromol. 21, 81–88. Beuling, E.E., van Dusschoten, D., Lens, P., van den Heuvel, J.C., van As, H., Ottengraf, S.P.P., 1998. Characterization of the diffusive properties of biofilms using pulsed field gradient-nuclear magnetic resonance. Biotechnol. Bioeng. 60 (3), 283 – 291. Beyenal, H., Seker, S., Tanyolac, A., Salih, B., 1997. Diffusion coefficients of phenol and oxygen in a biofilm of Pseudomonas putida. AIChE 43, 242–250. Callaghan, P.T., 1991. Principles of Nuclear Magnetic Resonance Microscopy. Oxford University Press, Oxford. Casu, B., 1985. Nuclear magnetic resonance studies of polysaccharide structure and interactions. In: Atkins, E.D.T. (Ed.), Polysaccharides: Topics in Structure and Morphology. VCH, Weinheim, pp. 1–39. Chen, X., Steward, P.S., 1996. Chlorine penetration into artificial biofilm is limited by a reaction-diffusion interaction. Environ. Sci. Technol. 30, 2078–2083. Colin, F., Gazbat, S., 1995. Distribution of water in sludges in relation to their mechanical dewatering. Water Res. 29, 2000 – 2005. Colquhoun, I.J., Defernez, M., Morris, V.J., 1995. NMR studies of acetan and the related bacterial polysaccharide, CR1/4, produced by a mutant strain of Acetobacter xylinum. Carbohydr. Res. 269, 319–331. de Beer, D., Stoodley, P., Roe, F., Lewandowski, Z., 1994a. Effects of biofilm structures on oxygen distribution and mass transport. Biotechnol. Bioeng. 43, 1131–1138. de Beer, D., Stoodley, P., Lewandowski, Z., 1994b. Liquid flow in heterogeneous biofilms. Biotechnol. Bioeng. 53, 151 – 158. de Beer, D., Stoodley, P., 1995. Relation between the structure of an aerobic biofilm and transport phenomena. Water Sci. Tech. 32, 11 – 18. de Beer, D., Stoodley, P., Lewandowski, Z., 1997. Measurement of local diffusion coefficients in biofilms by microinjection and confocal microscopy. Biotechnol. Bioeng. 53, 151 – 158. Grobe, S., Wingender, J., Tru¨per, H.-G., 1995. Characterisation of mucoid Pseudomonas aeruginosa strains isolated of technical water systems. J. Appl. Bacteriol. 79, 94–102. Hinton, D.P., Johnson, C.S. Jr, 1993. Diffusion ordered 2D NMR spectroscopy of phospholipid vesicles: determination of vesicle size distributions. J. Phys. Chem. 97, 9064–9072. Ka¨rger, J., Ruthven, D.M., 1992. Diffusion in Zeolites and Other Microporous Solids. John Wiley, New York. Katsiris, N., Kouzeli-Katsiri, A., 1987. Bound water content of biological sludges in relation to filtration and dewatering. Water Res. 21, 1319–1327.
145
La Heij, E.J., Kerkhoff, P.J.A.M., 1996. Determining porosity profiles during filtration and expression of sewage sludge by NMR imaging. AIChE 42, 959 – 959. La Heij, E.J., Kerkhoff, P.J.A.M., Herwijn, A.J.M., Coumans, W.J., 1996. Fundamental aspects of sludge filtration and expression. Water Res. 30, 697 – 703. Latour, L.L., Mitra, P.P., Kleinberg, R.L., Sotak, C.H., 1993. Time dependent diffusion coefficient of fluids in porous media as a probe of surface-to-volume ratio. J. Magn. Res. A 101, 342 – 346. Lemercinier, X., Jones, C., 1996. Full 1H-assignment and detailed O-acetylation patterns of capsular polysaccharides from Neisseria meningitidis use in vaccine production. Carbohydr. Res. 296, 83 – 96. Lens, P., Pol, L.H., Lettinga, G., van As, H., 1997. Use of 1H NMR to study transport processes in sulfidogenic granular sludge. Water Sci. Tech. 36, 157 – 163. Lens, P., Vergeldt, F., Lettinga, G., van As, H., 1999. 1H NMR characterisation of the diffusional properties of methanogenic granular sludge. Water Sci. Tech. 39, 187 – 194. Lewandowski, Z., Stoodley, P., Altobelli, S., Fukushima, E., 1994. Hydrodynamics and kinetics in biofilm systems-recent advances and new problems. Water Sci. Tech. 29, 223 – 229. Manz, B., Seymour, J.D., Callaghan, P.T., 1997. PGSE NMR measurement of convection in a capillary. J. Magn. Res. 125, 153 – 158. Mayer, C., Moritz, R., Kirschner, C., Borchard, W., Maibaum, R., Wingender, J., Flemming, H.-C., 1999. The role of intermolecular interactions: studies on model systems for bacterial biofilms. J. Biol. Macromol. 26, 3 – 16. Moonen, C.T.W., van Zijl, P.C.M., 1990. Highly effective water suppression for in vivo proton NMR spectroscopy. J. Magn. Res. 88, 28 – 41. Ohtsuka, A., Watanabe, T., 1996. The network structure of gellan gum hydrogels based on the structural parameters by the analysis of the restricted diffusion of water. Carbohydr. Polym. 30, 135 – 140. Ohtsuka, A., Watanabe, T., Suzuki, T., 1994. Gel structure and water diffusion phenomena in starch gels studied by pulsed field gradient stimulated echo NMR. Carbohydr. Polym. 25, 95 – 100. Okabe, S., Yasuda, T., Watanabe, Y., 1997. Uptake and release of inert fluorescent particles by mixed population biofilms. Biotechnol. Bioeng. 53, 459 – 469. Otting, G., 1997. NMR studies of water bound to biological molecules. Prog. Nucl. Magn. Reson. Spectrosc. 31, 259 – 285. Skja˚k-Bræk, G., Grasdalen, H., Larsen, B., 1986. Monomer sequences and acetylation patterns in some bacterial alginates. Carbohydr. Res. 154, 239 – 250. Smith, J.K., Vesilind, P.A., 1995. Dilatrometric measurement of bound water in waste water sludge. Water Res. 29, 2621 – 2626. Stejskal, E.O., Tanner, J.E., 1965. Spin diffusion measurements: spin echoes in the presence of a time-dependent field gradient. J. Chem. Phys. 42, 288 – 292.
146
M. Vogt et al. / Journal of Biotechnology 77 (2000) 137–146
Stilbs, P., 1987. Fourier transform pulsed-gradient spin-echo studies of molecular diffusion. Progr. NMR Spectr. 19, 1–45. Tanner, J.E., 1968. Transient diffusion in a system partitioned by permeable barriers. Applications to NMR measurements with a pulsed field gradient. J. Chem. Phys. 69, 1748 – 1754. Tanner, J.E., 1970. Use of stimulated echo in NMR diffusion studies. J. Chem. Phys. 52, 2523–2526. van As, H., van Dusschoten, D., 1997. NMR methods for imaging of transport processes in micro-porous systems. Geoderma 80, 389 – 403. van As, H., Palstra, W., Tallarek, U., van Duschoten, D., 1998. Flow and transport studies in (non)consolidated porous (bio)systems consisting of solid or porous beads by PFG NMR. Magn. Res. Imag. 16, 569–573. van den Heuvel, J.C., Beuling, E.E., van Dusschoten, D., Roosenschoon, O.L., Verschuren, P.G., 1997. Convective flow in methanogenic granules. Water Sci. Tech. 36, 311– 316.
.
van Zijl, P.C.M., Moonen, C.T.W., Faustino, P., Pekar, J., Kaplan, O., Cohen, J.S., 1991. Complete separation of intracellular and extracellular information in NMR spectra of perfused cells by diffusion-weighted spectroscopy. Proc. Natl. Acad. Sci. USA 80, 3228 – 3232. Volke, F., Eisenbla¨tter, S., Galle, J., Klose, G., 1994. Dynamic properties of water at phosphatidylcholine lipidbilayer surfaces as seen by deuterium and pulsed field gradient proton NMR. Chem. Phys. Lipids 70, 121 – 131. von Meerwall, E.D., 1983. Self-diffusion in Polymer Systems, Measured with Field-Gradient Spin Echo NMR Methods. Advances in Polymer Science, 54. Springer-Verlag, Berlin Heidelberg, p. 3. Xu, X., Stewart, P.S., Chen, X., 1996. Transport limitation of chlorine disinfection of Pseudomonas aeruginosa entrapped in alginate beads. Biotechnol. Bioeng. 49, 93 – 100. Zakhartchenko, N.L., Skirda, V.D., Valiullin, R., 1998. Selfdiffusion of water and oil in peanuts investigated by PFG NMR. Magn. Res. Imag. 16, 583 – 586.