Analysis of Bacteria on Steel Surfaces Using Reflectance Micro

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Anal. Chem. 2009, 81, 6467–6473

Analysis of Bacteria on Steel Surfaces Using Reflectance Micro-Fourier Transform Infrared Spectroscopy Jesu´s J. Ojeda,* Marı´a E. Romero-Gonza´lez, and Steven A. Banwart Cell-Mineral Research Center, Kroto Research Institute, The University of Sheffield, Broad Lane, Sheffield S3 7HQ, U.K. Reflectance micro-Fourier transform infrared (FT-IR) analysis has been applied to characterize biofilm formation of Aquabacterium commune, a common microorganism present on drinking water distribution systems, onto the increasingly popular pipe material stainless steel EN1.4307. The applicability of the reflectance micro-FTIR technique for analyzing the bacterial functional groups is discussed, and the results are compared to spectra obtained using more conventional FT-IR techniques: transmission micro-FT-IR, attenuated transmitted reflectance (ATR), and KBr pellets. The differences between the infrared spectra of wet and dried bacteria, as well as free versus attached bacteria, are also discussed. The spectra obtained using reflectance micro-FT-IR spectroscopy were comparable to those obtained using other FT-IR techniques. The absence of sample preparation, the potential to analyze intact samples, and the ability to characterize opaque and thick samples without the need to transfer the bacterial samples to an infrared transparent medium or produce a pure culture were the main advantages of reflectance micro-FT-IR spectroscopy. Microbial quality of drinking water is globally monitored. These analyses are important, apart from legislative requirements, to monitor biofouling that could affect the water taste and cause water discoloration,1-3 to detect the presence of pathogenic bacteria,4,5 to study the immobilization of particulate matter and heavy metals by bacteria,6-8 and to predict influence of these bacteria in the corrosion processes of metal pipes.9-11 More and more often water companies turn to analytical sciences in search of new tools * Corresponding author. Phone: +44 114 2225774. Fax: +44 114 2225701. E-mail: [email protected]. (1) Bays, L. R.; Burman, N. P.; Lewis, W. M. Water Treat. Exam. 1970, 19, 136–160. (2) HMSO. The microbiology of water 1994 Part I - Drinking Water. Report on Public Health and Medical Subjects No. 71; HMSO Books: London, 1994. (3) Ratsack, U. Neue Deliwa-Z. 1997, 5, 195–198. (4) Camper, A. K.; Jones, W. L.; Hayes, J. T. Appl. Environ. Microbiol. 1996, 62, 4014–4018. (5) LeChevallier, M. W.; Babcock, T. M.; Lee, R. G. Appl. Environ. Microbiol. 1987, 53, 2714–2724. (6) Gauthier, V.; Gerard, B.; Portal, J. M.; Block, J. C.; Gatel, D. Water Res. 1999, 33, 1014–1026. (7) Percival, S. L.; Knapp, J. S.; Edyvean, R. G. J.; Wales, D. S. Water Res. 1998, 32, 2187–2201. (8) Zacheus, O. M.; Lehtola, M. J.; Korhonen, L. K.; Martikainen, P. J. Water Res. 2001, 35, 1757–1765. (9) Beech, I. B.; Edyvean, R. G. J.; Cheung, C. W. S.; Turner, A. European Federation of Corrosion, Portugal, 1994; pp 328-337. 10.1021/ac900841c CCC: $40.75  2009 American Chemical Society Published on Web 07/06/2009

to speed up water quality measurements in order to safeguard public health. A potential rapid and inexpensive method to detect the presence of bacteria on surfaces and to identify the functional groups present in the cell wall is Fourier transform infrared spectroscopy (FT-IR).12-16 Infrared spectroscopy is a wellestablished technique to identify functional groups in organic molecules based on their vibration modes at different infrared wave numbers and has already been applied in monitoring of drinking water.14,15,17-19 The presence or absence of functional groups, their protonation states, or any changes due to new interactions can be monitored by analyzing the position and intensity of the different infrared absorption bands.20-24 Additionally, infrared spectroscopy is nondestructive and can be used to monitor the chemistry of living cells as well. Despite the complexity of the spectra, the elucidation of functional groups on gramnegative and gram-positive bacteria have already been wellestablished by infrared spectroscopy.12-16,22,24,25 FT-IR microspectroscopy is a novel tool that combines the FT-IR spectroscopy with microscopy.26 When micro-FT-IR spectroscopy is used, infrared bands corresponding to proteins, lipids, polysaccharides, (10) Keevil, C. W. Water Sci. Technol. 2004, 49, 91–98. (11) Kobrin, G.; Lamb, S.; Tuthill, A. H.; Avery, R. E.; Selby, K. A. Microbiologically influenced corrosion of stainless steels by water used for cooling and hydrostatic testing, 1997 International Water Conference, Pittsburgh, PA, NiDI Technical Series No 10 085. (12) Dittrich, M.; Sibler, S. J. Colloid Interface Sci. 2005, 286, 487–495. (13) Jiang, W.; Saxena, A.; Song, B.; Ward, B. B.; Beveridge, T. J.; Myneni, S. C. B. Langmuir 2004, 20, 11433–11442. (14) Ojeda, J. J.; Romero-Gonzalez, M. E.; Bachmann, R. T.; Edyvean, R. G. J.; Banwart, S. A. Langmuir 2008, 24, 4032–4040. (15) Schmitt, J.; Flemming, H. C. Int. Biodeterior. Biodegrad. 1998, 41, 1–11. (16) Yee, N.; Benning, L. G.; Phoenix, V. R.; Ferris, F. G. Environ. Sci. Technol. 2004, 38, 775–782. (17) Delille, A.; Quiles, F.; Humbert, F. Appl. Environ. Microbiol. 2007, 73, 5782–5788. (18) Park, S. J.; Lee, C. G.; Kim, S. B. Colloid Surf., B: Biointerfaces. 2009, 68, 79–82. (19) Raichlin, Y.; Marx, S.; Gerber, L.; Katzir, A. In Infrared fiber-optic evanescent wave spectroscopy and its applications for the detection of toxic materials in water, in situ and in real time, Conference on Optically Based Biological and Chemical Sensing for Defence, London, England, Oct 25-28, 2004; Carrano, J. C.; Zukauskas, A., Eds. London, England, 2004; pp 145-153. (20) Dubois, J.; Shaw, R. A. Anal. Chem. 2004, 76, 360 A367 A. (21) Frankel, D. S. Anal. Chem. 1984, 56, 1011–1014. (22) Conley, R. T. Infrared Spectroscopy; Allyn and Bacon: Boston, 1972. (23) Skoog, D. A.; Leary, J. J. Principles of Instrumental Analysis; Saunders College Publishing: Philadelphia, 1992. (24) Wade, L. G. Organic Chemistry; Prentice-Hall: Upper Saddle River, NJ, 1995. (25) Ede, S. M.; Hafner, L. M.; Fredericks, P. M. Appl. Spectrosc. 2004, 58, 317–322. (26) Koenig, J. L.; Wang, S.-Q.; Bhargava, R. Anal. Chem. 2001, 73, 360 A369 A.

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polyphosphate groups, and other carbohydrate functional groups can now be identified and compared along different microorganisms even without the need to produce a pure culture.27-31 MicroFT-IR analyses of bacteria can be performed either in transmission or reflectance mode. Transmission mode gives high quality spectra but requires infrared transparent surfaces and transfer of the colonies from the agar plate.30-32 This sample preparation sometimes can be difficult to standardize and difficult to establish as a routine protocol. Additionally, it limits the possibility to analyze intact larger samples and the utilization of thick or opaque surfaces. Recent advances in detector sensitivity have allowed the use of reflectance micro-FT-IR spectroscopy as an important analytical tool to analyze raw materials without the need of previous treatment, and it has become increasingly popular in fields such as geochemistry and coal structure analysis.33-36 However, the applicability of micro-FT-IR analysis in reflectance mode for biofilm samples has not yet been studied. This paper presents, to our knowledge, the first evaluation of the applicability of reflectance micro-FT-IR spectroscopy to bacterial cells and compares the obtained results with more conventional FT-IR techniques, such as transmission microFT-IR, attenuated transmitted reflectance (ATR), and KBr pellets, as well as the differences between the infrared spectra of wet and dried bacteria. The advantage that reflectance microFT-IR offers to the characterization of Aquabacterium commune, a predominant member of various European drinking water distribution systems37,38 in the increasingly popular pipe material stainless steel EN1.4307,39 is also discussed. EXPERIMENTAL SECTION Preparation and Purification of A. commune. A. commune (DSMZ-11901) was grown in modified R2A medium, according to the procedures described by Kalmbach et al. and Bachmann and Edyvean,38,40 containing (per liter of water): 0.5 g of yeast extract, 0.5 g of proteose peptone No.3, 0.5 g of casamino acid, 0.3 g of sodium pyruvate, 1.2 mL of Tween 80, 0.3 g of K2HPO4, and 0.1 g of MgSO4 · 7H2O. The modified R2A medium was sterilized by autoclaving at 121 °C for 20 min. All chemicals were purchased from Sigma-Aldrich and Fisher (U.K.). (27) Naumann, D.; Helm, D.; Labischinski, H. Nature 1991, 351, 81–82. (28) Stehfest, K.; Toepel, J.; Wilhelm, C. Plant Physiol. Biochem. 2005, 43, 717– 726. (29) Wenning, M.; Seiler, H.; Scherer, S. Appl. Environ. Microbiol. 2002, 68, 4717–4721. (30) Wenning, M.; Theilmann, V.; Scherer, S. Environ. Microbiol. 2006, 8, 848– 857. (31) Orsini, F.; Ami, D.; Villa, A. M.; Sala, G.; Bellotti, M. G.; Doglia, S. M. J. Microbiol. Methods 2000, 42, 17. (32) Mossoba, M. M.; Al-Khaldi, S. F.; Kirkwood, J.; Fry, F. S.; Sedman, J.; Ismail, A. A. Vib. Spectrosc. 2005, 38, 229–235. (33) Hacura, A.; Wrzalik, R.; Matuszewska, A. Anal. Bioanal. Chem. 2003, 375, 324–326. (34) Mastalerz, M.; Bustin, R. M. Fuel 1995, 74, 536–542. (35) Mastalerz, M.; Bustin, R. M. Int. J. Coal Geol. 1996, 32, 55–67. (36) Burgula, Y.; Khali, D.; Kim, S.; Krishnan, S. S.; Cousin, M. A.; Gore, J. P.; Reuhs, B. L.; Mauer, L. J. J. Rapid Methods Autom. Microbiol. 2007, 15, 146–175. (37) Kalmbach, S.; Manz, W.; Bendinger, B.; Szewzyk, U. Water Res. 2000, 34, 575–581. (38) Kalmbach, S.; Manz, W.; Wecke, J.; Szewzyk, U. Int. J. Syst. Bacteriol. 1999, 49, 769–777. (39) Broo, A. E.; Berghult, B.; Hedberg, T., Water Science and Technology: Water Supply 2001, 1 (3), 117-125. (40) Bachmann, R. T.; Edyvean, R. G. J. Int. Biodeterior. Biodegrad. 2006, 58, 112–118.

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A. commune cultures were grown at room temperature in 5 L of autoclaved modified R2A medium with stirring. Bacterial growth was monitored by measuring the optical density by light absorption in a WPA Lightwave II UV/vis spectrophotometer at a wavelength of 600 nm. Cells were harvested at the late-log growth phase by centrifugation at 4200 rpm (3000g) for 30 min at 10 °C in a Beckman centrifuge. The biomass was washed by resuspending it in ultra high quality (UHQ) water (18 MΩ) and by vortexing and centrifuging in a Hermle Z231 microcentrifuge at 8000 rpm (5000g) for 10 min at 10 °C three times. Pembrey et al.41 have pointed out that centrifugation at 5000g was found to be the protocol that least disrupted the structural integrity of E. coli cells (a gram-negative bacteria); therefore, these centrifugation steps should not induce significant damage to the cell walls. Sample Preparation. For transmitted FT-IR spectroscopy, aqueous suspensions of A. commune were analyzed using a liquid IR flow cell (Sigma-Aldrich) with CaF2 windows. For KBr pellets, the bacterial suspensions were air-dried, and the solid samples in powder form were carefully mixed with KBr (Sigma, UK) and molded into pellets under high pressure using a Specac evacuable pellet die (13 mm diameter). Reflectance micro-FT-IR spectra were obtained from the airdried bacterial suspensions. Studies of A. commune cells attached to stainless steel were made using autoclaved stainless steel EN1.4307 slides. The autoclaved slides were left on the culture medium with A. commune to be colonized at pH 7 for 100 h, according to the procedure published by Bachmann and Edyvean.40 After the formation of a biofilm, the stainless steel slides were rinsed with UHQ water and scanned using reflectance micro-FT-IR. Techniques. In order to evaluate of the applicability of reflectance micro-FT-IR spectroscopy to bacterial cells, conventional FT-IR techniques (such as KBr pellets, ATR-FT-IR, and transmission micro-FT-IR) were applied and the obtained spectra were compared with reflectance micro-FT-IR spectroscopy. All FT-IR measurements were performed on a Perkin-Elmer Spotlight FT-IR imaging system and on a Perkin-Elmer Spectrum One fourier transformation infrared spectrometer. For liquid samples in transmission mode, bacterial suspensions were monitored using a liquid IR flow cell (Sigma-Aldrich) with CaF2 windows. ATR-FT-IR measurements were performed using a Specac Silver Gate essential single reflection ATR accessory, consisting of a germanium crystal at a fixed angle of 45°. For liquid suspensions, one drop of bacterial suspension was placed on the top of the crystal and the spectrum was recorded. Micro-FT-IR spectra were collected over the 4000-700 cm-1 wavenumber range, at a resolution of 4 cm-1 and a beam diameter of 6.25 µm. RESULTS AND DISCUSSION Reflectance micro-FT-IR spectroscopy has not been used routinely to analyze bacterial samples; therefore, initial basic operating parameters were assessed following the approach published by Mastalerz and Bustin.34 (41) Pembrey, R. S.; Marshall, K. C.; Schneider, R. P. Appl. Environ. Microbiol. 1999, 65, 2877–2894.

Figure 2. Comparison between reflectance micro-FT-IR, transmission micro-FT-IR, and ATR-FT-IR spectra of air-dried Aquabacterium commune. (a) Full range spectra. (b) Spectra between 1800 and 750 cm-1. Approximate band positions are indicated in the figure.

Figure 1. Quality of spectra of a biofilm of Aquabacterium commune on stainless steel, as a function of scan number and aperture sizes using micro-FT-IR spectroscopy in reflectance mode. Aperture of 10 µm (a), 30 µm (b), and 50 µm (c).

Signal-to-Noise Ratio as a Function of the Number of Scans and Aperture Size. A comparison between spectra of A. commune on stainless steel using reflectance micro-FT-IR spectroscopy with 5, 10, 50, 100, 500, and 1500 coadded scans with aperture sizes of 10, 30, and 50 µm is presented in Figure 1. It can be seen that there is a significant improvement in the signalto-noise ratio when increasing the aperture size from 10 to 30 µm; however, the quality of the spectra is not significantly different when aperture sizes of 30 or 50 µm were used, regardless of the number of scans. In all cases, there was a significant improvement in the signal-to-noise ratio between 5 and 100 scans; however, after 100 scans, the improvement was less evident. Therefore, for subsequent analysis, 100 coadded scans were used. Eight minutes were required to obtain 100 coadded scans, whereas for 5, 10,

50, 500, and 1500 coadded scans, 0.1, 0.5, 5, 45, and 90 min were needed, respectively. There were no changes in the band positions or ratios between peaks regardless of the aperture size or the number of scans. Comparison between Reflectance Micro-FT-IR, Transmission Micro-FT-IR, and ATR-FT-IR Spectra. A comparison between air-dried A. commune using ATR-FT-IR, transmission micro-FT-IR, and reflectance micro-FT-IR is shown in Figure 2. For ATR-FT-IR and reflectance micro-FT-IR, the samples were scanned using a wavenumber range from 4000 to 750 cm-1. For transmittance mode, the sample was placed between CaF2 windows and the wavelength ranged between 4000 and 950 cm-1. It can be seen from Figure 2b that the spectra of A. commune obtained by ATR-FT-IR, transmission micro-FT-IR, and reflectance micro-FT-IR were not significantly different, and no shift was observed in any of the absorption bands. Previous studies employed a Kramers-Kronig transformation when reflectance FTIR on flat/polished organic samples was used.33-35 In this study, the A. commune biofilm scanned using reflectance micro-FT-IR did not require this transformation as the band peak positions were the same in the transmission and the reflectance mode and both spectra were not different. This could be because, on reflectance mode, the bacterial samples formed a thin biofilm lying on a steel slide, preventing the abnormal dispersion resulting from highly specular surfaces of polished or shiny materials. Analytical Chemistry, Vol. 81, No. 15, August 1, 2009

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Table 1. Infrared Absorption Bands of the Aquabacterium commune Functional Groups wavenumber (cm-1) 1739

1647 1548 1405 1453 1387 1305 1300-1250 1262

1225 1200-950

1085

976

functional group assignmenta stretching CdO of ester functional groups from membrane lipids and fatty acids; stretching CdO of carboxylic acids stretching CdO in amides (amide I band) N-H bending and C-N stretching in amides (amide II band) symmetric stretching for deprotonated COO-group bending CH2/CH3 (scissoring) symmetric stretching of COO-; bending CH2/CH3 vibration C-N from amides vibrations of C-O from esters or carboxylic acids vibrations of -COOH and C-O-H; double bond stretching of >PdO of general phosphoryl groups and phosphodiester of nucleic acids stretching of PdO in phosphates. asymmetric and symmetric stretching of PO2- and P(OH)2 in phosphates; vibrations of C-OH, C-O-C, and C-C of polysaccharides stretching PdO of phosphodiester, phosphorylated proteins, or polyphosphate products symmetric stretching vibration of phosphoryl groups

a Based on Dittrich et al.,12 Jiang et al.,13 Ojeda et al.,14 Schmitt et al.,15 Yee et al.,16 Conley,22 and Wade.24

The observed infrared bands of the spectra of A. commune obtained by ATR-FT-IR, transmission micro-FT-IR, and reflectance micro-FT-IR corresponded to the presence of proteins, lipids, polysaccharides, polyphosphate groups, and other carbohydrate functional groups. The functional groups assigned to the infrared bands and the corresponding frequencies for A. commune are summarized in Table 1. The region between 3000 and 2800 cm-1 shown in Figure 2 exhibited the C-H stretching vibrations (νC-H) corresponding to the CH3 and >CH2 functional groups present in the fatty acids and lipids (2800 cm-1). The O-H stretching signal (νO-H) corresponding to the presence of hydroxyl groups in the bacterial cell is observed as a broadband around 3000 cm-1. Complementary information to support the presence of the C-H peaks can be found on the region between 1470 and 1300 cm-1, where bending vibrations of C-H, >CH2, and -CH3 groups could be observed. The peak at 1453 cm-1 corresponds to the amine III group, and the peak at 1387 cm-1 can be attributed to the symmetric stretching C-O of carboxylate groups (νC-O). The amide I and II bands appeared at 1647 and 1548 cm-1, respectively, the first one being due to stretching CdO (νCdO) of amides associated with proteins and the latter one being due to a combination of bending N-H (δN-H) of 6470

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amides and also contributions from stretching CdN (νCdN) groups. The signal around 1739 cm-1 is actually a combination of two peaks: a signal corresponding to the vibrational CdO stretching (νCdO) of carboxylic acids at 1739 cm-1 and another peak at 1725 cm-1 corresponding to the stretching CdO of ester functional groups from membrane lipids and fatty acids. The double bond stretching of >PdO of general phosphoryl groups and phosphodiester of nucleic acids was observed at 1260 cm-1. Vibrations of -COOH and C-O-H are located here as well. The stretching of PdO groups of polyphosphate products, nucleic acid phosphodiesters, and phosphorylated proteins is present around 1084 cm-1. The region between 1200 and 950 cm-1 showed the C-O-C and C-O-P stretching of diverse polysaccharides groups. The results obtained when comparing the A. commune spectra using reflectance micro-FT-IR or transmittance micro-FT-IR with the ATR-FT-IR system showed essentially identical results. The penetration depth of the infrared beam on reflectance micro-FTIR, for organic samples, ranges from 2 to 5 µm,42 which is higher than the typical average cell size of the A. commune: 0.5 × 2-4 µm.38 Hence, the spectra obtained using microreflectance and microtransmittance FT-IR modes did not show any distinction between cell wall and inner cell components. ATR-FT-IR analysis is essentially a surface sensitive technique. The penetration depth of the infrared beam on ATR-FT-IR measurements depends on the sample, wavenumber, crystal used, and angle of incidence. In the case of bacterial cells, the penetration depth of the infrared beam can be around 339 nm at 1000 cm-1 and 188 nm at 1800 cm-1.13 Because the average thickness of bacterial cell walls ranges from 20 to 50 nm, a contribution from the interior of the cells can also be expected on the ATR-FT-IR spectra. This could explain why the results obtained using ATR-FT-IR were very similar to the ones obtained using microreflectance and microtransmittance FT-IR modes. Comparison between Reflectance Micro-FT-IR Spectra with KBr Pellet Techniques and Transmission FT-IR of Live Bacteria Using a Flow Cell. A. commune spectra obtained by reflectance micro-FT-IR and KBr techniques are compared in Figure 3a. Spectral band locations using these two methods were similar and no shifts were observed at any of the observed peak wavenumbers. In order to test differences between the infrared spectra of air-dried and wet samples, live bacterial suspensions were scanned using a liquid IR flow cell with CaF2 windows. The absorption bands on spectra obtained using these two methods were similar and no shifts were evident. These results are also comparable with results obtained by reflectance microFT-IR and KBr pellets. The main difference between the spectra obtained by reflectance micro-FT-IR and the liquid flow cell (Figure 3b) is the presence of a broadband around 3000 cm-1 due to the OH stretching of the water molecules in the flow cell. This signal masked the peaks due to C-H stretching around 2800 cm-1 that are more prominent on the spectra using reflectance micro-FT-IR. Additionally, the band appearing at 1647 cm-1, attributed to the stretching CdO in amides (amide I band), was also showing contributions from OH bending (42) Mitchell, G. D.; Davis, A.; Chander, S. Int. J. Coal Geol. 2005, 62, 33–47.

Figure 3. Comparison between infrared spectra of Aquabacterium commune (a) using reflectance micro-FT-IR and KBr pellet of airdried samples and (b) using reflectance micro-FT-IR and a liquid flow cell.

vibrations due to the water molecules present when the liquid flow cell was used. This can also be observed when comparing the insets in panels a and b of Figure 3, where the band at 1647 cm-1 is clearly more intense in the FT-IR spectra of the liquid flow cell, when compared with the spectra obtained using reflectance micro-FT-IR and the KBr pellet. In the first case (liquid flow cell), this band is the result of the combination of the amide I band and δOH from water, whereas the contribution from δOH at 1647 cm-1 is less evident when the cells were scanned using KBr pellets or micro-FT-IR, as the cells in those cases were dried. It can be seen from Figures 2 and 3 that the infrared spectra obtained using ATR-FT-IR, transmission micro-FT-IR, transmission FT-IR of wet bacteria using a flow cell, KBr pellets, and reflectance micro-FT-IR are very similar, and no shift was observed in any of the absorption bands. The similarities of the spectra highlight the advantage of using reflectance micro-FT-IR spectroscopy. This technique requires no sample preparation, allows the analysis of intact larger samples, and permits the characterization of opaque and thick samples without the need to transfer bacterial samples to an infrared transparent medium or produce a pure culture. Application of Reflectance Micro-FT-IR Spectroscopy for the Study of Biofilm Formation of A. commune on Stainless Steel. The main advantage of reflectance micro-FT-IR spectroscopy is the possibility of studying bacterial aggregations on thick solid surfaces, in situ, without the need of culturing the micro-

Figure 4. Application of reflectance micro-FT-IR spectroscopy for the study of biofilm formation of Aquabacterium commune on stainless steel. (a) Optical image of stainless steel and Aquabacterium commune biofilm. (b) Full range (4000 to 700 cm-1) false-color image of the sample scanned using reflectance micro-FT-IR spectroscopy. (c) False-color image showing the location of molecules with absorption bands intrinsic of amide I and II (3550-3200 and 1690-1540 cm-1). (d) False-color image showing the location of molecules with absorption bands intrinsic of C-H, CdO, and C-O (3200-2700, 1725-1700, and 1320-1211 cm-1). (e) False-color image showing the location of molecules with absorption bands intrinsic of phosphorylated proteins, polyphosphates, and phosphodiester groups (1270-1220, 1100-1070, and 1000-950 cm-1). (f) False-color image showing the location of molecules with absorption bands intrinsic of hydroxyl groups (3600-3200 cm-1).

organisms separately and transferring them from agar plates to infrared transparent surfaces. An immediate application of reflectance micro-FT-IR spectroscopy for bacterial samples is the study of biofilms in thick solid surfaces such as stainless steel water pipes. In this study, stainless steel slides colonized by A. commune were analyzed under the infrared microscope in reflectance mode. The results can be seen in Figure 4. Under the optical microscope, the biofilm looks like a diffuse darker area deposited on the stainless steel surface (Figure 4a). However, when the sample is irradiated using IR light, the falsecolor image obtained (Figure 4b) shows the presence of infrared absorbing molecules in a clearer and more evident way than on the optical image. The absorbance scale on the right approximately indicates an abundance of molecules absorbing on a specific area. The higher absorbance values at any specific wavenumber range can be related to a higher abundance of biofilm in that area. However, not all the species absorbing infrared radiation correspond to A. commune cells but may also come from organic molecules released by the bacterium itself. The areas showing infrared absorption bands intrinsic of amide groups (3550-3200 and 1690-1620 cm-1)12-16,22-24 were separated and are displayed in Figure 4c. These bands intrinsic of amide groups are associated with proteins and are ubiquitous in every FT-IR spectra of bacterial cells.12,13,15,16,36 Therefore, the Analytical Chemistry, Vol. 81, No. 15, August 1, 2009

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absence of amide groups in other infrared absorbing regions could be used to discard the presence of bacterial cells. The presence of molecules due to cell lyses or the secretion of extracellular polymeric substances (EPS) by bacteria could also give a signal related to proteins; however, the intensity of these bands would be lower compared to the signal produced by whole cells. Moreover, there is evidence that the infrared spectra of EPS and whole bacterial cells are different.43-48 Therefore, the more intense the false red color is on the image, the more accumulation of bacterial biofilm is on the surface. The false-color image in Figure 4d shows the infrared absorption regions intrinsic of C-H, CdO, and C-O bands (3200-2700, 1725-1700, and 1320-1211 cm-1).12-16,22-24 The biofilm on stainless steel was formed at pH 7. It has been reported that the pKa of carboxylic groups usually varies from 2 to 6.12,16,49-52 Therefore, most of the carboxylic groups should be deprotonated, forming a carboxylate anion. The signal observed from Figure 4d is likely to be from peptides, amino acids, and esters. There is no possibility using this technique to distinguish between the cell surface and the cytoplasmic components inside the cell. Thus, the spectra shown in Figure 4 correspond to the entire cell and its EPS as well. This also occurs due to the limitation on penetration depth of the infrared beam (between 2 and 5 µm42). Panels e and f of Figure 4 show the presence of hydroxyl groups (associated to sugars and polysaccharides) and phosphorylated groups and phosphodiesters (associated with nucleic acids), respectively. It seems from these figures that phosphorylated compounds, sugars, and polysaccharides are covering a slightly larger area on the stainless steel than what can be attributed to bacterial cells, on the basis of the presence of more “dark blue” areas (nonabsorbing molecules) in Figure 4c. This could be because EPS is also formed by phospholipids and phosphate groups liberated during nucleic acid degradation.53 However, the most intense red false color on these images correlates well with the position of amide bands characteristic of the presence of a bacterial cell wall (Figure 4c). There is a consistent presence of bacterial functional groups sensitive to IR light on the obtained micrographs, as evidenced by the intense red false color on all of the presented figures. This presence is also comparable with the accumulation of cells observed on the microphotograph. In this work, A. commune was used as a model microorganism for the study of biofim formation on steel surfaces. Previous studies using more conventional FT-IR techniques, such as ATRFT-IR or transmission FT-IR, have shown that different bacterial (43) Eboigbodin, K. E.; Biggs, C. A. Biomacromolecules 2008, 9, 686–695. (44) Eboigbodin, K. E.; Ojeda, J. J.; Biggs, C. A. Langmuir 2007, 23, 6691– 6697. (45) Kawaguchi, T.; Decho, A. W. J. Cryst. Growth 2002, 240, 230–235. (46) Kumar, C. G.; Joo, H. S.; Kavali, R.; Choi, J. W.; Chang, C. S. World J. Microbiol. Biotechnol. 2004, 20, 837–843. (47) Muralidharan, J.; Jayachandran, S. Process Biochem. 2003, 38, 841–847. (48) Richert, L.; Golubic, S.; Le Guedes, R.; Ratiskol, J.; Payri, C.; Guezennec, J. Curr. Microbiol. 2005, 51, 379–384. (49) Fein, J. B.; Daughney, C. J.; Yee, N.; Davis, T. A. Geochim. Cosmochim. Acta 1997, 61, 3319–3328. (50) Guine, V.; Spadini, L.; Sarret, G.; Muris, M.; Delolme, C.; Gaudet, J. P.; Martins, J. M. F. Environ. Sci. Technol. 2006, 40, 1806–1813. (51) Haas, J. R.; Dichristina, T. J.; Wade, R. Chem. Geol. 2001, 180, 33–54. (52) Ngwenya, B. T.; Sutherland, I. W.; Kennedy, L. Appl. Geochem. 2003, 18, 527–538. (53) Beech, I. B. Int. Biodeterior. Biodegrad. 2004, 53, 177–183.

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Figure 5. Comparison between infrared spectra of Aquabacterium commune attached to stainless steel and the free bacteria.

cells can give different FT-IR spectra54-58 and also that the infrared spectra of the same strain can change when changing the nutrients surrounding it.44 Based on this, there is a potential to use reflectance micro-FT-IR to discriminate different bacterial populations on thick surfaces, without sample preparation, by comparing the FT-IR spectra obtained in different areas of the scanned surface. The use of algorithms for data analysis, such as principal component analysis (PCA), has been successfully applied to vibrational spectroscopy to allow the identification of key chemical moieties that distinguish between different strains.59-63 The use of fluorescent probes on bacteria or their intrinsic fluorescence properties64,65 can also be combined with FT-IR microspectroscopy for bacterial identification and characterization. However, care must be taken to avoid the confusion between bands from bacterial cells and bands intrinsic to the fluorescent probes used. Differences between Attached and Free A. commune Cells. By comparing the spectra of free bacteria with the spectra obtained on bacteria attached to solid surfaces, some differences were observed, especially in the signals corresponding to carbonyl groups and carboxylate anions. Figure 5 shows the reflectance micro-FT-IR spectrum of A. commune when the bacteria are attached to stainless steel and the spectrum from “free” (not (54) Bosch, A.; Minan, A.; Vescina, C.; Degrossi, J.; Gatti, B.; Montanaro, P.; Messina, M.; Franco, M.; Vay, C.; Schmitt, J.; Naumann, D.; Yantorno, O. J. Clin. Microbiol. 2008, 46, 2535–2546. (55) Curk, M. C.; Peladan, F.; Hubert, J. C. FEMS Microbiol. Lett. 1994, 123, 241–248. (56) Garip, S.; Gozen, A. C.; Severcan, F. Food Chem. 2009, 113, 1301–1307. (57) Kirschner, C.; Maquelin, K.; Pina, P.; Thi, N. A. N.; Choo-Smith, L. P.; Sockalingum, G. D.; Sandt, C.; Ami, D.; Orsini, F.; Doglia, S. M.; Allouch, P.; Mainfait, M.; Puppels, G. J.; Naumann, D. J. Clin. Microbiol. 2001, 39, 1763–1770. (58) Savic, D.; Jokovic, N.; Topisirovic, L. Dairy Sci. Technol. 2008, 88, 273– 290. (59) Harz, A.; Rosch, P.; Popp, J. Cytometry, Part A 2009, 75A, 104–113. (60) Huang, W. E.; Griffiths, R. I.; Thompson, I. P.; Bailey, M. J.; Whiteley, A. S. Anal. Chem. 2004, 76, 4452–4458. (61) Huang, W. E.; Hopper, D.; Goodacre, R.; Beckmann, M.; Singer, A.; Draper, J. J. Microbiol. Methods 2006, 67, 273–280. (62) Al-Qadiri, H. M.; Al-Holy, M. A.; Lin, M.; Alami, N. I.; Cavinato, A. G.; Rasco, B. A. J. Agric. Food Chem. 2006, 54, 5749–5754. (63) Krafft, C.; Steiner, G.; Beleites, C.; Salzer, R. J. Biophotonics 2009, 2, 13– 28. (64) Ammor, M. S. J. Fluoresc. 2007, 17, 455–459. (65) Leblanc, L.; Dufour, E. Sci. Aliments 2004, 24, 207–220.

attached) bacterial cells, obtained from planktonic suspensions on the same culture and under the same conditions. It can be seen from Figure 5 that, when bacteria are attached to stainless steel, the IR band corresponding to the CdO stretching of carbonyl groups from membrane lipids and fatty acids (1739 cm-1) is only a shoulder masked by the peak at 1725 cm-1. However, a stronger CdO band can be seen on the spectrum corresponding to the “free” bacterial sample resulting from the combination of the peaks at 1739 cm-1 and 1725 cm-1 (stretching CdO from esters and carboxylic groups).13,14,22 Figure 5 also shows that the spectrum of nonattached bacteria exhibited a unique broadband around 1387 cm-1 (asymmetric stretching CdO from carboxylate anions),13,14,22 whereas an additional band around 1405 cm-1 was observed on the spectrum corresponding to the bacteria attached to stainless steel. Extensive studies on metal complexes of carboxylic acids have assigned the appearance of these bands to the formation of inner sphere complexes between carboxylate anions and metals66-68 or to the absorption of monocarboxylates and amino acids on surfaces mediated by hydrogen bonding.69,70 It appears that there are some interactions taking place between the A. commune cells and stainless steel similar to the ones observed when model molecules are used. Although it is unclear whether an inner sphere complex or a hydrogen bonding mechanism is taking place between the bacteria and the surface, it is clear that the carbonyl groups in the biofilm are playing a role in the adhesion onto stainless steel. This has also been observed when monitoring biofilm formation of Pseudomonas putida on mineral surfaces using ATR-FT-IR flow cells.71 Also, it can be noted from Figure 5 that the region corresponding to the polysaccharides, phosphoryl groups, and polyphosphates in the infrared spectra is different when bacteria are free or attached (around 1200-950 cm-1). These changes may also indicate that phosphoryl groups (-PdO) could be involved in cell attachment to stainless steel. In order to corroborate these (66) Chu, H. A.; Hillier, W.; Debus, R. J. Biochemistry 2004, 43, 3152–3166. (67) Deacon, G. B.; Phillips, R. J. Coordin. Chem. Rev. 1980, 33, 227–250. (68) Nakamoto, K. Infrared and Raman Spectra of Inorganic and Coordination Compounds, Part B, 5th ed.; John Wiley & Sons: New York, 1997. (69) Nore´n, K.; Loring, J. S.; Persson, P. J. Colloid Interface Sci. 2008, 319, 416–428. (70) Nore´n, K.; Persson, P. Geochim. Cosmochim. Acta 2007, 71, 5717–5730. (71) Ojeda, J. J.; Romero-Gonzalez, M. E.; Pouran, H. M.; Banwart, S. A. Mineral. Mag. 2008, 72, 101–106.

finding, further studies using other spectroscopic techniques such as extended X-ray absorption fine structure (EXAFS) may be required. The evidence presented also demonstrates that reflectance micro-FT-IR can be used to monitor the potential of bacterial suspensions to form biofilm on water pipes, as well as the removal of biofilm from surfaces after treatment. CONCLUSIONS The application of reflectance micro-Fourier transform infrared spectroscopy as a convenient tool for the study of biofilm in opaque solid surfaces has been evaluated. The obtained results were compared with more conventional FT-IR techniques, such as transmission micro-FT-IR, attenuated transmitted reflectance (ATR-FT-IR), and KBr pellets. Reflectance micro-FT-IR has been shown to be a suitable technique for studying the functional groups in biofilms in opaque or thick samples, such as stainless steel, without sample preparation. This technique compares favorably with ATR-FT-IR, transmitted micro-FT-IR, and KBr pellets and was found to be a very convenient and precise analytical tool for biofilm analysis. Reflectance micro-FT-IR spectra of A. commune biofilms on stainless steel showed several differences when compared with “free” (not attached) bacteria (from planktonic suspensions). The obtained spectra suggested that carboxylate and phosphoryl groups in the biofilm could be playing an important role in the adhesion onto stainless steel. However, additional spectroscopic evidence is needed to verify these hypotheses. ACKNOWLEDGMENT This work has been funded by the U.K. Engineering and Physical Sciences Research Council (EPSRC) and the Biotechnology and Biological Sciences Research Council (BBSRC), as part of the Cell-Mineral Interface Research Program (GR/S72467/ 01(P)). J.J.O. acknowledges funding from the EPSRC Platform Grant strategic funding (GR/S87416/01) and the Sorby Nano Investigation Center (http://www.sorbynano.org). Robert T. Bachmann, from the Department of Chemical and Process Engineering, University of Sheffield, is thanked for culturing the A. commune samples used in this work. Received for review April 20, 2009. Accepted June 18, 2009. AC900841C

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