Differentiation of Mesophilic and Thermophilic Bacteriawith Fourier Transform Infrared Spectroscopy

SEBNEM GARIP, FARUK BOZOGLU, and FERIDE SEVERCAN*Department of Biology, Middle East Technical University, 06531 Ankara, Turkey (S.G., F.S.); and Department of Food Engineering, Middle East

Technical University, 06531 Ankara, Turkey (F.B.)

In the present study the characterization and differentiation of mesophilic

and thermophilic bacteria were investigated by using Fourier transform

infrared (FT-IR) spectroscopy. Our results showed significant differences

between the FT-IR spectra of mesophilic and thermophilic bacteria. The

protein-to-lipid ratio was significantly higher for thermophiles compared

to mesophiles. The absorption intensity of the CH3 asymmetric stretching

vibration was higher in thermophilic bacteria, indicating a change in the

composition of the acyl chains. The higher intensity/area observed in the

CH2 symmetric stretching mode at 2857 cm�1, and the CH2 bending

vibration band at 1452 cm�1, indicated a higher amount of saturated lipids

in thermophilic bacteria. The lipid C¼O stretching vibration at 1739 cm�1,

which was observed in the mesophilic group, was not observed clearly in

the thermophilic group, indicating a difference in packing that is

presumably due to the decreased proportion of unsaturated acyl chains

in thermophilic bacteria. In addition, the carbonyl groups become

hydrogen bonded and the cellular DNA content was lower in thermophilic

bacteria. Moreover, in the 1000–400 cm�1 frequency region, the spectra of

each bacterial species belonging to both the mesophilic and thermophilic

bacterial groups, showed characteristic differences that were discriminat-

ed via dendrogram using cluster analysis. The curent study implies that

FT-IR spectroscopy could be succesfuly applied for the rapid comparison

of bacterial groups and species to establish either similarities or

discrepencies, as well as to confirm biochemical or physiological

characteristics.

Index Headings: Mesophilic; Bacteria; Differentiation; Fourier trasnform

infrared spectroscopy; FT-IR spectroscopy; Thermophilic.

INTRODUCTION

Identification of bacteria is very important in clinicalmicrobiology and in food analysis because of an increasingprevalence of infectious diseases and food poisoning.1

Bacterial classification and differentiation techniques are usedin various fields such as clinical, environmental, and foodmicrobiology.2

There are some traditional methods for the identification ofbacterial species such as 16S rDNA sequencing analysis.3

Besides 16S rDNA sequencing analysis, some other techniqueshave been used for differentiation of different bacterial speciessuch as denaturing gradient gel electrophoresis and sequenc-ing4 and PCR with DNA–DNA hybridization analysis.5 For theidentification of gram (þ) lactic acid bacteria, the techniquecalled RAPD-PCR has been used.6 Another technique that hasbeen used for the identification of bacteria is florescence in situhybridization (FISH). The most commonly used method for thedifferentiation and identification of bacteria is using selectiveand differential media. Identification of Enterobacter sakazakiiwith a chromogenic medium (Druggan-Forsythe-Iversen agar,DFI) is an example of this method in the literature.7

Differing from all of these traditional techniques, Fouriertransform infrared (FT-IR) spectroscopy is used as ananalytical tool for the identification and differentiation ofbacteria because it is a rapid and easy method.2 Temperature isone of the most important environmental factors affecting theactivity and evolution of living organisms. Since all theprocesses of growth depend on the biochemical reactions thatare affected by temperature, it has a significant role in thegrowth of microorganisms.8 It is possible to distinguish fourgroups of bacteria in relation to their temperature optima;psychrophiles (�10–25 8C), mesophiles (20–45 8C), thermo-philes (50–70 8C), and hyperthermophiles (�75 8C).

In the current study, four thermophilic bacterial species(Thermoanaerobacter ethanolicus, Clostridium thermohydro-sulfuricum, Thermobrachium celere, and Geobacillus caldox-ylosilyticus) and four mesophilic bacterial species(Lactobacillus plantarum, Escherichia coli, Pseudomonasaeruginosa, and Micrococcus luteus) were investigated.Thermophilic microorganisms have a biotechnological signif-icance in food, chemical, and pharmaceutical industries and inenvironmental biotechnology.9 Lactic acid bacteria play asignificant role in the food industry, as they aid in various foodprocesses and possess unique characteristics that allow them tobe potential probiotic organisms.10 They are used industriallyfor the production of yogurt, sauerkraut, pickles, and otherfermented foods, such as silage.11 Escherichia coli has foundextensive use as a vehicle for the preparation of biologicalpolymers, including polypeptide hormones, proteins, carbohy-drates, etc.12 E. coli can also cause enteric infections,extraintestinal infections, and animal infections.13,14 Pseudo-monas aeruginosa has medical importance and causes humaninfections, such as urinary tract infections, cystic fibrosis, andnosocomical infections.15 Micrococcus luteus is useful for theeconomic production of long-chain (C21-C34) aliphatichydrocarbons, which few bacteria other than micrococciproduce. These may be useful as lubricating oils and may besubstitutes for equivalent petroleum products.16 M. luteus hasalso been used as a test organism for the assay of antibiotics inbody fluids, animal feeds, milks, and pharmaceuticals.

This study aims to investigate the similarities and differencesbetween thermophilic and mesophilic bacteria groups andbetween bacterial species with FT-IR spectroscopy. Althoughthere are several bacterial identification studies using FT-IRspectroscopy,17 to the best of our knowledge, there is no studyreported in the literature on differentiation of thermophilic andmesophilic bacteria.

MATERIALS AND METHODS

Organisms. Thermophilic bacteria Thermoanaerobacterethanolicus (JW200), Clostridium thermohydrosulfuricum(DSM 2247), and Thermobrachium celere were provided by

Received 18 February 2006; accepted 20 November 2006.* Author to whom correspondence should be sent. E-mail: feride@metu.edu.tr.

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� 2007 Society for Applied Spectroscopy

Sedat Donmez (Ankara University, Food Engineering). Theother thermophilic bacterium, Geobacillus caldoxylosilyticus(HBB-D4), was provided by Halil Bıyık (Adnan MenderesUniversity, Biology). Mesophilic bacteria Escherichia coli,Micrococcus luteus, and Pseudomonas aeruginosa wereobtained from Refik Saydam Hıfzıssıhha Merkezi (ANKARA).Lactobacillus plantarum was provided by Candan Gurakan(METU, Food Engineering).

Growth Medium. Mesophilic microorganisms were grownin 200 mL nutrient broth at 37 8C in an incubator overnight.

Four thermophilic microorganisms were obtained separatelyin 60 mL TR1 media in four 100 mL serum bottles. Onemilliliter (1 mL) samples from each culture with visibleturbidity caused by massive growth were taken and transferredto Eppendorf tubes.

Sample Preparation. Bacterial cells were collected fromthese liquid inoculations at their late exponential phase bycentrifugation (Sorvall) at 10 000 rpm (SS34 Rotor) for 10minutes. After removing the supernatants, pellets were washedtwo times with phosphate buffer (pH 7; 1 mM Na2HPO4/NaH2PO4 buffer). After a second wash in phosphate buffer,samples were lyophilized (Labconco FreeZonet, Model 77520).

Sample Preparation for Fourier Transform InfraredSpectroscopy Studies. The samples were first ground into fineparticles using a mortar and pestle. One milligram (1 mg) ofeach sample was then mixed with 100 mg KBr, which wasextensively dried in microfuge tubes using a lyophilizer. Thismixture was then lyophilized in the same microfuge tubes. KBrbased pellets were prepared by establishing pressure of 100kg/cm2 (1200 psi) for about 6 minutes. Also, 1 mg phosphatebuffer was dried and mixed with 100 mg KBr to prepare a pellet.

Fourier Transform Infrared Spectroscopy and DataAnalysis. Infrared spectra were obtained by scanning theprepared pellets with a Perkin Elmer Spectrum One Spectrom-eter (Norwalk, CT). The spectrum of air was recorded asbackground and subtracted automatically using the SpectrumOne software program. Atmospheric vapor was also automat-ically subtracted. FT-IR spectra of bacterial samples wererecorded in the 4000–400 cm�1 region at room temperature.One hundred scans were taken for each interferogram at 4 cm�1

resolution. Recording and analysis of the spectral data wereperformed using the Spectrum One software from Perkin Elmer.

First the phosphate buffer’s spectrum was recorded to showwhere the phosphate absorption occurs. Then this spectrumwas subtracted from all bacterial spectra to prevent absorptions

from the inorganic phosphate. The band positions weremeasured according to center of weight. The averages of thespectra belonging to the same experimental groups, baselinecorrection, normalization, and the band areas were obtained byusing the same software program. The average spectra andnormalization process were applied only for visual represen-tation of the differences; however, for the determination of thespectral parameters and calculation of mean values andstatistical analysis, each baseline-corrected original spectrumwas taken into consideration.

Finally, cluster analysis was applied that classifies objects,via a tree diagram (dendrogram) calculated using the Ward’salgorithm. Constructed with the OPUS 5.5 software (BrukerOptics), the dendrogram graphically represents the clusteranalysis groups.

Statistics. The results were expressed as mean 6 standarddeviation values. The differences in the means of thethermophilic and mesophilic bacteria were compared usingthe Mann–Whitney U-test. A p value of less than 0.05 wasconsidered significant (p , 0.05*, p , 0.01**, p , 0.001***).

RESULTS

We have carried out FT-IR spectroscopic studies onthermophilic and mesophilic bacterial groups and comparedthe spectral differences and similarities between both thegroups and the bacterial species.

A general representative FT-IR spectrum of a bacterium isshown in Fig. 1. As seen from the spectrum, it is a complexspectrum containing several bands. The band assignments aregiven in Table I. Figure 2A shows the average FT-IR spectra offour thermophilic bacteria in 4000–1000 cm�1 region. As seenfrom this figure, these four thermophilic bacteria show identicalFT-IR spectra so we used the average spectra of thesemicroorganisms when comparing with mesophiles in this

FIG. 1. A general representative FT-IR spectrum of a bacteria.

TABLE I. General band assignments of bacteria in the literature.a

Peaknumbers

Wavenumbers(cm�1) Definition of the spectral assignment

1 3307 N–H and O–H stretching vibration:polysaccharides, proteins

2 2959 CH3 asymmetric stretch: methyl groups in fattyacids

3 2927 CH2 asymmetric stretch: methylene groups infatty acids

4 2876 CH3 symmetric stretch: methyl groups in fattyacids.

5 2857 CH2 symmetric stretch: methylene groups infatty acids

6 1739–1744 Ester C¼O stretch: lipid, triglycerides7 1657 Amide I (protein C¼O stretching): a helices8 1541 Amide II (protein N–H bend, C–N stretch): a

helices9 1452 CH2 bending: lipids

10 1391 COO� symmetric stretch: aminoacid sidechains, fatty acids

11 1236 PO�2 asymmetric stretching: mainly nucleicacids with the little contribution fromphospholipids

12 1152 CO–O–C asymmetric stretching: glycogen andnucleic acids

13 1080 PO�2 symmetric stretching: nucleic acids andphospholipids; C–O stretch: glycogen

14 969 C–Nþ–C stretch: nucleic acids

a See Refs. 18–27.

APPLIED SPECTROSCOPY 187

region. Also as seen from Fig. 2B, four mesophilic bacteriashow identical FT-IR spectra so the average spectra of thesebacteria was used in the comparison of thermophiles andmesophiles in the 4000–1000 cm�1 region. On the other hand,in the 1000–400 cm�1 frequency region, all the thermophilicand the mesophilic bacterial species show different spectra fromeach other and are differentiated by cluster analysis (Fig. 3).

Comparison of Mesophilic and Thermophilic BacterialGroups. Figure 4 shows the normalized infrared spectra ofthermophilic bacteria and mesophilic bacteria samples in the3000–2800 cm �1 region. The bands centered at 2927 cm�1 and2857 cm�1 correspond to the stretching mode of asymmetricaland symmetrical CH2 vibrations due to methylene groups infatty acids.22,24–27 The band centered at 2959 cm�1 and 2876cm�1 corresponds to the stretching mode of asymmetrical andsymmetrical CH3 vibrations due to methyl groups in fattyacids.27

The frequency and intensity values of both thermohiles andmesophiles are listed in Table II. As seen from the table, thewavenumber of the CH3 asymmetric stretching vibration at2959 cm�1 of thermophilic bacteria (2962.01 6 0.30) wassignificantly lower than mesophilic bacteria (2962.51 6 0.80)(p , 0.001). However, the absorption intensity of this bandwas significantly higher for thermophilic bacteria (0.38 6

0.12) compared to mesophilic bacteria (0.25 6 0.05) (p ,0.001). Moreover, the absorption intensity of the CH2

asymmetric stretching band at 2927 cm�1 was higher (p ,

0.01) for the thermophilic group (0.38 6 0.24) than for themesophilic group (0.26 6 0.04), as shown in Table II.

As seen from Fig. 4 and Table II, the wavenumber of the CH3

symmetric stretching band at 2876 cm�1 is significantly higher(2875.30 6 0.22) for thermophilic bacteria than mesophilicbacteria (2872.20 6 0.98) (p , 0.001). Also, the intensity ofthis band is higher (p , 0.01) for the thermophilic group (0.336 0.12) compared to the mesophilic group (0.24 6 0.05).

It is also observed that the intensity of the band at 2857cm�1, which is assigned as CH2 symmetric stretching, showeda higher value (p , 0.01) for thermophilic bacteria. Thefrequency of this band was significantly higher (p , 0.001) forthermophilic bacteria (2854.29 6 0.60) compared to meso-philic bacteria (2853.24 6 0.40), indicating an increase in thenumber of gauche conformers, e.g., an increase in disorderstatus of the acyl chains.22,24,26–28

The signal intensity and, more accurately, the area under thepeaks give information about the concentration of the functionalgroups responsible for the corresponding band.26,27,29–32 Fromthe FT-IR spectrum, a precise protein-to-lipid ratio can bederived by calculating the ratio of the areas of the bands arisingfrom lipids and proteins whose amounts in the membranes arean important factor affecting the membrane structure anddynamics.33 It is seen from Table III that the ratio of the sumarea of the CH2 and CH3 symmetric and asymmetric bands inthe 3000–2800 cm�1 region to the area of amide I þ amide IIgives information about the protein-to-lipid ratio,27 which wassignificantly higher (p , 0.01) for the thermophilic group (3.206 0.05) than for the mesophilic group (2.75 6 0.06).

Figure 5 shows the 1800–400 cm�1 region of the normalizedFT-IR spectra of thermophilic and mesophilic bacteria. Theband centered at 1739 cm�1 is mainly assigned to the .C¼Oester stretching vibration in phospholipids.22,24 As is illustratedin Fig. 3, the C¼O ester stretching vibration band at 1739 cm�1

was not observed in the thermophilic group, which wasdifferent from that of the mesophilic one, indicating adifference in packing of the ester groups.21

The bands centered at 1657 cm�1 corresponding to thestretching C¼O and bending C–N (amide I) vibrational modesof the polypeptide and protein backbone and at 1541 cm�1 areassigned to the bending N–H and stretching C–N (amide II).24

The amide I region is useful for determination of proteinsecondary structure. The frequency of vibration is verysensitive to changes in the nature of hydrogen bonds indifferent types of protein secondary structures.34 As can beseen from Fig. 5 and Table II, the frequency value of the amideI band is lower (1653.22 6 0.53) for the thermophilic groupcompared to the mesophilic group (1656.05 6 0.41) (p ,0.001). However, the intensity of amide I was higher (p ,0.001) for the thermophilic group (0.59 6 0.07) than for themesophilic group (0.29 6 0.05). Both the absorbance and thebandwidth of the amide II band (at 1541 cm�1) wassignificantly higher (p , 0.001) for the thermophilic groupcompared to the mesophilic group, as seen from Table IV.Moreover, the frequency of the amide II band was lower for thethermophilic group (1536.09 6 0.01) compared to themesophilic group (1540.23 6 0.07) (p , 0.001).

The intense band at 1452 cm�1 is assigned to the CH2

bending mode of lipids.24,35 As seen from Fig. 5, a highervalue of the intensity for thermophilic bacteria (0.34 6 0.12)than mesophilic bacteria (0.23 6 0.05) (p , 0.01) at 1452cm�1 is due to the bending vibration of CH2 in the lipids. It canbe seen from Table II that there is a lower frequency of thisband for the thermophilic group (1452.89 6 0.77) compared tothe mesophilic group (1455.72 6 0.80) (p , 0.001). The band

FIG. 2. (A) The average spectra of (a) T. celere (n¼ 20), (b) T. ethanolicus (n¼20), (c) C. thermohydrosulfuricum (n¼20), and (d) G. caldoxylosilyticus (n¼20) samples in the 4000–1000 cm�1 region. (B) The average spectra of (a) E.coli (n ¼ 20), (b) M. luteus (n ¼ 20), (c) L. plantarum (n ¼ 20), and (d) P.aeruginosa (n ¼ 20) samples in the 4000–1000 cm�1 region.

188 Volume 61, Number 2, 2007

FIG. 3. Hierarchical cluster analysis performed on the first-derivative spectra of bacterial samples and resulting from Ward’s algorithm. The study was conducted inthe 1000–400 cm�1 spectral region.

APPLIED SPECTROSCOPY 189

at 1391 cm�1 is due to COO� symmetric stretching vibration ofamino acid side chains and fatty acids.21,24 In the thermophilicspectrum (0.35 6 0.11) the intensity of the COO� symmetricstretching vibration band at 1395 cm�1 was higher (p , 0.01)compared to the mesophilic group (0.23 6 0.03). In addition,the frequency value of this band was significantly lower forthermophiles (1396.55 6 0.29) compared to mesophiles(1402.73 6 0.44) (p , 0.001) (Table II).

The relatively strong bands at 1236 and 1080 cm�1 aremainly due to the asymmetric and symmetric stretching modesof phosphodiester groups in nucleic acids rather than inphospholipids, respectively.20,24 Study of these bands revealedsignificant differences in the infrared spectra between thermo-philic and mesophilic bacteria. The intensity of the phosphateasymmetric stretching band was significantly higher (p , 0.01)for thermophilic bacteria (0.35 6 0.12) compared to meso-philic bacteria (0.24 6 0.03). Furthermore, the frequency ofthis band was also significantly higher for thermophiles(1236.70 6 1.26) compared to mesophiles (1232.64 6 0.86)(p , 0.001), as shown in Fig. 5. The frequency of thephosphate symmetric stretching band also showed a highervalue for thermophilic bacteria (1077.13 6 1.20) than formesophilic bacteria (1073.85 6 1.91) (p , 0.001) (Table II).The peak area ratio of bands at 1087 cm�1 and 1540 cm�1

(A1087/A1540) is often used to illustrate the change of the DNA/protein content of the cells.36–39 A higher value of this ratioimplies a higher DNA content.36–39 The results shown in TableIII suggest that the cellular DNA content was lower for

thermophilic bacteria (0.30 6 0.10) compared to mesophilicbacteria (1.16 6 0.50) (p , 0.001).

Comparison of Thermophilic Bacterial Species. Thermo-philic bacterial species show different FT-IR spectra in the1000–400 cm�1 frequency region. As seen from Fig. 6, therewere characteristic bands for each species in this region. Forthree thermophilic species there were two bands, at 964 and914 cm�1, but in G. caldoxylosilyticus, instead of these twobands, there was another band at 925 cm�1. Moreover, therewere characteristic peaks at 851 and 529 cm�1 that were onlyseen in G. caldoxylosilyticus. Another difference was the peakat 798 cm�1, which was observed in three thermophiles exceptfor T. ethanolicus (Fig. 6).

Comparison of Mesophilic Bacterial Species. Mesophilicbacterial species show different FT-IR spectra in the 1000–400cm�1 frequency region. There were characteristic peaks forboth Lactobacillus plantarum and Pseudomonas aeruginosa,as seen from Fig. 7. The band at 966 cm�1 was observed in E.coli and P. aeruginosa species, while it was not seen in theother two mesophilic bacteria. Moreover, the band at 935 cm�1

was sharper in P. aeruginosa than in the other bacterial species.There were characteristic bands for L. plantarum at 505 and420 cm�1 (Fig. 7).

DISCUSSION

High temperatures lead to an increase in the fluidity of thecellular membrane and to maintain optimal membrane fluidity,the composition of the membrane is altered in some cells.40 Ascan be seen from Fig. 2 and Table II, the absorption intensity ofthe CH3 asymmetric stretching vibration at 2959 cm�1 was

FIG. 4. The average spectra of the mesophilic (n¼ 80) and thermophilic (n¼80) bacteria samples in the 3000–2840 cm�1 region (the spectra werenormalized with respect to the CH2 asymmetric stretching band).

TABLE II. The band frequencies and intensity values of various functional groups in the mesophilic (n ¼ 80) and thermophilic (n ¼ 80) bacterialsamples.

Functional groups

Frequency Intensity

Mesophilic (n ¼ 80) Thermophilic (n ¼ 80) p values Mesophilic (n ¼ 80) Thermophilic (n ¼ 80) p values

CH3 asymmetric stretching 2962.50 6 0.80 2962.01 6 0.30 ,0.001*** 0.25 6 0.05 0.38 6 0.12 ,0.001***CH2 asymmetric stretching 2928.52 6 1.00 2926.44 6 0.30 ,0.001*** 0.26 6 0.04 0.38 6 0.24 ,0.01**CH3 symmetric stretching 2872.20 6 0.98 2875.30 6 0.22 ,0.001*** 0.24 6 0.05 0.33 6 0.12 ,0.01**CH2 symmetric stretching 2853.24 6 0.40 2854.29 6 0.60 ,0.001*** 0.24 6 0.05 0.32 6 0.12 ,0.01**Amide I 1656.05 6 0.41 1653.22 6 0.53 ,0.001*** 0.29 6 0.05 0.59 6 0.07 ,0.001***Amide II 1540.23 6 0.07 1536.09 6 0.01 ,0.001*** 0.26 6 0.04 0.49 6 0.20 ,0.001***CH2 bending 1455.72 6 0.80 1452.89 6 0.77 ,0.001*** 0.23 6 0.05 0.34 6 0.12 ,0.01**COO symmetric stretching 1402.73 6 0.44 1396.55 6 0.29 ,0.001*** 0.23 6 0.03 0.35 6 0.11 ,0.01**PO2 asymmetric stretching 1232.64 6 0.86 1236.70 6 1.26 ,0.001*** 0.24 6 0.03 0.35 6 0.12 ,0.01**PO2 symmetric stretching 1073.85 6 1.91 1077.13 6 1.20 ,0.001***

FIG. 5. The average spectra of the mesophilic (n¼ 80) and thermophilic (n¼80) bacteria samples in the 1800–400 cm�1 region (the spectra were normalizedwith respect to the amide I band).

190 Volume 61, Number 2, 2007

higher in thermophilic bacteria, indicating a change in thecomposition of acyl chains in the thermophilic group.19,24,26

Growth-temperature-dependent changes of the lipid composi-tion of the cytoplasmic membranes of bacteria were alsoreported previously.41–43

As seen from Table II, the higher value of the frequency ofthe CH2 symmetric stretching mode at 2857 cm�1 for thethermophilic group (p , 0.001) indicated a reorganization ofthe membrane in an ordered direction to an average bilayerstructure.44,24 In addition to this difference, the intensity of thisband was higher (p , 0.01) in thermophilic bacteria comparedto mesophilic bacteria. These results indicate a higherconcentration of saturated fatty acyl chains in thermophilicbacteria. This was confirmed by the higher value of the intensityof the CH2 bending vibration band at 1452 cm�1 in thermophilicbacteria (Fig. 4 and Table II). These results were confirmed bythe study of Chan et al.,45 which suggested that notabledifferences were that the thermophiles contained a highercontent of saturated straight- and branched-chain fatty acids.

Moreover, the lipid C¼O stretching vibration at 1739 cm�1,which was observed in the mesophilic group, was not clearlyobserved in the thermophilic group. This band suggests anincreased concentration and difference in packing of the estergroups in mesophilic bacteria21 (Fig. 5), which is in agreementwith Chan et al.45 The melting points of saturated straight-chain and iso-fatty acids range from 51 to 107 8C, whereasunsaturated, anteiso fatty acids have melting points rangingfrom below 0 to 40 8C.45 According to our results, thethermophiles contain a preponderance of higher melting fattyacids. The general pattern indicated that organisms grown athigher temperatures contain a higher percentage of saturatedacids with comparatively higher melting points.45 Vossenberget al.43 investigated the phase transition behavior of the lipidsderived from mesophilic B. subtilis and thermophilic B. subtiliscells grown at 13 and 50 8C by differential scanningcalorimetry (DSC) and the viscosity of the membranes byTMA-DPH fluorescence anisotropy (r) and lifetime (sh)measurements. They indicated that the most significant effectof growth temperature was a drastic decrease in the anteisofatty acids and an increase in the iso-branched fatty acids thatwas in accordance with our results.

It is seen from Table III that the protein-to-lipid ratio washigher (p , 0.01) in the thermophilic group compared to themesophilic group. Generally, the higher value of this ratiosuggests a high protein content or low lipid content orboth.24,46 In our case, the high protein content in thermophilicbacteria was supported by the higher value of the area of theamide I and amide II bands, while the low lipid content was

supported by the lower value of the area of the CH2 and theCH3 asymmetric stretching vibration band in the thermophilicbacterial samples.

In thermophilic microorganisms, thermostability is acquiredby thermostable proteins. The amino acid composition ofproteins from mesophilic and thermophilic organisms iscommonly assumed to reflect the mechanism of molecularadaptation to extremes of physical conditions.48 Thermostabil-ity seems to be a property acquired by a protein through manysmall structural modifications obtained with the exchange ofsome aminoacids.49–51 The advantages of the amino acidexchanges most frequently reported in thermophilic proteins arethat a higher number of hydrogen bonds may be formed.49 Fromthe comparative analysis of the X-ray structures available forseveral families of proteins, including at least one thermophilicstructure in each case, it appears that thermal stabilization isaccompanied by an increase in hydrogen bonds.49,52,53 Ourresults confirmed these studies since we found a significantdecrease in the frequency of the carbonyl group, indicating anincrease in the number of hydrogen bonds. The lower DNA/protein ratio might suggest that the cellular DNA content waslower for thermophilic bacteria36–39 (Table III).

In the comparison of the thermophilic bacterial species, inthe 1000–400 cm�1 region they showed different FT-IRspectra. There were characteristic bands for each species inthis region and they can be used for the discrimination of thesebacteria (Fig. 6). The bands at 964 and 914 cm�1, which aredue to the vibration of C–O–C ring deoxyribose,54,55 were seenin three thermophilic species, while in G. caldoxylosilyticus,instead of these two bands, there was another deoxyriboseband55,56 at 925 cm�1. There were characteristic peaks at 851and 529 cm�1 for G. caldoxylosilyticus. The peak at 798 cm�1,which is due to adenine, was not observed in T. ethanolicus.56

In the comparison of the mesophilic bacterial species, theyalso showed different FT-IR spectra in the 1000–400 cm�1

frequency region (Fig. 7). There were characteristic peaks forLactobacillus plantarum at 505 and 420 cm�1, and these bandscan be used for the discrimination of this mesophilic bacteria.The band at 966 cm�1 that is due to the vibration of C–O–C

FIG. 6. The average spectra of T. celere (n¼ 20), T. ethanolicus (n¼ 20), C.thermohydrosulfuricum (n¼ 20), and G. caldoxylosilyticus (n¼ 20) samples inthe 1000–400 cm�1 region (the spectra were normalized with respect to theamide I band).

TABLE III. The band area ratios of some functional groups in the thermophilic (n¼ 80) and mesophilic (n¼ 80) samples.

Ratio of peak areas Functional groups Mesophilic (n ¼ 80) Thermophilic (n ¼ 80) p values

Protein/lipid Amide I þ Amide II/CH3 Sym.þCH3 Asym.þCH2 Sym.þ CH2 Asym. 2.75 6 0.06 3.20 6 0.05 ,0.01**DNA/protein PO2 Symmetric/Amide II 1.16 6 0.50 0.30 6 0.10 ,0.001***

TABLE IV. Changes in the bandwidth of various functional groups inthe thermophilic (n¼ 80) and mesophilic (n¼ 80) samples.

Functional groups:bandwidth

Mesophilic(n ¼ 80)

Thermophilic(n ¼ 80) p values

Amide II 3.8 6 0.5 4.3 6 0.4 ,0.001***

APPLIED SPECTROSCOPY 191

ring deoxyribose was only observed in the E. coli and P.aeruginosa species.54,55 Moreover, another deoxyribose bandat 935 cm�1 was sharper in P. aeruginosa than in the othermesophilic species.54

CONCLUSION

In conclusion, our results show that there were significantspectral differences between mesophilic and thermophilicbacteria, indicating that the protein concentration was high;however, lipid concentration, the level of triglycerides, and theunsaturated acyl chains were low in thermophilic bacteriacompared to the mesophilic bacteria studied. Moreover, it wasalso found that the number of hydrogen bonds was high inthermophiles. In addition to that, there were characteristicpeaks for both thermophilic and mesophilic species that can beused for the discrimination of these different bacterial species.

ACKNOWLEDGMENTS

This study was supported by Middle East Technical University (METU)research fund BAP-2005-07-02-00-12.

1. D. Naumann, ‘‘Infrared Spectroscopy in Microbiology’’, in Encyclopediaof Analytical Chemistry, R. A. Meyers, Ed. (2002), pp. 102–131.

2. D. Naumann, ‘‘FT-IR and FT-NIR Raman Spectroscopy in BiomedicalResearch’’, in ‘‘Fourier Transform Spectroscopy: 11th InternationalConference’’ AIP Conference Proceedings, J. A. Haseth, Ed. (Woodbury,New York, 1998), 430, pp. 96–109.

3. C. Teyssier, E. Jumas-Bilak, H. Marchandin, H. Jean-Pierre, J. L Jeannot,G. Dusart, V. Foulongne, and M. Simeon de Buochberg, PathologieBiologie 51, 5 (2003).

4. J. F. Siqueira, Jr., I. N. Rocas, and A. S. Rosado, J. Endodontics 31, 775(2005).

5. P. Scheldeman, L. Herman, J. Goris, P. De vos, and M. Heyndrickx, J.Appl. Microbiol. 92, 983 (2002).

6. F. B. Elegado, M. A. R. V. Guerra, R. A. Mayacan, H. A. Mendoza, andM. B. Lirazan, Int. J. Food Microbiol. 95, 11 (2004).

7. C. Iversen, P. Druggan, and S. Forsythe, Int. J. Food Microbiol. 96, 133(2004).

8. J. Tortora, R. B. Gerard, and C. L. Case, Microbiology. An Introduction(The Benjamin Cummings Publishing Company, San Francisco, CA,1995), 5th ed., pp. 125–127.

9. F. Niehaus, C. Bertoldo, M. Kohler, and G. Antranikian, Appl. Microbiol.Biotechnol. 51, 711 (1999).

10. R. F. Vogel and M. Ehrmann, Biotechnol. Annu. Rev. 2, 123 (1996).11. C. N. Jacobsen, V. R. Nielsen, A. E. Haydorf, P. L. Moller, K. F.

Michaelsen, A. Paerregaard, B. Sandstrom, M. Tvede, and M. Jakobsen,Appl. Environ. Microbiol. 65, 4949 (1999).

12. J. R. Birch and Y. Onakunle, Methods. Mol. Biol. 308, 1 (2005).

13. F. Qadri, A. M. Svennerholm, A. S. Faruque, and R. B. Sack, Clin.Microbiol. Rev. 18, 465 (2005).

14. E. B. Breitschwerdt, C. DebRoy, A. M. Mexas, T. T. Brown, and A. K.Remick, J. Am. Vet. Med. Assoc. 226, 2016 (2005).

15. A. B. Aguilar, J. Perpinan, P. Ramos, M. Peris, L. Lorente, and A. Zuniga,J. Hosp. Infect. 6, (2006) [Epub ahead of print].

16. E. Bieszkiewich, H. Boszczyk-Maleszak, A. Wlodarczyk, and M. Horoch,Acta Microbiol. Pol. 51, 285 (2002).

17. L. Mariey, J. P. Signolle, C. Amiel, and J. Travert, Vib. Spectrosc. 26, 151(2001).

18. D. Naumann, D. Helm, and H. Labischinski, Nature (London) 351, 81(1991).

19. H. Takahashi, S. M. French, and P. T. T. Wong, Clin. Exp. Res. 15, 219(1991).

20. P. T. T. Wong, E. D. Papavassiliou, and B. Rigas, Appl. Spectrosc. 45,1563 (1991).

21. M. Jackson, B. Ramjiawan, M. Hewko, and H. H. Mantsch, Cell Mol. Biol.44, 89 (1998).

22. A. M. Melin, A. Perromat, and G. Deleris, Bioploymers (Biospectroscopy)57, 160 (2000).

23. L. Chiriboga, H. Yee, and M. Diem, Appl. Spectrosc. 54, 480 (2000).24. G. Cakmak, I. Togan, C. Uguz, and F. Severcan, Appl. Spectrosc. 57, 835

(2003).25. N. Toyran and F. Severcan, Chem. Phys. Lipids 123, 165 (2003).26. G. Cakmak, I. Togan, and F. Severcan, Aquatic Toxicol. 77, 53 (2006).27. J. Kneipp, M. Beekes, P. Lasch, and D. Naumann, J. Neurosci. 22, 2989

(2002).28. C. Schultz and D. Naumann, FEBS Lett. 294, 43 (1991).29. T. J. Bruno, Appl. Spectrosc. Rev. 34, 91 (1999).30. A. Femenia, S. Sanchez, and C. Rosello, J. Agric. Food Chem. 46, 271

(1998).31. N. Toyran, F. Zorlu, G. Donmez, K. Oge, and F. Severcan, Eur. Biophys. J.

33, 549 (2004).32. A. Dogan, G. Siyakus, and F. Severcan, Food Chem. 100, 1106 (2007).33. B. Szalontai, Y. Nishiyama, Z. Gombos, and N. Murata, Biochim.

Biophys. Acta 1509, 409 (2000).34. P. I. Haris and F. Severcan, J. Mol. Catal. B: Enzymatic 7, 207 (1999).35. R. Manoharan, J. J. Baraga, P. R. Rava, R. R. Dasari, M. Fitzmaurice, and

M. S. Feld, Atherosclerosis 103, 181 (1993).36. J. R. Mourant, Y. R. Yamada, S. Carpenter, L. R. Dominique, and J. P.

Freyer, Biophys. J. 85, 1938 (2003).37. A. Perromat, A. Melin, C. Lorin, and G. Deleris, Biopolymers

(Biospectroscopy) 72, 207 (2003).38. J. Zhou, Z. Wang, S. Sun, M. Liu, and H. Zhang, Biotechnol. Appl.

Biochem. 33, 127 (2001).39. E. Taillendier and J. Liquier, Methods Enzymol. 211, 307 (1992).40. L. J. Rothschild and R. L. Mancinelli, Nature (London) 409, 1092 (2001).41. R. Garu and D. de Mendoza, Mol. Microbiol 8, 535 (1993).42. J. Svobodova and P. Svoboda, Folia Microbiol. (Praha.) 33, 161 (1988).43. J. L. C. M. Vossenberg, A. J. M. Driessen, M. S. Costa, and W. N.

Konings, Biochim. Biophys. Acta 1419(9), 97 (1999).44. E. N. Lewis, I. W. Lewin, and C. J. Steer, Biochim. Biophysics. Acta 986,

161 (1989).45. M. Chan, R. H. Himes, and J. M. Akagi, J. Bacteriol. June, 876 (1971).46. M. Jackson, M. G. Sowa, and H. H. Mantsch, Biophys. Chem. 68, 109

(1997).47. K. F. M. Fung, M. Senterman, P. Eid, W. Faught, Z. N. Mikhael, and P. T.

T. Wong, Gynocol. Oncol. 66, 10 (1997).48. G. Bohm and R. Jaenicke, Int. J. Pept. Protein Res. 43, 97 (1994).49. R. Scandurra, V. Consalvi, R. Chiaraluce, L. Peliti, and P. C. Engel,

Biochimie 80, 993 (1998).50. P. Argos, M. G. Rossman, U. M. Grau, H. Zuber, G. Frank, and J. D.

Tratschin, Biochemistry 18, 5698 (1979).51. G. L. Warren and G. A. Petsko, Protein Eng. 8, 905 (1995).52. E. Querol, J. A. Perez-Pons, and A. Mozo-Villarias, Protein Eng. 9, 265

(1996).53. G. Vogt, S. Woell, and P. Argos, J. Mol. Biol. 269, 631 (1997).54. M. Ghomi, R. Lettellier, J. Liquier, and E. Taillandier, Int. J. Biochem. 22,

691 (1990).55. G. I. Dovbeshko, O. P. Gnatyuk, V. I. Chegel, Y. M. Shirshov, D. V.

Kosenhov, E. A. Andreev, H. A. Tajmir-Riahi, and P. M. Lytvyn,Semicond. Phys., Quant. Electron. Optoelectron. 7, 318 (2004).

56. E. Taillandier, J. Liquier, and J. A. Taboury, Adv. Infrared RamanSpectrosc. 65 (1985).

FIG. 7. The average spectra of E. coli (n ¼ 20), M. luteus (n ¼ 20), L.plantarum (n¼ 20), and P. aeruginosa (n¼ 20) samples in the 1000–400 cm�1

region (the spectra were normalized with respect to the amide I band).

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