Fourier transform infrared detection of pyruvic acid assimilation by E

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Anal, Chem. 1985, 5 7 , 2487-2491

Fourier Transform Infrared Detection of Pyruvic Acid Assimilation by E . coli Robert L. White*' and Dean E. Roberts Mattson Instruments, 6333 Odana Road, Madison, Wisconsin 53719

Michael C. Attridge Gibco Division, Life Technologies, Inc., 2801 Industrial Drive, Madison, Wisconsin 53713

A flow cell FTIR interface is used to monitor changes in fermentation broth caused by E . coli digestion of pyruvk acid. Infrared spectral variations are attributed to pyruvate concentratlon decrease and phosphate buffer changes. With infrared measurements, pyruvate concentration is found to decline by 0.016 M from an inltial concentration of 0.023 M afler 9 h of fermentation. Pyruvate absorbance band intensity variations as small as 0.001 18 A are detected during fermentation with a measurement precision of f0.000 04 A .

Attenuated total reflectance ( A m ) techniques have greatly simplified infrared analysis of aqueous solutions. With some difficulty, short path length transmission cells have been constructed for aqueous solution analysis. These cells can be difficult to fill and internal pressure fluctuations can change cell path length. On the other hand, ATR liquid cell designs can provide highly reproducible path lengths facilitating removal of water bands fTom infrared spectra with absorbance subtraction methods ( 1 ) . In addition, ATR cells can be filled easily and can be adapted for flow cell applications. Recombinant DNA research has led to development of bacterial strains capable of producing substances which can serve as precursors for synthesis of antibiotics (2). Anaerobic fermentation is a method of producing fuels such as ethanol and methane from biomass digestion (3). Recently, biotechnology companies have been formed to market bacteria-derived products and fermentation technology has been scaled to a commercial level (4). Optimized fermentation efficiency requires extreme care in preparation of cell media. Fermentation broths must contain nutrients in proper proportion and contaminants known to have a detrimental effect on bacteria must be eliminated. Fermentations typically require several hours to produce significant quantities of bacterial product. T o maintain fermentation reactor efficiency, real-time monitoring techniques measuring changes in fermentation broth composition are needed. Previous studies have demonstrated the capability of FTIR spectrometers with ATR accessories for quantitative analysis of fermentation broth components with detection limits below 0.1% (5,6). In this study, digestion of pyruvic acid by Escherichia coli k802 bacteria was detected with a flow cell ATR accessory coupled with an FTIR. Cell growth and phosphate buffer concentration changes were monitored and could be attributed to aerobic assimilation of pyruvic acid by the bacteria.

'Present address: Department of Chemistry, University of Oklahoma, 620 Parrington Oval, Norman, OK 73019. 0003-2700/85/0357-2487$01.50/0

EXPERIMENTAL SECTION Apparatus. A Mattson Inetruments (Madison, WI) Sirius 100 FTIR was used for fermentation broth infrared measurements. The FTIR was equipped for mid-infrared operation with a Ge/KBr beam splitter, water-cooled globar source, and a wideband MCT detector (475 cm-' cutoff). A Barnes/Spectra Tech (Stamford, CT) cylindrical internal reflection (CIR) cell with a ZnSe rod 0.635 cm in diameter and 8 cm in length was employed as a flow cell interface to an Airlift fermentation reactor (Bethesda Research Laboratories-Division of Life Technologies, Inc.). The fermentation reactor and flow cell were connected with rubber tubing and a peristalic pump was used to maintain a flow rate of 0.8 L/min through the CIR cell. No attempt was made to separate cell material from broth prior to passing the solution through the flow cell. Aerobic metabolism was ensured by regulating a flow rate of air into the reactor chamber at 2 L/min. When fermentation was stopped, it was discovered that the temperature probe of the fermentation reactor was defective. As a result, the temperature of the fermentation broth during the experiment was not regulated. The temperature of the fermentation broth at the end of the fermentation experiment was measured with a thermometer and found to be 42 "C. Data Collection. By use of a macro program written in UNIX C-shell language, infrared spectra were obtained in 6.5-min intervals. The FTIR interferometer scan velocity was 1.27 cm/s and 600 scans were signal averaged using dual direction scanning at 8-cm-l resolution. All single-beam iqfrared spectra collected during fermentation were ratioed to a previously collected reference single beam spectrum measured using an empty CIR flow cell. Two thousand scans were signal averaged to generate the reference single-beam spectrum. At various intervals during the fermentation experiment, aliquots of broth were extracted from the fermentation reaction vessel for colony counting to determine cell concentration. Infrared spectra of pure-component aqueous solutions were obtained by acquiring 64 signal averaged scans for both reference and sample at 4 cm-l resolution. All infrared spectra were measured while the FTIR was purged with dry nitrogen. A nitrogen purge rate of 1.5 scfm was maintained for the duration of fermentation. Reagents. Fermentation broth and E. coli k802 were obtained from Gibco Division, Life Technologies, Inc. (Madison, WI). Two liters of fermentation media was added to the fermentation reactor. The fermentation broth was a Blattner media and consisted of 32 components dissolved in water. Sodium pyruvate was present in this mixture at a concentration of 0.25% (0.023 M). RESULTS AND DISCUSSION Pyruvate Fermentation. The bacterial strain selected for fermentation studies was Escherichia coli k802. This particular strain of E. coli is known to assimilate pyruvic acid but will not digest lactose. A strain incapable of fermenting lactose was chosen because both pyruvic acid and lactose were present in the growth media. Aerobic assimilation of pyruvic acid by E. coli is equivalent to combustion to form carbon dioxide and water. CHSCOCOOH

+ 5/202 + 3co2 + 2Hz0

0 1985 American Chemical Society

(1)

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ANALYTICAL CHEMISTRY, VOL. 57, NO. 13, NOVEMBER 1985

CELL GROWTH

i

140

I I\

0.0100

I

P

+-Q

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100

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036i

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669

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1229

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l

1

1

1

5

6

7

8

9

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(HOURS)

Figure 1. Variation of fermentation broth E. coli cell concentration with

time. In the absence of oxygen, fermentation proceeds via an anaerobic mechanism yielding acetic acid and formic acid (7).

CH3COCOOH

asee

+ HgO + CH3COOH + HCOOH

(2)

The experiments described here were a feasibility study. Every attempt was made to minimize unexpected changes in broth composition during fermentation. Conditions were adjusted for aerobic fermentation of pyruvic acid by continuously bubbling air through the fermentation reactor at a rate of 2 L/min during fermentation. However, the possibility of anaerobic metabolism as cells passed through the flow cell and connecting tubing did exist. A relatively high flow rate through the flow cell (0.8 L/min) was maintained to minimize the length of time cells spent in these areas and reduce the possibility of anaerobic fermentation. Prior to start of the fermentation process, the reactor and connecting tubing were sterilized. The flow cell was not subjected to sterilization because the effect of heat on the ZnSe rod was uncertain. Instead, the ZnSe rod was washed thoroughly with acetone and dried in an oven at 60 "C. The fermentation process was initiated by inoculating the growth media with a concentrated solution of E. coli grown the previous day. Fermentation was allowed to proceed for 9 h. During this period, 83 infrared spectra were obtained at equal time intervals. Also, 16 aliquots of broth were removed at various times. Colony counting methods were used to determine cell concentration for each of the aliquots. Figure 1 depicts cell concentration increase as a function of time derived fram colony counting measurements. As expected, cell concentration increase was exponential. A plot of the natural logarithm of cell concentration vs. time yielded a straight line (correlation coefficient 0.989) with a slope of 0.72 In [celI]/h. Spectral Changes. Spectral changes occurring during the fermentation experiment were qualitatively examined by subtracting the first infrared spectrum acquired ( t = 0) from the last spectrum measured (t = 9 h). The resulting difference spectrum is illustrated in Figure 2. Substances responsible for difference peaks were identified by comparing peak location in the difference spectrum with corresponding peak locations measured in spectra of individual broth components (Table I). Solvent interactions significantly broaden absorbance bands and in some instances introduce peak shifts. For valid comparison, pure component spectra used for reference were obtained from solutions of pure substances dissolved in water. Spectral changes noted in Figure 2 can be attributed to the expected decrease in pyruvic acid concentration and changes in [H2P04-]/ [HP042-]buffer concentrations. Contributions from acetic acid and formic acid expected

5

Figure 2. Absorbance difference spectrum showing changes in component concentrations due to fermentation of pyruvlc acid.

Table I. Absorbance Peak Locations for Substances Dissolved in Water component

peak location, cm-l

CH&OCOOH

1707 1601 1424 1396 1356 1176

39 100 22 27 27 69

H2P0i

1155 1076 939

100

HP02-

'

re1 intensity, %

65 54

1076 989

100

CH3COOH

1710 1390 1276 1051 1014

100 60 99 20 35

HCOOH

1712 1396 1209 1072

100

48

34

71 14

for anaerobic fermentation were not observed. A peak at 1261 cm-' increased in intensity during fermentation but could not be assigned by comparison with pure component spectra. This peak was not observed in data obtaiped from a second fermentation using similar conditions. Consequently, this peak was attributed to an unknown impurity present either in the growth media or in the inoculating solution prior to fermentation. The difference peak at 1088 cm-l was attributed to changes in phosphate buffer concentrations. Spectra obtained from H2P0, and HP042-pure component solutions did not contain peaks with maxima at 1088 cm-l. However, both pure component spectra contained bands with maxima at 1076 cm-l. The peak width of the 1076-cm-l band in the HP04'spectrum was larger than the corresponding peak width of the H2P04-band. In addition, the HP042-1076-cmV1peak was found to be'asymmetric when superimposed on the H2P041076-cm-' peak. The 1088-cm-l difference peak was most likely an artifact generated because of this asymmetry. In fact, a curve similar to the difference spectrum was constructed by subtracting pure component H2P04-and HPOd2-spectra (Figure 3). Data Manipulation. In order to compare fermentation broth infrared spectra visually, spectral contributions from intense water bands were removed. This was accomplished by subtracting a reference spectrum of distilled water from each of the fermentation broth spectra. The water spectrum

ANALYTICAL CHEMISTRY, VOL. 57, NO. 13, NOVEMBER 1985 0.0440

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Figure 5. Pyruvate concentration variation as measured by changes in 1176-cm-' absorbance intensity.

Wavenumbers

PYRUVATE

(1356 crri' 1

b

I

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110,

1051

I 100

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Wavenumbers

Flgure 3. (a) Expanded reglon of the difference spectrum in Figure 2 suspected to represent phosphate buffer changes. (b) Absorbance difference spectrum generated by subtracting the H2P04- pure component spectrum from the HP042-spectrum. FERMENTATIOY 0.060 0.055

Flgure 6. Pyruvate concentration variation as measured by changes in 1356-cm-' absorbance intensity. PHOSPHATE VARIATION

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Figure 4. Typical fermentation broth spectrum after removal of water bands by absorbance subtraction. was obtained under the same conditions as were fermentation broth spectra. The subtraction factor by which the water reference spectrum was multiplied to eliminate water bands by spectral subtraction was the ratio of 1640-cm-' band intensities for broth and water reference spectra. To eliminate base line drift contributions in 1640-cm-' band intensity measurements, the absorbance of the 1640-cm-l band was computed relative to an arbitrarily selected base line reference point a t 1900 cm-l (Le., A = A(1640) - A(1900)). After subtracting water band absorbances, difference spectra reveal

TIME (HOURS)

Figure 7. Phosphate buffer concentration fluctuations observed during the fermentation experiment. Concentrations were Calculated from infrared absorbance measurements. information for all 32 media components and interactions between these components with each other and aqueous solvent (Figure 4). Intensities for pyruvic acid and phosphate buffer bands were calculated from difference spectra derived from the 83 infrared measurements after water reference subtraction. Changes in band absorbance were plotted as a function of time (Figures 5-7). Absorbance of unresolved bands was calculated relative to local base lines- computed using points on each side of peak center. For dilute solutions, Beer's law is used to convert spectral absorbance variations to concentration changes if absorbance cell path length and molar extinction coefficient are known.

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ANALYTICAL CHEMISTRY, VOL. 57, NO. 13, NOVEMBER 1985

Table 11. Variations of Fermentation Broth Components

component

peak location, cm-'

Aabsorbance ( A )

std dev

pyruvate pyruvate

1176 1356 939 989

-0.0035 -0.0012 -0.0030 0.0033

0.000 08 0.000 04

HZPOc

HP02-

0.00009

0.000 04

Table 111. Logarithmic Linear Regression Results

slope peak location,

component

cm-'

pyruvate pyruvate

1176 1356 939 989

HZPO4HP0,2'

(In I[component]l/ h) 0.76

0.80 0.68 0.66

corr coeff 0.99 0.98 0.99 0.99

For CIR analysis, path length (depth of radiation penetration) is determined by the relative refractive indexes of ZnSe and sample material and by the angle of incident radiation (8). For the flow cell used in this study, angle of incidence was fixed by the alignment of the FTIR optics and was not changed for any of the infrared measurements. CIR flow cell path length was calibrated for dilute aqueous solution measurements using distilled water. The extinction coefficient for water at 1655 cm-l was derived by Centeno (9) using normal incidence reflectance measurements. With his extinction coefficient value (0.1372) and Beer's law, CIR flow cell path length was calculated to be 6 pm. This path length calculation should be valid for dilute solutions such as the fermentation broth used in this study. Dissolved solutes in concentrated solutions significantly alter the refractive index of the solvent and would change CIR cell path length. Aqueous solutions of known concentration were analyzed by using the CIR cell and molar extinction coefficients for pyruvic acid and phosphate absorbance bands were determined (Table 11). Known concentration solutions used for molar extinction coefficient determination were prepared with pure component concentrations equal to the concentration of each species in the growth media before fermentation. Figures 5 and 6 depict variations of pyruvic acid concentration during fermentation. As expected, pyruvate uptake was inversely proportional to cell concentration increase. Concentration values in Figures 5 and 6 were computed from absorbance measurements using Beer's law and calculated molar extinction coefficients. Inflection points in the curves for the 1176-cm-1and 1356-cm-l bands occurred after 5 h of fermentation. This agrees with cell concentration increase shown in Figure 1. Logarithmic plots of concentration variations as a function of time derived from data acquired during the last hour of fermentation yielded straight lines. Linear regression slopes and correlation coefficients for these graphs are compiled in Table 111. Concentration slopes computed for pyruvate 1176-cm-l and 1356-cm-*bands should in theory be equal and were indeed very similar (0.76 vs. 0.80). Either of these bands could be used to monitor pyruvate consumption. However, the 1176-m-l band would be preferred because the molar extinction Coefficient for this bahd was larger than the molar extinction coefficient for the 1356-cm-l band. A plot of peak intensity vs. time for the most intense pyruvate band at 1601 cm-l also produced an exponentially declining curve, but the scatter of data points about the best fit curve was significantly greater than for data points obtained from 1176-cm-' and 1356-cm-l measurements. The 1601-cm-' band was not well resolved from spectral interferences and was located on the side of a major absorbance band in broth difference spectra (see Figure 4).

ext coeff, L/(mol em)

A[component], M

std dev

364 156 465 552

-0.016 -0.013 -0.011 0.010

0.0004 0.0004 0.0001 0.0003

Figure 7 shows changes in phosphate buffer concentration during fermentation. To some extent, these changes can be explained in terms of multiple equilibria. Phosphate buffer was employed to maintain the cell growth medium at pH 6. During the fermentation, pH remained relatively constant. Toward the end of the experiment, fluctuations in pH were observed but these changes were minor and random. At pH 6, pyruvic acid is primarily in a dissociated form ([CH,COCOO-]/ [CH,COCOOH] = 3200). Removal of pyruvic acid from the fermentation broth by cell metabolism would cause a concomitant reduction in hydrogen ion concentration. To compensate, H2P04- species would dissociate creating hydrogen ions and HP042-anions. Spectral changes shown in Figure 7 are in qualitative agreement with expected changes in buffer species concentrations. Slopes of best fit lines computed from logarithmic plots of phosphate concentration vs. time were similar for both phosphate species (0.68 and 0.66). It is noteworthy that slopes computed for phosphate species concentration changes were lower than the slopes computed for pyruvate consumption. It is possible that the unknown species responsible for the 1261-cm-' absorbance increase during fermentation had an effect on hydrogen ion concentration. This effect would have to be considered to fully understand changes in phosphate concentration. In addition to equilibrium shift, phosphate buffer concentrations decreased because phosphate is a nutrient for growing cells. It has been estimated that 3% of E. coli dry weight is comprised of phosphorus (IO). Peak intensity variations for each of the analyte bands in Figures 5-7 is complied in Table 11. Change in absorbance during fermentation was typically a few thousandths of an absorbance unit. The inflection point in Figure 1and for each of the curves in Figures 5-7 occurred at approximately 5 h into the fermentation experiment. Prior to this point, plots were linear. Cell concentration changes on the order of 1 X lo7 cells/mL were required to produce perceptible changes in analyte band intensities. An estimate of concentration measurement precision was computed as the standard deviation for concentration values derived from spectra acquired during the first 4 h of fermentation. Standard deviations better than fO.001 M were obtained in every case. Although infrared absorbance changes were minimal, measurement precision was sufficient to permit monitoring these changes as a function of time.

CONCLUSION This study establishes the feasibility of using FTIR spectrometry to monitor E . coli assimilation of pyruvic acid in a fermentation reactor. During the 9-h fermentation experiment, absorbance peak variations of a few thousandths of an absorbance unit were detected with a precision better than f0.0001 A. Measurement precision might be improved further with a more sensitive narrow-band MCT detector in place of the wide-band MCT detector used for this study. In fact, a narrow band detector would be preferred for these studies because the ZnSe ATR material used for the flow cell interface was opaque below 800 cm-l. Results presented here show a qualitative link between changes in fermentation broth nutrient concentration and cell growth. A quantitative relationship based on conservation

249 1

Anal. Chem. 1985, 57, 2491-2499

of mass could be derived from measurements of dry cell weight instead of cell concentration. Unfortunately, our laboratory was not equipped for these measurements. The biological system selected for this study was simple. Even so, three broth components were found to change significantly during fermentation. More complicated biological systems may generate several products from a variety of precursors. By use of the techniques described here, it should be possible to monitor products and precursors in real time. By association of spectral changes with specific substances, on-line monitoring methods could be developed for complex fermentation reactions. Due to the complexity of spectra obtained from fermentation broth measurements, deconvolution methods may prove useful for future investigations of more involved biological systems.

LITERATURE CITED Rein, A.; Wilks, P. Am. Lab. (Fairfield, Conn.) 1982, 74 (lo),197. McAleer, W. J.; Buynak, E. 6.; Maigetter, R. Z.; Wampler, D. E.; Miller, W. J.; Hilleman, M. R. Nature (London) 1984, 307, 178-180. Haggin, J.; Krieger, J. H. Chem. Eng. News 1983, 67 (ll),28-30. Webber, D. Chem. Eng. News 1984, 62 (16),11-19. Wong, J. S.; Rein, A. J.; Wilks, P.; Wilks, D. Appl. Spectrosc. 1984, 38,32-35. (6) Kuehl, D.; Crocombe, R. Appl. Spectrosc. 1984, 38, 907-909. (7) Stephenson, M. I n "Bacterlal Metabolism"; The MIT Press: Cambridge, MA, 1966;p 79. (8) Harrick, N. J. I n "Internal Reflection Spectroscopy"; Harrick Scientific Corp.: Ossining, NY, 1979;p 30. (9) Centeno, M. J . Opt. SOC. Am. 1941, 3 7 , 245-248. (10) Ingraham, J. L.; Maaloe, 0.; Neidhardt, F. C. I n "Growth of the Bacterial Cell"; Slnauer Associates, Inc.: Sunderland, MA, 1983; p 3.

RECEIVED for review May 14,1985. Accepted June 24,1985.

Lower Limit of the Thickness of the Measurable Surface Layer by Fourier Transform Infrared Attenuated Total Reflection Spectrometry Koji Ohta and Reikichi Iwamoto*

Government Industrial Research Institute, Osaka, Midorigaoka 1, Ikeda, Osaka 563, Japan

Investlgatlon has been made of the mlnlmum thickness of the surface layer on an Infrared absorblng polymer film that can be measured by spectral subtraction In Fourler transform Infrared attenuated total reflectlon (FT-IR-ATR) spectrometry. Spectral separability of a surface layer was examined for polymer fllms coated wlth thin layers of polystyrene or Langmulr-Blodgett (LB) fllms of known thicknesses. Absorptlon bands of polystyrene were recognized even for the thlckness of 12 A. For the LB fllms, even a group of weak bands was Isolated for the thickness of 55 A. To give the observable limit In FT-IR-ATR spectrometry, the concept of detectable least signal increment (DLSI) was Introduced. The DLSI value was found to be 2-4 times the noise level. Observablllty of a surface layer of a thlckness Is predictable by comparlng the expected absorbances of the bands of the surface layer wlth the DLSI value obtainable from the noise level of a spectrometer.

Fourier transform infrared attenuated total reflection (FT-IR-ATR) spectrometry is one of the most frequently used tools for surface characterization of polymer materials (1,2). Its advantage as a surface analytical method is the ability to give much information on a surface such as chemical composition and chemical structure (l), orientation and conformation (3),crystallinity ( 3 , 4 ) ,hydrogen bonding (5), etc. In addition, vastly accumulated infrared data on the bulk and chemical structures add value to usefulness of the method (6, 7). The capability of ATR spectrometry for surface analysis is attributed to the penetrating nature of the light on total reflection from the internal reflection element (IRE)-sample interface into the sample (8). Although ATR infrared spectrometry has been familiar as a convenient surface analytical method for chemists, they have usually applied the method for qualitative purposes and have

not extensively examined its capability to analyze very thin surface layers. The method has not been generally considered as powerful as sophisticated surface analytical techniques like ESCA, SIMS, Auger electron spectroscopy, etc., which are useful to analyze thin surface layers as 10-50 8,. These methods, however, give information basically of atoms or atomic groups but not on molecular structures of higher order and their arrangement. If it is possible to measure very thin surface layers by FT-IR-ATR spectrometry, it should give much richer information on material surfaces. However, the evanescent wave, which picks up information on surfaces in ATR spectrometry, penetrates to the depth of from a few tenths to a few micrometers (2) and it has generally been considered that it is difficult to characterize such a thin surface layer as 50 8, or so especially when the underlying base layer is infrared absorbing. On the other hand, the evanescent wave has the nature that the amplitude is larger a t a shorter distance from the IRE-sample interface and, therefore, a thinner surface layer has a larger contribution per thickness to the total absorption intensity of a band. If we utilize this nature of the evanescent wave and subtract the contribution of a deeper layer from the FT-IR-ATR spectrum, it may be possible to obtain the spectrum of a very thin surface layer on an infrared absorbing base layer. In the present study, we have examined the capability of FT-IR-ATR spectrometry as a tool to measure a very thin surface layer and it has been found that the surface layer of 50 8, or so can be observed without much difficulty if spectral subtraction is applicable. EXPERIMENTAL SECTION

A Nicolet 7199 FT-IR spectrometer and a Wilks Model 50 ATR attachment were used for FT-IR-ATR measurements as reported in a previous paper (I). A specially designed platform was used to fix the ATR attachment firmly in the FT-IR instrument (I). FT-IR-ATR measurements were made of thinly coated samples of a polyurethane film, in which the coating layer was of polystyrene, and also of a poly(4-methyl-1-pentene) (PMP, obtained in the form of films from Mitsui Petroleum Co., Ltd.) film coated

0003-2700/85/0357-2491$01.50/00 1985 Amerlcan Chemical Society