807
Anal. Chem. 1984, 56,807-808
CORRESPONDENCE Ion Chromatography for Determination of Metabolic Patterns of Sulfate-Reducing Bacteria Sir: Ion chromatography has been a powerful instrument in the field of water analysis. It is being used in water treatment control as well as in the food industries as an accurate and fast method for simultaneous detection of various ionic species in aqueous solutions. Recently, we explored the ability of this instrument to monitor ionic species in bacterial growth media in conjunction with microbiology research. This research is aimed at the study of a biological sulfate-reduction process for recovering sulfur from high-sulfate wastes. Metabolic patterns of the sulfate-reducing bacteria have already been explained by Postgate (I) and others over 30 years ago. However, the power of the ion chromatograph to relate the various components which are involved in the metabolic reactions opens a new perspective in the understanding of the bacterial process. Furthermore, the total analysis time is only 25 min. This is a major advantage of the method over those presently used which require several hours for a complete analysis. This paper explores growth characteristics of sulfate-reducing bacteria from Desulfovibrio genus, by means of ion chromatography. EXPERIMENTAL SECTION The ion chromatography system used is made by Dionex Corp. It is equipped with an ion chromatographyexclusion (ICE) column for organic acid analysis ( 2 , 3 ) ,a Dionex conductivity detector, and an HP3390A integrator. The ICE column is capable of simultaneous detection of sulfate, lactate, acetate, and carbonate ions, which play an important role in the metabolism of the sulfate-reducing bacteria. The difficulty in the detection process is the elution of the carbonate peak, since it has low conductivity and thus requires higher sensitivity than the other species. Fortunately, the carbonate elutes last, about 5 min after the acetate peak. By increasing the sensitivity of the detector shortly before the appearance of the carbonate peak and by using a weak (1m M HCl) eluent, we can detect the carbonate at concentrations as low as 5 mg/L. The conductivity detector was set to an output range of 30 hs for the detection of the sulfate, lactate, and acetate ions and was then set to 3 fis for the carbonate detection. The HP3390A integrator was operated in the internal standard mode of analysis. In this method, an additional component, not appearing in any sample, is added in a known amount to the calibration mixture. The concentrations of the solutes are calculated from their peak-height relationship to that of the internal standard. Throughout this experiment, a 20 mg/L formate spike was used as the internal standard. The samples were diluted with distilled water, so as to reach a concentration level of up to 50 mg/L for all the solutes involved, and were additionally spiked with the formate standard. This measurement provided reproducible results and thus required no further treatment of the samples. The growth characteristics of a mixed Desulfovibrio culture from sewage were studied in a 1-L reactor using a synthetic sodium sulfate growth media, as described in Table I. The reactor was incubated anaerobically at room temperature (25 "C) for a 24-h period. The initial and final ionic compositions were measured in the ion chromatograph in terms of the main compounds, as previously discussed. 0003-2700/84/0356-0807$0 1.50/0
Table I. Na,SO, Mediaa 6.0 g 1.1g
Na,SO, KH,PO, NH,CI yeast extract ascorbic acid 60% sodium lactate thiogl colate FeC1, distilled water
0.75 g 1.1g 0.15 g 15.0 mL 0.15 mL
x
0.1 g 1.5 L a
Final aH. 7.2.
Added after boiling.
Table 11. Experimental Data lactate
acetate
initialconcna finalconcna
2927 279
7727 1048
0
0
3906
1744
reductiona productiona
2648
6679
3906
1744
molar ratiob
1.00
2.39
1.05
In mg/L. solution. a
carbonateC
sulfate
In moles.
2.70
CO, portion then remains in
RESULTS AND DISCUSSION The chromatograms of both the initial and the final ion concentrations in the Na2S04growth media are shown in Figure 1. Table 11, which follows, presents the experimental data that were calculated from the chromatograms. The change in the concentration of those species (reduction or production) is interpreted, in the bottom line of Table 11, as a molar relationship between the four ionic species. The molar ratio shows that 2.70 mol of lactate are required to reduce 1.00 mol of sulfate to sulfide, while producing 2.39 mol of acetate and 1.05 mol of carbonate, under the specific conditions of the experiment. This information is essential to any engineering design of specific biological systems, such as desulforization, denitrification, etc. In such systems, the bacterial metabolism ratio will allow an optimal operation of the biological reactor and will further determine the economical feasibility of the process. Other ions are involved in the metabolic reactions; sulfide is produced in the sulfate reduction and the lactate, in the pairing reaction, is broken down to acetate and carbon dioxide. However, under the reaction conditions most of the COz will remain in solution as carbonate species, which can be analyzed in the ion chromatograph. The gas portion can be detected by gas chromatography or by other conventional methods. Furthermore, the carbonate portion by itself is an important factor in the design of various water systems (e.g., lime treatment). The sulfide ion concentration can be detected in the ion chromatograph, using an anion column and an electrochemical detector ( 4 ) . Other available methods include a sulfide ion selective electrode measurements and gravimetric methods (5, 6). 0 1984 American Chemical Society
aoa
Anal. Chem. 1984, 56,808-810
bolic reactions will allow fast and simple optimization of biological systems. ACKNOWLEDGMENT We wish to thank Jay Stern from Joy Manufacturing Co. for initiating and continuously encouraging this research. We also wish to express our appreciation to John Findley for his helpful remarks. Registry No. Sulfate, 14808-79-8;lactic acid, 50-21-5; acetic acid, 64-19-7; carbonate, 3812-32-6. LITERATURE C I T E D
0
5
io
15 minutes
20
25
30
(1) Postgate, J. R. “The Sulphate-Reducing Bacteria”; Cambridge University Press: Cambridge, 1979. (2) Rich, W.; Johnson, E.; Lois, L.; Kabra, P.; Stanford, B.; Marton, L. Clin. Chem. (W/flSfOn-S8/em,N.C.) 1980, 26, 1492. (3) Koppel, I. R., Paper Presented at the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, 1981; Paper 241. (4) Rockiin, R.; Johnson, E. Anal. Chem. 1983, 55, 4 (5) Ai-Hiili, I. K.; Moody, G. R.; Thomas, J. D. R. Analyst (London) 1983, 108, 43. (6) Ai-HiIIi, I. K.; Moody, G. R.; Thomas, J. D. R. Analyst (London) 1983, 708, 1209.
Figure 1. Ion chromatograph readings: (a) initial (b) final (Dionex 20001 with ICE column connected to HP3390A integrator).
The accuracy of the Dionex ion chromatography system was up to i 5 % for the detection of sulfate, lactate, and acetate species and &lo% for the carbonate. This is satisfactory for an engineering design. In conclusion, ion chromatography can successfully contribute to the understanding and the exploration of bacterial metabolism patterns. Its ability to simultaneously quantify the major ions which are involved in various bacterial meta-
Alon Lebel T e h Fu Yen* Department of Civil Engineering Environmental Engineering Program University of Southern California Los Angeles, California 90089
RECEIVED for review October 24, 1983. Accepted January 4, 1984. We thank Joy Manufacturing Co. for providing the funding for this research.
Definition of the Response Time of Ion-Selective Electrodes and Potentiometric Cells Sir: One of the critical limiting factors in the use of ionselective membrane electrodes, especially in routine analysis, is their so-called response time. Uemasu and Umezawa ( I ) pointed out the logical paradox involved in the internationally accepted definitions (2,3)oft, and t*, namely, that one cannot determine t , and t* values without knowing E , (equilibrium potential) or t,, because t , or t* is defined as time required for the ion-selective electrode to reach a% of its equilibrium potential (2)or to become equal to its steady-state value within 1mV (3),respectively, after a concentration step change in the sample. Since these definitions (2, 3) provide no aid to practical analytical work, Uemasu and Umezawa (1) defined a value, called differential quotient, t(At,aE),as a measure of practical response time and compared it with other conventional definitions. The differential quotient in fact is a limiting value of the slope of the potential-time curves and in this sense it was applied first by Lindner, Tdth, and Pungor (4) for characterizing and comparing the transient functions of ionselective electrodes. Pungor and Umezawa suggested a way to draw an unambiguous distinction between the response time of the potentiometric cell (containing an ion-selective electrode) and that of the ion-selective electrode itself ( 5 ) . These two response time values may become equal only under special experimental conditions (high mass transport
rate and low concentrations) (6) or in the case of ion-selective electrodes with relatively long response times (7-9). In Uemasu and Umezawa’s paper some characteristic features of the slope method suggested for determining response times have been summarized as follows: it has the advantage that “it is concentration independent when At is chosen properly short and AE is small” and “it has no direct relation to mathematical formulation of response curves like the time constant in an exponential” equation. The aim of the present paper is to discuss the above statements in more detail based on the simple diffusion model (7, IO), generally valid for describing the dynamic response of potentiometric cells under practical analytical conditions (low mass transport rate and high sample activities, i.e., relatively slow measuring setup and electrodes with short response times) ( 6 , I I ) . The diffusion model was selected to illustrate the fact that the differential quotient, Le., slope value has direct relation to the mathematical equation describing the response time curves as Uemasu and Umezawa worked under conditions fulfilling the diffusion model assumptions. In light of this, the activity dependence of the slope values will become obvious. Naturally the same treatment can be carried out for other types of mathematical equations also if different model assumptions are valid (e.g., for different types of electrodes (8) or other experimental conditions (13, 14)).
0003-2700/84/0356-0808$01.50/00 1984 American Chemical Society
