Jul 29, 1977 - tritiated glucose was used as a radioactively labeled substrate were collected, and ... Counts per minute were corrected for quench. (by external ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1978, p. 456-458
0099-2240/78/0035-0456$02.00/0 Copyright i 1978 American Society for Microbiology
Vol. 35, No. 2
Printed in U.S.A.
Respiration Correction for Microbial Heterotrophic Activity Assays That Use Tritium-Labeled Substrates ALLAN S. DIETZ AND LAWRENCE J. ALBRIGHT* Simon Fraser University, Burnaby, British Columbia, Canada V5A IS6 Sciences, Department of Biological
Received for publication 29 July 1977
Filtrates from microbial heterotrophic activity assay experiments in which tritiated glucose was used as a radioactively labeled substrate were collected, and their radioactivities were determined. These filtrates were subsequently evaporated to dryness to remove tritiated water generated by metabolism of the labeled glucose; the residue was suspended to original volume with distilled water, and the tritium levels were again assayed. In the water samples assayed, the amount of tritium label retained by the microorganism was about 75% of that respired. Tritiated organic substrates have recently been shown to be suitable substitutes for 4Clabeled ones in heterotrophic microbial activity assays of aquatic ecosystems (1, 2). This substitution has the advantage of allowing an investigator to add tritium-labeled nutrients to waters at concentrations that more closely approximate in situ nutrient conditions, but has the disadvantage of the respired substrate not being assayed. Hobbie and Crawford (3) reported a method to correct for respiration of 14CO2 from the utilization of "4C-labeled substrate by heterotrophic microorganisms. In this communication, we report on a respiration correction for heterotrophic microbial activity assays that use tritiated substrates. The technique is based upon assay of tritiated water respired by the ecosystem's microorganisms under aerobic conditions. The technique is not useful for anaerobic environments where respiration does not occur. Water samples were collected from the Simon Fraser University (Burnaby, Canada) reflecting pool from a depth of 20 cm by using a sterile, 2liter flask, maintained at in situ temperatures, and processed within 10 min after removal to the laboratory. Portions (100 ml) of sample water were added to each of four 250-ml Erlenmeyer flasks. Tritiated glucose (D-[6-3H]glucose; specific activity, 10 Ci/mmol; Amersham/Searle) was then added to the flasks so as to have final glucose concentrations of from 2 x 101o to 2 x 10' M glucose (0.2 to 1.0 ,uCi/100-ml water sample). After these additions, the samples were incubated at in situ temperature and duplicate 10-ml subsamples were removed from each of the flasks at 15-min intervals. These were filtered onto membrane filters (0.22-,um pore size, 47-mm diameter; Millipore Corp.). In each instance, filtrates were
collected in separate, clean filtration flasks containing 2 ml of 10% Formalin. The filtration head was then moved to a second filtration flask, and filters were rinsed with 10 ml of prefiltered sample water. Killed controls were prepared in the same manner, except that 2 ml of 10% Formalin was added before addition of radiotracer and no Formalin was added to the filtrate. After filtration, all filters were immediately placed in scintillation vials containing 15 ml of Aquasol scintillation cocktail with 10% ethyl acetate added to solubilize the cellulose nitrate of the filters. After clarification and partial dissolution, the tritium-labeled material retained on each filter was determined by using a Beckman LS-250 liquid scintillation spectrometer. Counts per minute were corrected for quench (by external standard) and machine efficiency and reported as disintegrations per minute. The washed (see above) radioactive material retained by the filters was considered to have been incorporated into a microbial biomass. A 1- and a 5-ml portion of each filtrate was added to a scintillation vial containing the scintillation cocktail described above and a culture tube, respectively. Each 5-ml portion was then evaporated to dryness at 100°C overnight and then suspended in 5 ml of distilled water with the aid of a 5-mim sonic treatment. A 1-ml portion from each of these tubes was added to a scintillation vial containing scintillation cocktail. The tritium label was then counted as described above, and the counts per minute were converted and reported as disintegrations per minute. Radioactive material lost during the evaporation procedure was considered to represent respired tritiated water. Upon application of this technique, incorporation of tritiated glucose by cells of Simon 456
VOL. 35, 1978
NOTES
Fraser University pool water was noted to be curvilinear as a function of time, whereas respiration was approximately linear (Fig. 1). The amount of substrate incorporated into cell mass was approximately 70 to 80% of that respired. This emphasizes the importance of a respiration correction for use with tritiated substrates. The loss of 3H material from the killed control filtrates after evaporation and subsequent suspension may have been due to leakage and activity of enzymes from killed cells. When several substrate concentrations were used, rates of incorporation and respiration were both linear functions of time (Fig. 2). Rates of respiration were somewhat greater than those for cellular incorporation. When the additional respiration data were incorporated in the approach and equation as previously developed (2), there was a marked reduction in glucose turnover time (Fig. 3). However, the estimated value for natural substrate concentration (S.) was not affected. We used a hot evaporation method, and certain unknown tritiated organic materials (which resulted from the metabolism of the tritiated glucose substrate) may have codistilled with the tritiated water. Thus, if these are present, lyophilization may be a better technique for assaying tritiated water since these compounds (as well as glucose) are less likely to sublime. However, neither of these separation techniques may
457
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60 0 20 INCUBATION TIME (min)
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FIG. 2. The rates of incorporation (A) and respiration (B) of tritiated material by the microflora of the Simon Fraser University reflectingpool at several concentrations of added tritiated glucose: 2 x 1-10 M(A), 5 x 10-10 M(A), 7x 10-10 M(0), and 1l-9M
(@).
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'H - GLUCOSE ADDED (x 10-"° M)
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FIG. 3. Turnover times of tritiated glucose by the microflora of the Simon Fraser University reflecting pool determined from rates of incorporation (0), respiration (0), and total utilization (incorporation plus respiration) (0). Incorporation and respiration rates were derived from the data presented in Fig. 2.
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INCUBATION TIME min)
FIG. 1. Incorporation (-) and respiration (0) of tritiated material by the microflora of the Simon Fraser University reflecting pool Tritiated glucose was added to a concentration of 4.3 x 10-10 M (0.43 ,iCi/100 ml of sample water). Controls were killed with 10% Formalin before addition of radiotracer.
be suitable for very volatile organic substrates, such as methane and acetate. The turnover time data presented here (Fig. 3) do not have the good linear correlation exhibited in previously reported data on the tritium-labeled system (2). We can only assume that these data result from activity within one of the "shift areas" previously postulated (see Dietz et al., 2). Nonetheless, these data demonstrate the need for a respiration correction to the heterotrophic activity technique when using tritium-labeled compounds. We feel that the technique described by Dietz et al. (2) modified with this respiration correction has advantages over the "4C-based system. The tritium-based system allows greater sensitivity, closer approximation of in situ nutrient conditions, and since
458
NOTES
reaction vessels need not be gas tight, a more natural and favorable gas equilibrium. We thank M. D. Morely for his technical assistance and acknowledge the financial assistance of the National Research Council of Canada. LITERATURE CITED 1. Azam, F., and 0. Holm-Hansen. 1973. Use of tritiated
APPL. ENVIRON. MICROBIOL. substrates in the study of heterotrophy in seawater. Mar. Biol. 23:191-196. 2. Dietz, A. S., L. J. Albright, and T. Tuominen. 1977. Alternative model approach for determining microbial heterotrophic activities in aquatic systems. Appl. Environ. Microbiol. 33:817-823. 3. Hobbie, J. E., and C. C. Crawford. 1969. Respiration corrections for bacterial uptake of dissolved organics in natural waters. Limnol. Oceanogr. 14:528-532.
