Sep 13, 1976 - Water samples were taken from Loon Lake, Uni- versity of British ... tail (New England Nuclear Corp.) ..... hold Publishing Corp., New York. 13.
APPLED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1977, Copyright © 1977 American Society for Microbiology
Vol. 33, No. 4 Printed in U.S.A.
p. 817-823
Alternative Model and Approach for Determining Microbial Heterotrophic Activities in Aquatic Systems A. S. DIETZ,' L. J. ALBRIGHT,*
AND
T. TUOMINEN
Department of Biologial Sciences, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6 Received for publication 13 September 1976
Increasing amounts of high-specific-activity tritiated organic compounds were added to samples of several natural waters such that in situ substrate concentrations might be approximated. The uptake responses by the native heterotrophic microflora suggested that (i) heterotrophic populations metabolize the added nutrients, but (ii) these responses are not necessarily a reflection of MichaelisMenten enzyme kinetics. The uptake kinetics appeared to be due to dilution of the naturally occurring metabolite by added radioactive substrate and physiological responses of the microflora to organic enrichment. Utilization rates of organic substrates by the heterotrophic microflora of natural aquatic systems have been studied in recent years mainly by the kinetic model originally described by Parsons and Strickland (5) and developed by Wright and Hobbie (14, 15) and Hobbie and Crawford (4). This technique involves the addition of increasing amounts of 14C-labeled substrate to several subsamples of a water sample, followed by incubation and subsequent analysis for incorporation and respiration of the radioactive substrate by the native microflora. For a more complete description of the heterotrophic potential technique, based upon MichaelisMenten enzyme kinetics equations, see Wright and Hobbie (14, 15) and Hobbie and Crawford (4). The heterotrophic potential technique has been used to obtain heterotrophic potentials (Vmax), turnover times (Tt), and Kt + Sn values for several eutrophic lakes (2, 4, 15) and coastal waters (C. C. Crawford, Ph. D. thesis, University of North Carolina, Raleigh, 1971). However, application of this technique to many organic-carbon moderate waters has often been difficult, if not impossible (3, 8, 9). Several investigators have referred to the need for an alternate explanation and/or approach for determining heterotrophic activities of aquatic microflora (9, 10). Azam and Holm-Hansen (1) noted that very high-specific-activity tritiated organic substrates may be substituted for 14C-labeled metabolites. Thus, added substrates may be used at concentrations that more nearly approximate those of the naturally occurring substrate. Their technique allows approximations of in situ turnover times, but determination of
absolute velocities of utilization requires separate laborious analysis of substrate concentrations, many of which are below thresholds of detection. In this communication we describe a simple, sensitive, and versatile alternative approach to assay of heterotrophic microbial activities of natural waters. This technique allows one to determine in situ substrate concentrations (S.), turnover times (Tn), and velocities of utilization (V5) of any substrate that can be suitably radioactively labeled. The model of this technique is based upon the simple dilution of the naturally occurring substrate by the radioactively labeled substrate.
MATERIALS AND METHODS Water samples were taken from Loon Lake, University of British Columbia Research Forest, from a depth of 1 m, using a clean Van Dorn sampler. This is an oligotrophic lake (organic-carbon poor), with a surface area of 44.7 hectares, a shoreline perimeter of 5.2 km, and volume of 1073.4 hectare meters. The maximum depth of Loon Lake is 57.9 m, and the mean depth is 23.5 m. Water samples were also removed from Georgia Strait and Barkley Sound from a depth of 1 m, using a clean Van Dorn sampler. Georgia Strait is an estuarine environment with an approximate salinity of 23vov, whereas Barkley Sound is an open coastal environment with an approximate salinity of 32%oo. The stations in Georgia Strait were in open water, whereas the station in Barkley Sound was close to beds of macroalgae. Georgia Strait and Barkley Sound are mesotrophic systems (organic-carbon rich). Water samples were taken from the dystrophic (organic-carbon rich) Simon Fraser University (SFU) reflecting pool from a depth of 0.2 m, using a sterile 1-liter Erlenmeyer flask. This is a concrete fish pool with approximate dimensions of 70 m in length by 20 m in width, with I Present address: Scripps Institution of Oceanography, an overall depth of 0.5 m. Water samples were treated within 0.5 h of removal to the laboratory. La Jolla, CA 92037. 817
818
DIETZ, ALBRIGHT, AND TUOMINEN
A portion of sample water was aspirated into a disposal plastic syringe, with no needle attached. This was immediately followed by addition of a selected tritium-labeled organic compound through the orifice of the syringe, using a microliter pipette. A hypodermic needle was then placed on the syringe, a small volume of air was aspirated to facilitate mixing, and the needle was capped with a neoprene stopper. The syringe was inverted three times to mix the sample. Duplicate experimental and control syringes were prepared for each incubation period and each substrate concentration. Controls consisted of samples poisoned with 5% glutaraldehyde before addition of radiotracer. In this study, tritiated glucose (D-[6-3H]glucose, specific activity 10 Ci/ mmol; Amersham/Searle Corp.) was added to yield a final concentration range in the various water samples offrom 0.001 ;LCi/ml (10-10 M glucose) to 10 ,uCi/ ml (10-6 M glucose). Generally, the lower concentration ranges were used. We also used tritiated amino acids (L-[2,3-3H]alanine, specific activity 41 Ci/ mmol; L- [2,5-3H]histidine, specific activity 47 Ci/ mmol; L-[4,5-3H]leucine, specific activity 53 Ci/ mmol; L-[3,4-(n)-3H]proline, specific activity 41 Ci/ mmol; L-[3,5-3H]tyrosine, specific activity 42 Ci/ mmol; all from Amersham/Searle Corp.) in the lower concentration ranges. Water samples were incubated at the ambient water temperature. Incubated samples were filtered through wetted membrane filters (0.22-,um pore size, 47-mm diameter; Millipore Corp.) at a vacuum head of 25 cm of Hg and rinsed with 10 ml of 00C, prefiltered sample water. The filters were then placed in scintillation vials containing 15 ml of Aquasol scintillation cocktail (New England Nuclear Corp.) with 10% ethyl acetate to solubilize the filters. Alternatively, scintillation vials contained 15 ml of Beckman FilterSolv scintillation cocktail. After the filters had clarified and partially dissolved, the samples were counted in a Beckman LS-250 liquid scintillation spectrometer. Counts per minute were corrected for quench (by the external standard method), machine efficiency, and half-life decay, and are reported as disintegrations per minute. Alternatively, incubated samples were filtered through glass fiber filters (Whatman GFC, 2.4-cm diameter) at a vacuum head of 10 cm of Hg. These filters were then placed in scintillation vials containing 1 ml of Protosol (New England Nuclear Corp.) and allowed to digest for at least 1 h at room temperature to remove the cellular material from the filter. After digestion, 10 ml of PPO/POPOP cocktail [2,5-diphenyloxazole (PPO), 4 g; 1,4-bis-(5-phenyloxazole)-benzene (POPOP), 0.05 g; in 1 liter of toluene] was added to each vial, and the disintegrations per minute in each vial, were determined as described above. The tritiated glucose was diluted in carbon-free water prepared by the method of Strickland and Parsons (7) and filtered through sterile membrane filters (0.22-,um pore size, 25-mm diameter; Millipore Corp.) before use.
THEORY Once the uptake (in disintegrations per minute) was known, the turnover time for each added con-
APPL. ENVIRON. MICROBIOL. centration was calculated by the equation T = Adtl Ud, were T is the turnover time, Ad is the disintegrations per minute added, t is the incubation time in hours, and Ud is the net uptake in disintegrations per minute. These turnover times are those for the amount of substrate added (A) as well as the substrate naturally present (S.), or (A + S.). If velocity of utilization is constant, since background amounts of substrate were added, then the turnover times should increase in proportion to the total amount of substrate, (A + S,). The relationship of turnover time as a function of increasing added substrate is linear only if velocity of utilization is constant. Any departure from constancy will introduce curvature or erratic points into the relationship. A plot of turnover time as a function of the amount of added substrate results in a straight line (Table 1, Fig. 1) similar to a Lineweaver-Burk plot. This plot intersects the ordinate at the in situ turnover time, Tn, and the abscissa on the negative side at the in situ substrate concentration, Sn. Furthermore, assuming constant velocity of utilization, a plot of uptake of radioactive counts as a function of the amount of added labeled substrate results in a quadratic hyperbola (Fig. 1), similar to the classic MichaelisMenten enzyme saturation curve. Data were analyzed by computer program to determine the line of best fit. To check the accuracy of estimation of substrate concentration by bioassay, Escherichia coli was grown at room temperature to stationary phase in minimal broth (NaCl, 5 g; MgSO4, 2 mg; K2HPO4, 1 mg; NH3PO4, 1 mg; in 1 liter of distilled water) with a growth-limiting concentration of glucose (10-5 M) as the sole carbon source. The culture was incubated for a further 24 h after it had become turbid to insure glucose exhaustion. Cells from this suspension were diluted to an approximate density of 1.6 x 104 colony-forming units per ml in fresh minimal broth with 2 x 10-8 M glucose as the sole carbon source. A 1:1,000 dilution was made directly from the stationary-phase culture into the fresh broth. This culture was incubated at room temperature, in the dark, for 2 h before analysis to allow cells to recover from inhibitory effects of resting in the stationary-phase culture. A further check on the accuracy of estimation of Sn involved addition of subtle amounts of unlabeled glucose to SFU pool water. Three subsamples were taken from a sample of SFU pool water. The first subsample was unaltered, and Sn was determined as described above. The second subsample was amended by adding nonradioactive 2 x 10-10 M glucose, and Sn was determined after a 20-min preincubation. The third subsample was amended by addition of nonradioactive 5 x 10-10 M glucose, and Sn was determined after a 30-min preincubation.
RESULTS Analysis for S,, of the E. coli suspension to which 2 x 10-8 M glucose was added indicated an Sn value of 1.9 x 10-8 M glucose, a T,, value of 28.3 h, and a Vn value of 6.7 x 10-10 mol/liter per h (Table 2). The data are easily interpreted,
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TABLE 1. Theoretical plots illustrating the relationship of net uptake and turnover time to increasing amounts of added radioactive substratea A units
units
Added dpm
Uptake in
Turnover
units
dpm
time (h)
2 4 6 8 10 15 20 25 30 40 50 60 70 80 90 100
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
12 14 16 18 20 25 30 35 40 50 60 70 80 90 100 110
20,000 40,000 60,000 80,000 100,000 150,000 200,000 250,000 300,000 400,000 500,000 600,000 700,000 800,000 900,000 1,000,000
1,667 2,857 3,750 4,444 5,000 6,000 6,666 7,143 7,500 8,000 8,333 8,571 8,750 8,889 9,000
12 14 16 18 20 25 30 35 40 50 60 70 80 90 100 110
S.
Total
9,090
Assume that the natural substrate concentration (S.) is 10 units, that additions of radioactively labeled substrate (A) contain 10,000 dpm per unit, that the heterotrophic microflora assimilates 1 unit of substrate per h, regardless of the amount of added radioactive substrate, and that the incubation period is 1 h. a
UNITS OF ADDED RADIOACTIVE SUBSTRATE
FIG. 1. Theoretical plot of net uptake (0) and turnover time (0) versus units of added radioactively labeled substrate. as can be seen in Fig. 2. was 95% of the known
The estimated S. value glucose, but when the amount of glucose that would have been used during the preincubation period was added to the indicated S,, value, the corrected S,, value was 102% of the known added glucose. When glucose was added to alter the S,, value of SFU pool water, the indicated increases in
the Sn values were only 85% of the amount of known increases (Table 2). The data were easily interpreted (Fig. 3). However, when the amount of glucose used during the preincubation periods was added to the indicated S,,, the corrected increases were 100 and 98% of the smaller and larger additions, respectively. The in situ turnover times (T5) did not increase in direct proportion to the increases in S, and the increases in S. appear to have stimulated increases in the rates of utilization (Table 2). The course of uptake over time was followed by using several concentrations of glucose in a water sample from the SFU pool. Use of a single incubation time may lead to erroneous results, since some points in Fig. 4 are clearly off the time course curve. Plots of uptake versus amount added at various substrate concentration should ideally be made after the general rate of uptake, i.e., the slope of a line, is determined. The time course of uptake over extended incubation periods was also followed in several aquatic systems (Fig. 5). It is evident that the uptake response is not always linear over longer periods of time and that shorter incubation periods are advisable. The dip in the curve for Loon Lake is due to an accidental reduction in incubation temperature. Several experiments in March 1976, using samples taken from Loon Lake, resulted in no uptake of [3H]glucose or several 3H-labeled amino acids (L-alanine, L-histidine, xleucine, L-proline, and L-tyrosine). Uptake was tested at
820
DIETZ, ALBRIGHT, AND TUOMINEN
APPL. ENVIRON. MICROBIOL.
TABLE 2. S,, Tn, and Vn analyses of two experimental systems Sample
E. coli culture SFU pool SFU pool SFU pool
added(M) added (M) S. (M)
Tn (h)
Vn~~~~~~~~~~~~~~~~per (mol/liter h)
2 x 10-8
28.3 6.6 8.4 9.4
6.7 x 9.0 x 9.1 x 10.8 x
1.90 x 10-8 5.95 x 10-"0 7.65 x 10-10 10.15 x 10-10
None 2 x 10-10 5 x 10-"0
10-10 10-11 10-11 10-11
10
uJ z
20 18 16 14 12 0
FIG. 2. Turnover time of glucose by an Escherichia coli culture versus added [3H]glucose.
10
20 30 40 INCUBATION TIME (min)
50
60
FIG. 4. Net uptake of glucose by the native micro-
flora of the SFU pool water versus incubation time. The symbols 0, 0, A, and O refer to added glucose concentrations of 1.2 x 10-9, 2.4 x 10-9, 6.0 x 10-9, and 1.2 x 10-8 M, respectively.
10
8
6
4
2
0 2 4 6 3H-GLUCOSE ADDED (X 10-'° M)
FIG. 3. Turnover time of glucose by the native
microflora of the SFU pool
water
versus
added
[3H]glucose. The symbols 0, 0, and O refer to unamended and 2 10-10 and 5 x 10-1° M glucosetreated water, respectively. x
10-min intervals over the period of 1 h, at several substrate concentrations. This failure to metabolize these substrates indicates that the heterotrophic processes of the native microflora were inactive. Uptake of tritiated glucose by the heterotrophic microflora of Loon Lake was initiated in mid-July 1975 and had ceased by mid-September 1975. In March 1976 we unsuccessfully attempted to induce an uptake response by amending a Loon Lake water sample with 5 x 10-10 M glucose. Figure 6 illustrates results characteristic of this assay. The curve of uptake versus glucose added (Fig. 6) was hyperbolic; the nature of the curve indicates that addition of radiotracer was at the lower end of the theoretical curve presented in Fig. 1. The relationship of turnover time to added glucose was linear (Fig. 6). The results of an experiment over a wide
VOL. 33, 1977
Q-
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HETEROTROPHIC ACTIVITY
20 80
16
16-
y
12 0
CL
0460 ~~~~~~~~a: LU
C 0 12-
LLJ
z
x
r
~~~~~~~~0
~~~~~~~z
10 60 0 18-4
4
3H-GLUCOSE ADDED (X 1 0-9 M) C
0
60
120 180 INCUBATION TIME (min)
240
FIG. 5. Net uptake of glucose versus time by the native microflora of (a) Loon Lake (5.6 x 10-10 M), (O) SFU pool (10-10 M), (A) Barkley Sound (10-9 M), and (O) Georgia Strait (10-9 M).
range of added [3H]glucose are illustrated in Fig. 7. Net uptake as a function of added substrate was a hyperbolic function, and sorption by killed controls increased in direct proportion to the amount of added [3H]glucose. However, the relationship of turnover time to amount of added substrate was not the linear function predicted in Fig. 1. An initial linear relationship at low substrate concentrations was interrupted and followed by a second linear relationship of lower slope. This may indicate that at higher substrate concentration there is (i) an increase in the numbers of microbes metabolizing the substrate or (ii) an increase in the rate of utilization by the microbes. The change in slope of plot of turnover time versus glucose added resulted in a change in the indicated S,, and Tn values. The microflora of Georgia Strait responded to addition of 3H-labeled amino acids in the predicted manner (Fig. 8). Alanine and leucine were taken up in water removed from 1- and 100-m depths, whereas tyrosine was metabolized only in water removed from 100 m (Table 3). Proline was not metabolized. The concentrations and rates of utilization of alanine and leucine were greater at 1 m than at 100 m. Alanine was present in greater concentrations than that of leucine.
FIG. 6. Net uptake (a) and turnover time (O) verby the native microflora of the SFU pool.
sus added [3H]glucose
DISCUSSION The precision with which this technique can determine known S., values as well as known increases in S,, values (Table 2, Fig. 2) is excellent. However, one major objection of this approach is that addition of substrate to the water sample causes an increase in the rate of metabolite utilization and is not corrected for by the basic equation. This increase in utilization rate after substrate addition may be observed in the data of Table 2 and has been noted by Wright (13). However, in the approach we describe in this communication, the increase in uptake of metabolite due to nutrient addition is small in comparison to the total incorporation rate of the 3H-labeled material. Since this nutrient-induced acceleration of activity does not appear to significantly alter the heterotrophic activity (i) over short time periods (
