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Comparison of leucine uptake methods and a thymidine incorporation method for measuring bacterial activity in sediment
ELSEVIER
Journal of Microbiological Methods 24 (1995) 125-134
Journal ofMicrobiological Methods
Comparison of leucine uptake methods and a thymidine incorporation method for measuring bacterial activity in sediment Liisa Tuominen* Department of Limnology (E-building),
and Environmental Protecting Section of Limnology, P.O. Box 27 FIN-ooo14, Universiry of Helsinki, Helsinki, Finland
Received 15 February 1995; accepted 1 June 1995
Abstract Three [14C]leucine uptake methods and a [‘Hlthymidine method for measuring bacterial activity in sediment were compared. For leucine uptake, a combustion method, in which prior to combustion sediment was washed with ethanol, yielded the most consistent results. The TCA extraction methods with a filtration step yielded very
variable and much lower results than the combustion method. A high concentration of leucine was needed to reach the saturation level (13.7 PM), but that was not found to enhance bacterial activity. No clear saturation level for thymidine incorporation was achieved for two of the four lake sediments tested. Therefore, and because of other weaknesses of the method, the thymidine incorporation underestimated the bacterial carbon production in sediment. Keywords:
Bacterial activity; Leucine uptake; Sediment;
1. Introduction Bacterial activity in aquatic systems has traditionally been estimated by measuring the in-
corporation of [3H]thymidine into bacterial DNA. Tobin and Anthony [l] were the first to invent a method to measure thymidine incorporation in sediment samples. The soluble material that was not hydrolyzed in dilute NaOH, but was hydrolyzed in hot dilute acid, was considered to be DNA. Later Pollard [2] compared the ex-
* Corresponding author. [email protected].
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Thymidine
incorporation
traction method of Tobin and Anthony with a dialysis method, and found that the dialysis method gave higher recoveries of DNA and that the non-diffusible material after dialysis did not contain macromolecules other than DNA. In general, thymidine incorporation into DNA is considered to be specific to bacteria, since other organisms have not been found to contain thymidine kinase, which is essential to the incorporation of thymidine into DNA [3]. However, the thymidine method has also disadvantages, especially when used in sediment samples. Thymidine is very easily bound to both organic and inorganic particles in the sediment [4]. Also, the assumption that thymidine uptake
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follows Michaelis-Menten kinetics has been disputed by Logan and Fleury [5]. They showed that thymidine uptake and incorporation could be fitted by a diffusion-based transport equation. Due to the continuous phosphorylation of thymidine in the cell, the concentration gradient between the cell and the environment is maintained, and therefore, no clear saturation level can be achieved. Thus, the result will rather be a function of the concentration of thymidine used than that of bacterial activity in the sample. It has also been shown that especially some anaerobic bacteria are not able to incorporate thymidine [6-81. Leucine incorporation, which measures bacterial protein synthesis, gives a more direct measurement of bacterial biomass change (or carbon production) than that given by thymidine incorporation in which average carbon content of bacterial cells should be known. However, there are only a few studies in which [‘“Cl- or [3H]leucine incorporation methods have been applied to sediment samples: a dialysis technique has been used by Tibbles et al. [9], extraction and precipitation by Meyer-Reil and Charfreitag [lo], and a combustion technique by Meyer-Reil (111 and Tuominen and Kairesalo [12]. However, no comparisons and recommendations of the different methods have been given earlier. As a small amino acid (molecular weight 131), leucine is easily assimilated by bacteria, but fungi and protozoa may also contribute significantly to the uptake of leucine in sediment. However, bacteria are obviously of principal importance to the leucine uptake in short term incubations ( < 30 min), since the uptake of small organic molecules is much faster by bacteria than other organisms. For instance, in bacterioplankton studies no leucine uptake by phytoplankton or cyanobacteria has been observed [13]. In this study, the applicability of a simple combustion method for sediment samples was evaluated by comparin 8 different [ i4C]leucine uptake methods and a [ Hlthymidine incorporation method.
Methods 24 (199.5) 125-134
2. Materials and Methods 2.1. Sediments Sediment samples from four different lakes were used: Lake Vesijarvi (The Enonselka Basin), Lake Tuusulanjarvi, Lake Paajarvi and Lake Hoytiiinen. Lake Vesijirvi (61”05’N, 25”30’E; see refs. [14,15] for a general description of the lake) and Lake Tuusulanjarvi (60”25’N, 25”03’E; [16,17]) are eutrophic lakes in Lake Paajijirvi (61”04’N, southern Finland, 25”08’E; [18,19]) is a polyhumic lake in southern Finland, and Lake Hoytiainen (62”45’N, 29”40’E; [20]) is an oligotrophic lake in eastern Finland. The topmost O-2 cm sediment layers were collected from about 10 m depth, except from Lake Hijytiiinen, from which a layer of O-4 cm was collected from 30 m depth. After sampling, many surface sediment samples from the same area were homogenized and stored in 0.5 1 polyethylene containers at + 2°C (Lake Hoytiiinen at + 4°C). The sediments differed in their C and N contents (Table 1) as well as visually: Lake Vesijtirvi and Lake Tuusulanjirvi sediments were grey in colour, Lake Plajlrvi sediment was reddish brown and Lake Hoytiainen sediment was chocolate brown. Lake Hoytilinen sediment had the coursest and Lake Tuusulanjarvi sediment the finest texture. All the results of bacterial activity in this study are expressed as mol leucine or thymidine incorporated h-’ 1-l of fresh sediment, since the expression per g of sediment dry mass distorts the
Table 1 The water contents (W, % of fresh mass)‘, losses on ignition (LOI, % of dry mass)“, C and N contents (% of dry mass) and C/N atomic ratios in the lake sediments Lake
W
LO1
c
N
C/N
Vesijtirvi TuusulanjBrvi pgSj..i
90 92 95 84
12 13 15 10
5.2 4.4 5.1 3.1
0.7 0.6 0.5 0.3
8.7 8.6 11.9 12.1
HijytiLinen a Dried at 60°C. ’ Ignited at 550°C.
L. Tuominen I Journal of Microbiological Methods 24 (1995) 125-134
results of different sediments with different mass per volume ratios [21] (Table 1).
dry
2.2. Leucine incorporation methods Three different methods to measure leucine incorporation in sediment samples were compared. The methodological comparisons were made with Lake Vesijirvi sediment. The incubations were done in 13 ml polypropylene tubes, in which 0.1 ml sediment was mixed with 0.9 ml filtered, deionized water (three replicates). The results of control samples (three replicates), which were killed by adding 0.9 ml of 4% formaldehyde to the tubes instead of water, were subtracted from the normal samples. Daily before use, the [14C]leucine (11.5 GBq mmol-‘, Amersham) was diluted 20 times with fresh unlabelled leucine solution of the same leucine concentration (1.61 x 10e4 mol 1-l). The leucine solution was added to the tubes and the incubation was performed at 20°C in darkness for an optimal time (1.5 min; see below). The procedure was continued as follows:
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fluorescence microscope at a magnification of 1250 x . The reliability of the combustion was tested by combusting internal standard capsules ( Wallac) . Method 2
Hot TCA method: This method was performed principally as used for water samples by Chin-Leo and Kirchman [23], and by Simon and Azam [24]. The incubation was terminated by inserting the tubes into ice and by adding 0.5 ml 15% ice-cold TCA (trichloroacetic acid; final concentration 5%). The samples were then heated for 15 min at 90°C cooled, filtered onto Sartorius 0.2 pm cellulose nitrate filters, and rinsed twice with 4 ml of ice-cold 5% TCA and once with 4 ml of 80% ethanol. The filters were left to dissolve in a mixture of 0.2 ml ethylenglycol-monomethylether and 10 ml Lumagel (Lumac LSC B.v.) for 24 h, and measured in a liquid scintillation counter. Different amounts of Lumagel were tested in order to achieve a liquid or a gel. Method 3.
Method 1
Combustion: This method was performed principally according to Tuominen and Kairesalo [12]. Briefly, the incubation was terminated by adding 4 ml of 80% ethanol containing 100 mg 1-l unlabelled leucine and centrifugating for 5 min at 5000 g. The supernatant was then discarded, and the procedure was repeated twice more. The pellet was dried at 60°C and combusted at 900°C (Junitek Oxidizer). The evolved 14C02 was adsorbed into Lumasorb II, and the scintillation liquid used was Carboluma (both Lumac LSC B.v.). The sufficiency of the three ethanol washings to remove the unincorporated leucine was tested by counting subsamples of the supernatants in a liquid scintillation counter after each washing step; in this test a fourth washing step was added. To ensure that bacteria were not discarded with the supernatants, subsamples of the supernatants were fixed with 4% formaldehyde, stored in + 4°C and later stained with acriflavine [22] and counted with a Nikon epi-
Cold TCA method (principally water samples by Kirchman [25]): tion the samples were poured into funnels, filtered, and rinsed twice ice-cold 5% TCA and once with ethanol. The filters were dissolved as in method 2.
as used for After incubacold filtration with 4 ml of 4 ml of 80% and counted
2.3. Thymidine incorporation The incubation was performed as in the leucine methods, except that the substrate used was [methyl-3H]thymidine (185 GBq mmol-‘, Amersham), which was diluted 10 times with filtered, deionized water. The thymidine incorporated into DNA was extracted with the dialysis method of Moriarty [3] and Moriarty and Pollard [26]. The incubation was terminated by addition of 4 ml of 80% ethanol which included 100 mg 1-l unlabelled thymidine . The samples were then centrifuged for 5 min (5000 g), and the ethanol washing step was repeated. The pellets
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were suspended in 2 ml of 0.3 M NaOH and heated at 90°C for 40 min. After centrifugation the supernatants were poured into the Spectra/ Por Membrane nr. 1 dialysis tube (molecular weight cut-off 6000-8000; Spectrum), and dialysed overnight (minimum 16 h; [2]). After dialysis a further step to separate the DNA was performed by heating the samples in 5% TCA at 90°C for 40 min, and after centrifugation a subsample from the supematant was measured with Lumagel (Lumac LSC B.v.) in a liquid scintillation counter.
The saturation levels were tested by incubating the samples with increasing leucine (from 4.8 PM to 21.6 PM; combustion method) or thymidine (from 250 nM to 3333 nM) concentrations. The concentration where the incorporation turned to be horizontal was taken as the saturation level. The optimal incubation time was determined as the shortest time when incorporation was high enough to get a clear difference between the normal and the control samples, but when it was still linear. This was analyzed by incubating the samples for 10, 15 and 20 min.
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The possibility that the leucine additions enhanced bacterial activity in the samples was tested. This was done by measuring the bacterial activity of Lake Vesijtirvi sediment with the [3H]thymidine incorporation in samples in which unlabelled leucine (from 0 to 17.8 PM) was added together with [3H]thymidine.
3. Results 3.1. Saturation levels and optimal incubation times
2.4. Saturation levels and optimal incubation times
2.5
Methods 24 (1995)
The leucine saturation level was 13.7 PM for all the sediments (Fig. 1). By contrast, no clear saturation level was found for thymidine incorporation in Lake Vesijlrvi and Lake Hbytilinen sediments, although a slight level was perceived between 952 and 1818 nM thymidine additions (Fig. 2). The thymidine saturation level for Lake Tuusulanjarvi was 1818 nM and for Lake Paajarvi 952 nM (Fig. 2). The optimal incubation time was 15 min (at 20°C). After that, the incorporation of both leucine and thymidine started to be non-linear and the variation between replicates tended to increase as well.
5-
1
Lake Vesijhrvi
+
Lake TuusulanjBrvi
2
P
t
Yz
Lake Hijytiainen
.
07
i 0
6
10
15
20
Leucine added, pM
25
5
10
16
20
25
Leucine added, pM
Fig. 1. The saturation curves of leucine uptake (combustion method). Points represent minimum and the maximum values of three replicates. Curves fitted by hand.
the average values and crosses the
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Lake VaSijirVi
Methods 24 (1995) 125-134
129
O.‘T
+
I
Lake Tuusulanjiirvi +
0.03 -
I
+
Lake Haytllinen Laka
Piiiijiirvi +
i boo
0
*OS
,500
lhyrnidine
2000
0
2500 3000 3600
600
added, nM
1000
1300 2000 2600 3000
I 3sao
Thymidineadded, nM
Fig. 2. The saturation curves of thymidine incorporation. Points represent the average values and crosses the minimum and the maximum values of three replicates. Curves fitted by hand.
3.2. Leucine incorporation methods Both the hot TCA method and the cold TCA method yielded much lower results than the combustion method (Fig. 3). When using these methods, the variation between replicates was much greater than when using the combustion method, and also the results measured with the hot TCA method on consecutive days differed a lot. With the cold TCA method an unexpected descent was observed when 17.8 PM leucine was added. This reflects the unreliability of the
2.5 L
method. When comparing the results of gel and liquid scintillation samples to samples in which the filters were combusted, samples with partitles bound to a gel yielded on average 81%) and samples with particles settled to the bottom on average 37% of the combusted samples. The three centrifugations in the combustion method removed the unincorporated leucine effectively. The disintegrations per minute (DPM) in the third supematant were only 0.56% of those in the first supernatant (Table 2). An additional fourth centrifugation step lowered
T
+
Oxidation -
Min
-
Max
+
Hot TCA +
+
+ +
Min Max Cold TCA
x
Min
x
Max
15 leucine added, VM
Fig. 3. Leucine uptake in Lake Vesijiirvi sediments measured by combustion, hot TCA and cold TCA methods (see text) as a function of added leucine concentration (averages + minimum and maximum values).
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Table 2 The radioactivity in the supernatants of the combustion method after each centrifugation step compared to the first supernatant Centrifugation step
% Of the radioactivity of the 1st supematant
1st 2nd 3rd 4th
100 5.04 0.56 0.16
these figures a little, but because the prekilled controls are always subtracted from the normal samples, there is no need for a lower percentage. Sediment bacteria were very efficiently settled by the centrifugation at 5000 g, since no bacteria could be found in any of the stained supernatants. The combustion process is also very reliable (at least with the Junitek Oxidizer), and on average 97% of the combusted activity can be found in the scintillation samples. The leucine addition (from 0 to 17.8 PM) did not enhance the bacterial activity as measured by thymidine incorporation. No significant differences were found between any samples (ANOVA, p-value 0.27).
4. Discussion 4.1. Leucine incorporation methods The problem when measuring sediment samples with a method in which filters are dissolved in a scintillation cocktail is, that although the filter itself dissolves, the sediment particles remain. The particles can then be bound to scintillation gel or settle to the bottom of the bottle. In the first case, the particles cause quenching, and in the second case, the counting geometry is biased. In order to eliminate these problems, combustion is currently the only technically correct method to handle sediment samples for liquid scintillation counting: it results in a homogenous sample with no particles. Using filters to terminate incubation can also possess other problems. If the bacteria in the
Methods 24 (1995) 125-134
sample are very small, part of them can be lost through the filter. The use of a filter with a pore size smaller than 0.2 pm can be unusable for sediment samples because of slow flow rate. Kairesalo and Saukkonen [27] have also noted, when measuring thymidine incorporation in water samples, that the edge of the filter (situated under the filtration funnel) could absorb part of the sample which was then not rinsed properly. This resulted in highly variable replicates. They solved the problem by rinsing the filters before use in unlabelled thymidine solution to block the absorption of labelled thymidine to the filter. Leucine has been found to label proteins almost exclusively (90%) [13]. Therefore, there is no need to extract proteins from the cells. The ethanol rinses in the combustion method, however, can remove leucine adsorbed to lipids [28,29] especially from the cell membrane. The simple combustion method with three ethanol rinses was the most reliable method for measuring leucine uptake in sediment samples. Very high leucine additions were needed to reach the saturation level in sediment samples. Although it was shown that 17.8 PM leucine did not enhance sediment bacterial activity, the high leucine additions may enable the uptake of leucine by other organisms, especially fungi. Fallon and Newell [30] have observed that axenic fungal cultures were capable of incorporating leucine . Also, the prymnesiophyte Emiliania huxleyi was found to grow with leucine as source of N, but in mixed cultures with bacteria, the bacteria outcompeted the algae in amino acid uptake [31]. Glaser [32] observed that ciliated protozoa Tetrahymena pyriformis was able to take up amino acids (glycine and histidine), but he speculated that ciliates may contribute significantly to amino acid dynamics only in benthic environments with high levels of amino acids. Kirchman et al. [13] observed no evidence of leucine uptake by phytoplankton or cyanobacteria. It was most likely that bacteria were of principal importance to the leucine uptake in the current study, since the short incubation time (15 min) benefits the bacterial uptake of dissolved substances compared to other organisms.
L. Tuominen I Journal of Microbiological Methods 24 (1995) 125-134
We have earlier observed that the gradual emergence of bacterial activity after fixing with dilute formaldehyde [33,34] was much faster when measured with leucine uptake than with thymidine incorporation. After a 6-day incubation thymidine incorporation had risen to about l/3 of that in the live control samples, while leucine uptake already was higher than in the control samples [33]. This suggests that bacterial protein production and DNA replication are not balanced in that kind of situation. It could also be possible, that organisms other than bacteria (e.g. fungi) or a specific bacterial strain produced the steep leucine uptake curve. 4.2. The use of leucine and thymidine incorporation methods in sediment samples Leucine incorporation into proteins is a straight forward method for measuring bacterial biomass production, because ca. 50% of bacterial dry mass consists of proteins [35]. The molar proportion of leucine compared to other amino acids has been found to be rather constant in natural bacterioplankton assemblages [24]. Simon and Azam [24] noted that smaller bacteria were more ‘concentrated’ and contained more protein and carbon per volume (w/v) than larger bacteria. The ratio of cellular protein and carbon remained, however, constant throughout the size classes from 0.026 to 0.400 pm3. Therefore bacterial carbon production (BCP) can be calculated as [24]: BCP(g) = (mol 131.2 x 0.86
leucine,,,)
X (100/7.3) X
(1)
where mol leucineinc = moles of leucine incorporated; 7.3 = mol% of leucine in protein; 131.2 = formula weight of leucine; 0.86 = a conversion factor to convert a gram of protein produced to a gram of carbon. Thymidine incorporation is principally very well known [3, 36,371. The main problems with this method are the assumptions needed to calculate bacterial carbon production, and the
131
question if all bacterial strains incorporate exogenous thymidine. At least some chemolithotrophic bacteria, sulphate reducers, and methane bacteria have been found to be unable to utilize exogenous thymidine [6-81. This problem seems to be more pronounced in anaerobic than in aerobic environments. When calculating the results based on thymidine incorporation, several assumptions have to be made [38]: (1) the proportion of the four bases that thymidine constitutes; (2) the amount of DNA in each cell; and if the results are converted to bacterial carbon production (3) the amount of carbon per bacterial cell. For water samples the conversion factor to convert moles of thymidine incorporated to number of bacterial cells produced can also be determined empirically. This is, however, not possible in sediment samples, because of the problems with recording changes in numbers of bacteria by microscopy. If equation 1 is used to calculate the bacterial carbon production based on the leucine uptake at the saturation levels of the lakes (the moles of leucine taken up being first corrected for the 10% non-protein uptake of leucine; [13]), and if the thymidine results are calculated with a conversion factor of 5 x 10” cells produced per mole thymidine incorporated [3], and the thymidine results are further corrected for the recovery of DNA in the dialysis method (71%, [2]), and if an average bacterial size of 0.026 pm3 (unpublished results for Lake Vesijarvi) and C/cell of 10.4 x lo-l5 g [24] are supposed, the leucine method yields much higher bacterial carbon production values than the thymidine method (Table 3). The values based on the thymidine results are, however, underestimates, since no clear saturation levels for thymidine were achieved for Lake Vesijarvi and Lake Hdytiainen, and the isotope dilution may therefore be substantial. Also, the activity of bacteria in the anaerobic microsites in the sediments was not included in the thymidine method [6-81. The differences in the results could, however, partly be real. This can be due to unbalanced DNA and protein production or uptake of leucine by organisms other than bacteria. Therefore, it can be concluded that the level of the bacterial
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Table 3 Bacterial carbon production based on the leucine uptake (BCP,,,) and on the thymidine incorporation sediments Lake Vesijarvi Tuusulanjarvi Piajlrvi Hiiytilinen
13.7 13.7 13.7 13.7
Cr,, and C,,, = the concentrations ’ Unclear saturation level.
952” 1818 952 952”
(BCP,,,)
in four lake
BCP,,, (mg C h-’ 1-l)
BCP,,, (mg C h-’ 1-l)
BCP,,, /BCP,,,
2.8 5.7 1.5 1.9
0.16 0.57 0.09 0.12
17.0 10.0 16.1 15.3
of the added leucine and thymidine, respectively (cf. Figs. 1 and 2).
carbon production can be measured with both methods, but the value based on the thymidine incorporation is most probably an underestimate. The correlation of thymidine incorporation and leucine uptake was linear, except for the results from Lake Tuusulanjarvi (Fig. 4). The thymidine incorporation was higher at the same
leucine uptake level in Lake Tuusulanjarvi sedito Lake Vesijarvi. Lake ment compared Tuusulanjirvi sediment differed from the other sediments by being very fine in structure with very much colloidal clay. Also, bacterial metabolism might have been different. However, a more detailed explanation remains unclear and calls for further investigations.
0.16
0.12 E ‘ij 2
0.1
0 ,P 00 .s
0.08
e ._ 9 E, 0.06 i-” 0.04
0 0
1
2
3
4
5
6
Leucine uptake
Fig. 4. The correlation between thymidine incorporation and leucine uptake (both expressed as pmol h-’ I-’ of fresh sediment) in four lake sediments. The results from Lake Tuusulanjarvi are not included in the equation.
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I Journal of Microbiological
Acknowledgements I am grateful to Antti Uusi-Rauva and Kaj Hurme (from the isotope section of The Faculty of Agriculture and Forestry, University of Helsinki) for help and advice in the laboratory, to Timo Kairesalo for comments on the manuscript, and to Tiina Ristola and Heli Vahtera for sending me the sediment samples from Lake Hoytiiinen and Lake Tuusulanjarvi.
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[29] Hollibaugh, J.T. and Wong, P.S. (1992) Ethanol-extractable substrate pools and the incorporation of thymidine, L-leucine, and other substrates by bacterioplankton. Can. J. Microbial. 38, 605-613. [30] Fallon, R.D. and Newell, S.Y. (1986) Thymidine incorporation by the microbial community of standing dead Spartina alterniflora. Appl. Environ. Microbial. 52, 1206-1208. [31] Ietswaart, T., Schneider, P.J. and Prins R.A. (1994) Utilization of organic nitrogen sources by two phytoplankton species and a bacterial isolate in pure and mixed cultures. Appl. Environ. Microbial. 60, 15541560. [32] Glaser, D. (1988) Simultaneous consumption of bacteria and dissolved organic matter by Tetrahymenn pyriformis. Microb. Ecol. 15, 189-201. [33] Kairesalo, T., Tuominen, L., Hartikainen, H. and Rankinen, K. (1994) The role of bacteria in the nutrient exchange between sediment and water in a flow-through system. Microb. Ecol. 5, 129-144.
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[34] Tuominen, L., Kairesalo, T. and Hartikainen, H. (1994) Comparison of methods for inhibiting bacterial activity in sediment. Appl. Environ. Microbial. 60, 3454-3457. [35] Fenchel, T. and Blackbum T.H. (1979) Bacteria and mineral cycling. Academic Press, London. ISBN O-12252750-X. [36] Moriarty, D.J.W. (1986) Measurement of bacterial growth rates in aquatic systems from rates of nucleic acid synthesis. Adv. Microb. Ecol. 9, 245-292. [37] Robarts, R.D. and Zohary, T. (1993) Fact or fictionbacterial growth rates and production as determined by [mefhyl-3H]-thymidine? Adv. Microb. Ecol. 13, 371425. 1381 Moriarty, D.J.W. (1988) Accurate conversion factors for calculating bacterial growth rates from thymidine incorporation into DNA: elusive or illusive? Arch. Hydrobiol. Beih. Ergebn. Limnol. 31, 211-217.
Journal of Microbiological Methods 24 (1995) 125-134
Journal ofMicrobiological Methods
Comparison of leucine uptake methods and a thymidine incorporation method for measuring bacterial activity in sediment Liisa Tuominen* Department of Limnology (E-building),
and Environmental Protecting Section of Limnology, P.O. Box 27 FIN-ooo14, Universiry of Helsinki, Helsinki, Finland
Received 15 February 1995; accepted 1 June 1995
Abstract Three [14C]leucine uptake methods and a [‘Hlthymidine method for measuring bacterial activity in sediment were compared. For leucine uptake, a combustion method, in which prior to combustion sediment was washed with ethanol, yielded the most consistent results. The TCA extraction methods with a filtration step yielded very
variable and much lower results than the combustion method. A high concentration of leucine was needed to reach the saturation level (13.7 PM), but that was not found to enhance bacterial activity. No clear saturation level for thymidine incorporation was achieved for two of the four lake sediments tested. Therefore, and because of other weaknesses of the method, the thymidine incorporation underestimated the bacterial carbon production in sediment. Keywords:
Bacterial activity; Leucine uptake; Sediment;
1. Introduction Bacterial activity in aquatic systems has traditionally been estimated by measuring the in-
corporation of [3H]thymidine into bacterial DNA. Tobin and Anthony [l] were the first to invent a method to measure thymidine incorporation in sediment samples. The soluble material that was not hydrolyzed in dilute NaOH, but was hydrolyzed in hot dilute acid, was considered to be DNA. Later Pollard [2] compared the ex-
* Corresponding author. [email protected].
Fax:
+358-O-7085257; e-mail:
Thymidine
incorporation
traction method of Tobin and Anthony with a dialysis method, and found that the dialysis method gave higher recoveries of DNA and that the non-diffusible material after dialysis did not contain macromolecules other than DNA. In general, thymidine incorporation into DNA is considered to be specific to bacteria, since other organisms have not been found to contain thymidine kinase, which is essential to the incorporation of thymidine into DNA [3]. However, the thymidine method has also disadvantages, especially when used in sediment samples. Thymidine is very easily bound to both organic and inorganic particles in the sediment [4]. Also, the assumption that thymidine uptake
0167-7012/95/$09.50 @ 1995 Elsevier Science B.V. All rights reserved SSDI 0167-7012(95)00062-3
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follows Michaelis-Menten kinetics has been disputed by Logan and Fleury [5]. They showed that thymidine uptake and incorporation could be fitted by a diffusion-based transport equation. Due to the continuous phosphorylation of thymidine in the cell, the concentration gradient between the cell and the environment is maintained, and therefore, no clear saturation level can be achieved. Thus, the result will rather be a function of the concentration of thymidine used than that of bacterial activity in the sample. It has also been shown that especially some anaerobic bacteria are not able to incorporate thymidine [6-81. Leucine incorporation, which measures bacterial protein synthesis, gives a more direct measurement of bacterial biomass change (or carbon production) than that given by thymidine incorporation in which average carbon content of bacterial cells should be known. However, there are only a few studies in which [‘“Cl- or [3H]leucine incorporation methods have been applied to sediment samples: a dialysis technique has been used by Tibbles et al. [9], extraction and precipitation by Meyer-Reil and Charfreitag [lo], and a combustion technique by Meyer-Reil (111 and Tuominen and Kairesalo [12]. However, no comparisons and recommendations of the different methods have been given earlier. As a small amino acid (molecular weight 131), leucine is easily assimilated by bacteria, but fungi and protozoa may also contribute significantly to the uptake of leucine in sediment. However, bacteria are obviously of principal importance to the leucine uptake in short term incubations ( < 30 min), since the uptake of small organic molecules is much faster by bacteria than other organisms. For instance, in bacterioplankton studies no leucine uptake by phytoplankton or cyanobacteria has been observed [13]. In this study, the applicability of a simple combustion method for sediment samples was evaluated by comparin 8 different [ i4C]leucine uptake methods and a [ Hlthymidine incorporation method.
Methods 24 (199.5) 125-134
2. Materials and Methods 2.1. Sediments Sediment samples from four different lakes were used: Lake Vesijarvi (The Enonselka Basin), Lake Tuusulanjarvi, Lake Paajarvi and Lake Hoytiiinen. Lake Vesijirvi (61”05’N, 25”30’E; see refs. [14,15] for a general description of the lake) and Lake Tuusulanjarvi (60”25’N, 25”03’E; [16,17]) are eutrophic lakes in Lake Paajijirvi (61”04’N, southern Finland, 25”08’E; [18,19]) is a polyhumic lake in southern Finland, and Lake Hoytiainen (62”45’N, 29”40’E; [20]) is an oligotrophic lake in eastern Finland. The topmost O-2 cm sediment layers were collected from about 10 m depth, except from Lake Hijytiiinen, from which a layer of O-4 cm was collected from 30 m depth. After sampling, many surface sediment samples from the same area were homogenized and stored in 0.5 1 polyethylene containers at + 2°C (Lake Hoytiiinen at + 4°C). The sediments differed in their C and N contents (Table 1) as well as visually: Lake Vesijtirvi and Lake Tuusulanjirvi sediments were grey in colour, Lake Plajlrvi sediment was reddish brown and Lake Hoytiainen sediment was chocolate brown. Lake Hoytilinen sediment had the coursest and Lake Tuusulanjarvi sediment the finest texture. All the results of bacterial activity in this study are expressed as mol leucine or thymidine incorporated h-’ 1-l of fresh sediment, since the expression per g of sediment dry mass distorts the
Table 1 The water contents (W, % of fresh mass)‘, losses on ignition (LOI, % of dry mass)“, C and N contents (% of dry mass) and C/N atomic ratios in the lake sediments Lake
W
LO1
c
N
C/N
Vesijtirvi TuusulanjBrvi pgSj..i
90 92 95 84
12 13 15 10
5.2 4.4 5.1 3.1
0.7 0.6 0.5 0.3
8.7 8.6 11.9 12.1
HijytiLinen a Dried at 60°C. ’ Ignited at 550°C.
L. Tuominen I Journal of Microbiological Methods 24 (1995) 125-134
results of different sediments with different mass per volume ratios [21] (Table 1).
dry
2.2. Leucine incorporation methods Three different methods to measure leucine incorporation in sediment samples were compared. The methodological comparisons were made with Lake Vesijirvi sediment. The incubations were done in 13 ml polypropylene tubes, in which 0.1 ml sediment was mixed with 0.9 ml filtered, deionized water (three replicates). The results of control samples (three replicates), which were killed by adding 0.9 ml of 4% formaldehyde to the tubes instead of water, were subtracted from the normal samples. Daily before use, the [14C]leucine (11.5 GBq mmol-‘, Amersham) was diluted 20 times with fresh unlabelled leucine solution of the same leucine concentration (1.61 x 10e4 mol 1-l). The leucine solution was added to the tubes and the incubation was performed at 20°C in darkness for an optimal time (1.5 min; see below). The procedure was continued as follows:
127
fluorescence microscope at a magnification of 1250 x . The reliability of the combustion was tested by combusting internal standard capsules ( Wallac) . Method 2
Hot TCA method: This method was performed principally as used for water samples by Chin-Leo and Kirchman [23], and by Simon and Azam [24]. The incubation was terminated by inserting the tubes into ice and by adding 0.5 ml 15% ice-cold TCA (trichloroacetic acid; final concentration 5%). The samples were then heated for 15 min at 90°C cooled, filtered onto Sartorius 0.2 pm cellulose nitrate filters, and rinsed twice with 4 ml of ice-cold 5% TCA and once with 4 ml of 80% ethanol. The filters were left to dissolve in a mixture of 0.2 ml ethylenglycol-monomethylether and 10 ml Lumagel (Lumac LSC B.v.) for 24 h, and measured in a liquid scintillation counter. Different amounts of Lumagel were tested in order to achieve a liquid or a gel. Method 3.
Method 1
Combustion: This method was performed principally according to Tuominen and Kairesalo [12]. Briefly, the incubation was terminated by adding 4 ml of 80% ethanol containing 100 mg 1-l unlabelled leucine and centrifugating for 5 min at 5000 g. The supernatant was then discarded, and the procedure was repeated twice more. The pellet was dried at 60°C and combusted at 900°C (Junitek Oxidizer). The evolved 14C02 was adsorbed into Lumasorb II, and the scintillation liquid used was Carboluma (both Lumac LSC B.v.). The sufficiency of the three ethanol washings to remove the unincorporated leucine was tested by counting subsamples of the supernatants in a liquid scintillation counter after each washing step; in this test a fourth washing step was added. To ensure that bacteria were not discarded with the supernatants, subsamples of the supernatants were fixed with 4% formaldehyde, stored in + 4°C and later stained with acriflavine [22] and counted with a Nikon epi-
Cold TCA method (principally water samples by Kirchman [25]): tion the samples were poured into funnels, filtered, and rinsed twice ice-cold 5% TCA and once with ethanol. The filters were dissolved as in method 2.
as used for After incubacold filtration with 4 ml of 4 ml of 80% and counted
2.3. Thymidine incorporation The incubation was performed as in the leucine methods, except that the substrate used was [methyl-3H]thymidine (185 GBq mmol-‘, Amersham), which was diluted 10 times with filtered, deionized water. The thymidine incorporated into DNA was extracted with the dialysis method of Moriarty [3] and Moriarty and Pollard [26]. The incubation was terminated by addition of 4 ml of 80% ethanol which included 100 mg 1-l unlabelled thymidine . The samples were then centrifuged for 5 min (5000 g), and the ethanol washing step was repeated. The pellets
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were suspended in 2 ml of 0.3 M NaOH and heated at 90°C for 40 min. After centrifugation the supernatants were poured into the Spectra/ Por Membrane nr. 1 dialysis tube (molecular weight cut-off 6000-8000; Spectrum), and dialysed overnight (minimum 16 h; [2]). After dialysis a further step to separate the DNA was performed by heating the samples in 5% TCA at 90°C for 40 min, and after centrifugation a subsample from the supematant was measured with Lumagel (Lumac LSC B.v.) in a liquid scintillation counter.
The saturation levels were tested by incubating the samples with increasing leucine (from 4.8 PM to 21.6 PM; combustion method) or thymidine (from 250 nM to 3333 nM) concentrations. The concentration where the incorporation turned to be horizontal was taken as the saturation level. The optimal incubation time was determined as the shortest time when incorporation was high enough to get a clear difference between the normal and the control samples, but when it was still linear. This was analyzed by incubating the samples for 10, 15 and 20 min.
Y
125-134
The possibility that the leucine additions enhanced bacterial activity in the samples was tested. This was done by measuring the bacterial activity of Lake Vesijtirvi sediment with the [3H]thymidine incorporation in samples in which unlabelled leucine (from 0 to 17.8 PM) was added together with [3H]thymidine.
3. Results 3.1. Saturation levels and optimal incubation times
2.4. Saturation levels and optimal incubation times
2.5
Methods 24 (1995)
The leucine saturation level was 13.7 PM for all the sediments (Fig. 1). By contrast, no clear saturation level was found for thymidine incorporation in Lake Vesijlrvi and Lake Hbytilinen sediments, although a slight level was perceived between 952 and 1818 nM thymidine additions (Fig. 2). The thymidine saturation level for Lake Tuusulanjarvi was 1818 nM and for Lake Paajarvi 952 nM (Fig. 2). The optimal incubation time was 15 min (at 20°C). After that, the incorporation of both leucine and thymidine started to be non-linear and the variation between replicates tended to increase as well.
5-
1
Lake Vesijhrvi
+
Lake TuusulanjBrvi
2
P
t
Yz
Lake Hijytiainen
.
07
i 0
6
10
15
20
Leucine added, pM
25
5
10
16
20
25
Leucine added, pM
Fig. 1. The saturation curves of leucine uptake (combustion method). Points represent minimum and the maximum values of three replicates. Curves fitted by hand.
the average values and crosses the
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I Journal of Microbiological
Lake VaSijirVi
Methods 24 (1995) 125-134
129
O.‘T
+
I
Lake Tuusulanjiirvi +
0.03 -
I
+
Lake Haytllinen Laka
Piiiijiirvi +
i boo
0
*OS
,500
lhyrnidine
2000
0
2500 3000 3600
600
added, nM
1000
1300 2000 2600 3000
I 3sao
Thymidineadded, nM
Fig. 2. The saturation curves of thymidine incorporation. Points represent the average values and crosses the minimum and the maximum values of three replicates. Curves fitted by hand.
3.2. Leucine incorporation methods Both the hot TCA method and the cold TCA method yielded much lower results than the combustion method (Fig. 3). When using these methods, the variation between replicates was much greater than when using the combustion method, and also the results measured with the hot TCA method on consecutive days differed a lot. With the cold TCA method an unexpected descent was observed when 17.8 PM leucine was added. This reflects the unreliability of the
2.5 L
method. When comparing the results of gel and liquid scintillation samples to samples in which the filters were combusted, samples with partitles bound to a gel yielded on average 81%) and samples with particles settled to the bottom on average 37% of the combusted samples. The three centrifugations in the combustion method removed the unincorporated leucine effectively. The disintegrations per minute (DPM) in the third supematant were only 0.56% of those in the first supernatant (Table 2). An additional fourth centrifugation step lowered
T
+
Oxidation -
Min
-
Max
+
Hot TCA +
+
+ +
Min Max Cold TCA
x
Min
x
Max
15 leucine added, VM
Fig. 3. Leucine uptake in Lake Vesijiirvi sediments measured by combustion, hot TCA and cold TCA methods (see text) as a function of added leucine concentration (averages + minimum and maximum values).
130
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I Journal of Microbiological
Table 2 The radioactivity in the supernatants of the combustion method after each centrifugation step compared to the first supernatant Centrifugation step
% Of the radioactivity of the 1st supematant
1st 2nd 3rd 4th
100 5.04 0.56 0.16
these figures a little, but because the prekilled controls are always subtracted from the normal samples, there is no need for a lower percentage. Sediment bacteria were very efficiently settled by the centrifugation at 5000 g, since no bacteria could be found in any of the stained supernatants. The combustion process is also very reliable (at least with the Junitek Oxidizer), and on average 97% of the combusted activity can be found in the scintillation samples. The leucine addition (from 0 to 17.8 PM) did not enhance the bacterial activity as measured by thymidine incorporation. No significant differences were found between any samples (ANOVA, p-value 0.27).
4. Discussion 4.1. Leucine incorporation methods The problem when measuring sediment samples with a method in which filters are dissolved in a scintillation cocktail is, that although the filter itself dissolves, the sediment particles remain. The particles can then be bound to scintillation gel or settle to the bottom of the bottle. In the first case, the particles cause quenching, and in the second case, the counting geometry is biased. In order to eliminate these problems, combustion is currently the only technically correct method to handle sediment samples for liquid scintillation counting: it results in a homogenous sample with no particles. Using filters to terminate incubation can also possess other problems. If the bacteria in the
Methods 24 (1995) 125-134
sample are very small, part of them can be lost through the filter. The use of a filter with a pore size smaller than 0.2 pm can be unusable for sediment samples because of slow flow rate. Kairesalo and Saukkonen [27] have also noted, when measuring thymidine incorporation in water samples, that the edge of the filter (situated under the filtration funnel) could absorb part of the sample which was then not rinsed properly. This resulted in highly variable replicates. They solved the problem by rinsing the filters before use in unlabelled thymidine solution to block the absorption of labelled thymidine to the filter. Leucine has been found to label proteins almost exclusively (90%) [13]. Therefore, there is no need to extract proteins from the cells. The ethanol rinses in the combustion method, however, can remove leucine adsorbed to lipids [28,29] especially from the cell membrane. The simple combustion method with three ethanol rinses was the most reliable method for measuring leucine uptake in sediment samples. Very high leucine additions were needed to reach the saturation level in sediment samples. Although it was shown that 17.8 PM leucine did not enhance sediment bacterial activity, the high leucine additions may enable the uptake of leucine by other organisms, especially fungi. Fallon and Newell [30] have observed that axenic fungal cultures were capable of incorporating leucine . Also, the prymnesiophyte Emiliania huxleyi was found to grow with leucine as source of N, but in mixed cultures with bacteria, the bacteria outcompeted the algae in amino acid uptake [31]. Glaser [32] observed that ciliated protozoa Tetrahymena pyriformis was able to take up amino acids (glycine and histidine), but he speculated that ciliates may contribute significantly to amino acid dynamics only in benthic environments with high levels of amino acids. Kirchman et al. [13] observed no evidence of leucine uptake by phytoplankton or cyanobacteria. It was most likely that bacteria were of principal importance to the leucine uptake in the current study, since the short incubation time (15 min) benefits the bacterial uptake of dissolved substances compared to other organisms.
L. Tuominen I Journal of Microbiological Methods 24 (1995) 125-134
We have earlier observed that the gradual emergence of bacterial activity after fixing with dilute formaldehyde [33,34] was much faster when measured with leucine uptake than with thymidine incorporation. After a 6-day incubation thymidine incorporation had risen to about l/3 of that in the live control samples, while leucine uptake already was higher than in the control samples [33]. This suggests that bacterial protein production and DNA replication are not balanced in that kind of situation. It could also be possible, that organisms other than bacteria (e.g. fungi) or a specific bacterial strain produced the steep leucine uptake curve. 4.2. The use of leucine and thymidine incorporation methods in sediment samples Leucine incorporation into proteins is a straight forward method for measuring bacterial biomass production, because ca. 50% of bacterial dry mass consists of proteins [35]. The molar proportion of leucine compared to other amino acids has been found to be rather constant in natural bacterioplankton assemblages [24]. Simon and Azam [24] noted that smaller bacteria were more ‘concentrated’ and contained more protein and carbon per volume (w/v) than larger bacteria. The ratio of cellular protein and carbon remained, however, constant throughout the size classes from 0.026 to 0.400 pm3. Therefore bacterial carbon production (BCP) can be calculated as [24]: BCP(g) = (mol 131.2 x 0.86
leucine,,,)
X (100/7.3) X
(1)
where mol leucineinc = moles of leucine incorporated; 7.3 = mol% of leucine in protein; 131.2 = formula weight of leucine; 0.86 = a conversion factor to convert a gram of protein produced to a gram of carbon. Thymidine incorporation is principally very well known [3, 36,371. The main problems with this method are the assumptions needed to calculate bacterial carbon production, and the
131
question if all bacterial strains incorporate exogenous thymidine. At least some chemolithotrophic bacteria, sulphate reducers, and methane bacteria have been found to be unable to utilize exogenous thymidine [6-81. This problem seems to be more pronounced in anaerobic than in aerobic environments. When calculating the results based on thymidine incorporation, several assumptions have to be made [38]: (1) the proportion of the four bases that thymidine constitutes; (2) the amount of DNA in each cell; and if the results are converted to bacterial carbon production (3) the amount of carbon per bacterial cell. For water samples the conversion factor to convert moles of thymidine incorporated to number of bacterial cells produced can also be determined empirically. This is, however, not possible in sediment samples, because of the problems with recording changes in numbers of bacteria by microscopy. If equation 1 is used to calculate the bacterial carbon production based on the leucine uptake at the saturation levels of the lakes (the moles of leucine taken up being first corrected for the 10% non-protein uptake of leucine; [13]), and if the thymidine results are calculated with a conversion factor of 5 x 10” cells produced per mole thymidine incorporated [3], and the thymidine results are further corrected for the recovery of DNA in the dialysis method (71%, [2]), and if an average bacterial size of 0.026 pm3 (unpublished results for Lake Vesijarvi) and C/cell of 10.4 x lo-l5 g [24] are supposed, the leucine method yields much higher bacterial carbon production values than the thymidine method (Table 3). The values based on the thymidine results are, however, underestimates, since no clear saturation levels for thymidine were achieved for Lake Vesijarvi and Lake Hdytiainen, and the isotope dilution may therefore be substantial. Also, the activity of bacteria in the anaerobic microsites in the sediments was not included in the thymidine method [6-81. The differences in the results could, however, partly be real. This can be due to unbalanced DNA and protein production or uptake of leucine by organisms other than bacteria. Therefore, it can be concluded that the level of the bacterial
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Methods 24 (1995) 125-134
Table 3 Bacterial carbon production based on the leucine uptake (BCP,,,) and on the thymidine incorporation sediments Lake Vesijarvi Tuusulanjarvi Piajlrvi Hiiytilinen
13.7 13.7 13.7 13.7
Cr,, and C,,, = the concentrations ’ Unclear saturation level.
952” 1818 952 952”
(BCP,,,)
in four lake
BCP,,, (mg C h-’ 1-l)
BCP,,, (mg C h-’ 1-l)
BCP,,, /BCP,,,
2.8 5.7 1.5 1.9
0.16 0.57 0.09 0.12
17.0 10.0 16.1 15.3
of the added leucine and thymidine, respectively (cf. Figs. 1 and 2).
carbon production can be measured with both methods, but the value based on the thymidine incorporation is most probably an underestimate. The correlation of thymidine incorporation and leucine uptake was linear, except for the results from Lake Tuusulanjarvi (Fig. 4). The thymidine incorporation was higher at the same
leucine uptake level in Lake Tuusulanjarvi sedito Lake Vesijarvi. Lake ment compared Tuusulanjirvi sediment differed from the other sediments by being very fine in structure with very much colloidal clay. Also, bacterial metabolism might have been different. However, a more detailed explanation remains unclear and calls for further investigations.
0.16
0.12 E ‘ij 2
0.1
0 ,P 00 .s
0.08
e ._ 9 E, 0.06 i-” 0.04
0 0
1
2
3
4
5
6
Leucine uptake
Fig. 4. The correlation between thymidine incorporation and leucine uptake (both expressed as pmol h-’ I-’ of fresh sediment) in four lake sediments. The results from Lake Tuusulanjarvi are not included in the equation.
L. Tuominen
I Journal of Microbiological
Acknowledgements I am grateful to Antti Uusi-Rauva and Kaj Hurme (from the isotope section of The Faculty of Agriculture and Forestry, University of Helsinki) for help and advice in the laboratory, to Timo Kairesalo for comments on the manuscript, and to Tiina Ristola and Heli Vahtera for sending me the sediment samples from Lake Hoytiiinen and Lake Tuusulanjarvi.
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[121 Tuominen, L. and Kairesalo, T. (1992) A method for measuring the uptake of thymidine and leucine in sediment. Aqua Fenn. 22, 43-48. 1131 Kirchman, D., Knees, E. and Hodson, R. (1985) Leucine incorporation and its potential as a measure of protein synthesis by bacteria in natural aquatic systems. Appl. Environ. Microbial. 49, 599-607. iI41 Keto, J. (1982) The recovery of L. Vesijlrvi following sewage diversion. Hydrobiologia 86, 195-199. 1151 Keto, J. and Sammalkorpi, I. (1988) A fading recovery: a conceptual model for Lake VesijHrvi management and research. Aqua Fenn. 18, 193-204. [161 Pekkarinen, M. (1990) Comprehensive survey of the hypertrophic Lake Tuusulanjlrvi - nutrient loading; water quality and prospects of restoration. Aqua Fenn. 20, 13-25. [I71 Tolonen, K., Ilmavirta, V., Hartikainen, H. and Suksi, J. (1990) Paleolimnological investigation of the eutrophication history of Lake Tuusulanjarvi, southern Finland. Aqua Fenn. 20, 27-41. [I81 Ruuhijirvi, R. (1974) A general description of the oligotrophic lake Paajiirvi, southern Finland, and the ecological studies on it. Ann. Bot. Fennici 11, 95-104. [I91 Hakala, I. and Arvola, L. (1994) Alarming signs of eutrophication in Lake Plajiirvi. Lammi Notes 21, l-5. PO1 Simola, H., Sandman, 0. and Ronkko, J. (1987) A clay horizon indicating the lowering of Lake Hoytilinen AD 1859: a pre-industrial marker level for northern Lake Saimaa. Aqua Fenn. 17, 51-57. WI Schallenberg, M. and Kalff, J. (1993) The ecology of sediment bacteria in lakes and comparisons with other aquatic ecosystems. Ecology 74, 919-934. WI Bergstrom, I., Heininen, A. and Salonen, K. (1986) Comparison of acridine orange, acriflavine, and bisbenzimide stains for enumeration of bacteria in clear and humic waters. Appl. Environ. Microbial. 51, 664-667. 1231 Chin-Leo, G. and Kirchman, D.L. (1988) Estimating bacterial production in marine waters from the simultaneous incorporation of thymidine and leucine. Appl. Environ. Microbial. 54, 1934-1939. 1241 Simon, M. and Azam. F. (1989) Protein content and protein synthesis rates of planktonic marine bacteria. Mar. Ecol. Prog. Ser. 51, 201-213. 1251 Kirchman, D.L. (1992) Incorporation of thymidine and leucine in the subarctic Pacific: application to estimating bacterial production. Mar. Ecol. Prog. Ser. 82, 301-309. WI Moriarty, D.J.W. and Pollard, P.C. (1990) Effects of radioactive labelling of macromolecules, disturbance of bacteria and adsorption of thymidine to sediment on the determination of bacterial growth rates in sediment with tritiated thymidine. J. Microbial. Methods 11, 127-139. 1271 Kairesalo, T. and Saukkonen, P. (1990) Thymidine incorporation by littoral and pelagial bacterioplankton in a mesohumic lake. Verh. Internat. Ver. Limnol. 24, 677-681. WI Wicks, R.J. and Robarts, R.D. (1988) Ethanol extraction requirement for purification of protein labeled with [3H]leucine in aquatic bacterial production studies. Appl. Environ. Microbial. 54. 3191-3193.
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