CURRENT MICROBIOLOGY Vol. 47 (2003), pp. 125–128 DOI: 10.1007/s00284-002-3942-z
Current Microbiology An International Journal © Springer-Verlag New York Inc. 2003
Mercury Decreases Culturability of Pseudomonas frederiksbergensis JAJ 28 in Soil Microcosms Kaare Johnsen,1,2 Flemming Ekelund,3 Svend J. Binnerup,4 Lasse D. Rasmussen5 1
Geological Survey of Denmark and Greenland, Copenhagen, Denmark Danish Veterinary Institute, Bu¨lowsvej 27, DK-1790 Copenhagen, Denmark 3 Zoological Institute, University of Copenhagen, Copenhagen, Denmark 4 Department of Environmental Chemistry and Microbiology, National Environmental Research Institute, Roskilde, Denmark 5 Department of General Microbiology, University of Copenhagen, Copenhagen, Denmark 2
Received: 9 August 2002 / Accepted: 2 October 2002
Abstract. Mercury is a biologically potent heavy metal, which has been found to change the diversity of culturable bacteria. Therefore, we investigated whether Hg kills bacteria in soil or reduces culturability. Soil microcosms were inoculated with Pseudomonas frederiksbergensis JAJ 28 and were sampled regularly during 28 days. The total number of acridine orange-stained cells was relatively constant, and Hg reduced the number on only one sampling day. However, the fraction of culturable cells on 1/10 tryptic soy agar was lowered on days 6, 13, and 21. The number of microcolony forming units, which represents viable cells, was also affected by Hg, but this effect was delayed compared with the effects on CFUs. The amount of headspace CO2 per cell was overall increased by Hg, another indication of the toxic effects of Hg on the bacterial cells. Our results thus emphasize the need to take culturability into account when studying the effects of heavy metals on bacterial diversity.
Mercury is a biologically potent heavy metal, which has been shown to affect bacterial diversity in soil, partly owing to the selection for Hg-resistant bacteria [5, 11, 14, 15]. The Hg effects are associated with the bioavailable fraction of the metal, which is controlled by factors such as pH, amount of dissolved organic C, and clay content [3]. Hence, biosensors with the ability to measure the bioavailable fraction of mercury [Hg(II)] in soil have been constructed [16]. Torsvik et al. [18] used molecular techniques to show that the diversity of the total bacterial community was 200 times higher than the diversity of the fraction recovered on an agar medium. The fraction of so-called unculturable cells in soil is a heterogeneous blend of cells that belong to taxa unable to grow at the specific culture conditions and cells from normally culturable taxa, which are presently in an unculturable state because of physiological reasons such as stress [17]. To distinguish these from each other experimentally, it is feasible to inoculate sterile soils with pure cultures of bacteria that are normally culturable and then examine whether induced stress will reduce culturability. Correspondence to: K. Johnsen: email:
[email protected]
Following this approach, Ghezzi and Steck [7] found little effect of copper sulfate on culturability of Xanthomonas campestris in sterile soil, though there was a pronounced effect on culturability in liquid microcosms. This was possibly because the remaining cells in the soil microcosms had entered the viable-but-not-culturable (VBNC) state as measured by the live/dead staining kit for reasons other than the copper. Another way of measuring the number of VBNC cells is by the microcolony technique, in which cell suspensions are filtered onto a filter, incubated on a growth medium, stained by acridine orange, and examined under the microscope [4]. Hence, bacteria that are able to divide a few times on the filters represent viable cells. This raises the question whether Hg kills the bacterial cells in soil or to what extent the cells enter the VBNC state. When Hg effects on adaptation or on the diversity of the culturable bacterial community are studied, this knowledge is important. To study this, we used the soil bacterium Pseudomonas frederiksbergensis, which was isolated from a contaminated former coal gasification site [1, 2, 8]. Thus, our aim was to investigate whether the pres-
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CURRENT MICROBIOLOGY Vol. 47 (2003)
ence of Hg or the amoeba Hartmanella verniformis killed or reduced the culturability of P. frederiksbergensis JAJ 28. Materials and methods Microcosm setup. The experiments were carried out in a soil from Ringe, Denmark. A 7-kg soil sample was air dried, passed through a sieve (mesh size, 2 mm), and thoroughly mixed. Soil characteristics were: coarse sand (⬎200 m) 33.0%, fine sand (63–200 m) 29.2%, coarse silt (20 – 63 m) 7.8%, fine silt (2–20 m) 8.0%, clay (⬍2 m) 13.0%, humus 1.1%, CaCO3 7.9%, total C 1.59%, total N 0.08%, total P 404 mg kg⫺1, pH (H2O) 8.2. The soil was sterilized by gamma irradiation (25 kGy). Moistened, incubated soil gave no CFUs on 1/10 TSA (data not shown). The background total Hg content was 48.3 ⫾ 25.3 ng g⫺1 soil. Ten-g aliquots of dry soil were distributed in 117-mL crimp seal vials, and 2.5 mL amoeba saline [13] was distributed on top of the soil with a pipette. In Hg-treated microcosms, the amoeba saline contained HgCl2 to give a final concentration of 5.0 mg Hg kg⫺1 soil. Microcosms were left at 15°C overnight. A single colony of P. frederiksbergensis JAJ 28 (DSM 13022T) [8] was transferred to 25 mL 1/10 tryptic soy broth in an Erlenmeyer flask, incubated overnight at 20°C (150 rpm), washed (3000 g, 5 min), and resuspended in 10 mM phosphate buffer. An axenic strain of H. verniformis (ATCC 50237) was grown on a peptone-yeast medium [0.36 g KH2PO4, 0.5 g Na2PO4, 5.0 g peptone (Difco), 10.0 g yeast extract (Difco), 1 L distilled water] for 40 days at 15°C in the dark. On day 0, P. frederiksbergensis JAJ 28 (6 ⫻ 102 CFUs in 250 L 10 mM phosphate buffer) and amoebae (102 cells in 250 L peptone-yeast medium) were distributed evenly on the top of the soil with a pipette. Microcosms without protozoa were amended with 250 L peptone-yeast medium from the protozoan cultures filtered through a 0.2-m filter to avoid a hidden treatment effect of the medium. Analyses. On each sampling occasion, triplicate microcosms were destructively sampled by removing 1.4 g soil with a sterile spatula for analysis for bioavailable and total Hg. Each crimp seal vial was then filled with 77.4 mL amoeba saline, whirly mixed for 30 s, left to settle for 30 s, after which the upper 10-mL supernatant was transferred to a plastic tube and used for biological analyses. Samplings were on days 0, 1, 3, 6, 9, 13, 21, and 28 (though bioavailable Hg was measured only on days 3, 6, 13, 21, and 28). In some of the first samplings, measurements were below the detection limit (as shown on the figures by omission of data points). Bioavailable Hg was measured with a bacterial whole-cell mer-lux biosensor as described by Rasmussen et al. [16]. Total soil Hg was measured using a Jerome 431-x Hg vapor analyzer (Arizona Instruments, Phoenix, AZ, USA) by using soil method 2 as described by Krieger and Turner [9]. Headspace CO2 was measured by taking gas samples of 1 mL with a syringe and analyzing on a gas ˚ rhus, Denmark) with a TCD and chromatograph (Mikrolaboratoriet, A 1.8 m ⫻ 3 mm Porapak Q column operated at 35°C. For acridine orange direct counts (AODC), sample aliquots were fixed in 4% paraformaldehyde and were stored at 5°C. Appropriate dilutions of the suspensions were filtered through a 0.22-m mesh size polycarbonate filter (Osmonics, Minnetonka, MN). Filters were stained with acridine orange, destained in water, and immediately transferred to microscope slides and counted in a Zeiss Axioplane epifluorescence microscope (Carl Zeiss, Jena, Germany). Excitation wavelength was 450 –390 nm. Emitted light passed a 510-nm beam splitter and a 520-nm barrier filter. Agar plates were 1/10 TSA [3 g tryptic soy broth (Difco) and 18 g Agar Noble (Difco) L⫺1]. A series of 75-L of 10-fold dilutions in a 0.9%
Fig. 1. The amount of g bioavailable Hg⫹⫹ g soil⫺1 as measured by using a mer-lux biosensor. Error bars represent standard error of the mean.
NaCl solution was spread on 1/10 TSA for each replicate microcosm. 1/10 TSA agar plates were incubated at 15°C for 3 days and enumerated. Microcolony samples were filtered onto white polycarbonate membrane filters and incubated ⬃18 h on 1/10 TSA at 15°C. At the end of incubation, filters were stained with acridine orange, and microcolonies (mCFU) were counted by epifluorescence microscopy according to Binnerup et al. [4]. Data were analyzed by using Sigma Stat for Windows 2.03 (SPSS Inc., Chigaco, IL). AODC, CFUs, and mCFUs were log-transformed. The effect of Hg and the presence of protozoa were assessed by using the two-factor analysis of variance.
Results The total amount of Hg in the Hg-treated microcosms was 4.70 ⫾ 1.96 mg Hg kg soil⫺1 and did not change with time. However, the amount of bioavailable Hg (Fig. 1) decreased significantly from day 0 to day 3, when the Hg was incorporated into the soil matrix or immobilized by the large amount of free organic matter present in the soil following irradiation. Thereafter, it increased significantly from day 3 to day 6 as organic matter was degraded and remained constant. There was no effect of protozoa on AODC, CFU culturability, or mCFU culturability, hence the results from treatments with and without protozoa were pooled in the figures. The presence of Hg significantly depressed AODC when looking at the experiment as a whole, but day 9 was the only single day with a significant effect of Hg (Fig. 2A). The CFU culturability of P. frederiksbergensis JAJ28 was low immediately after inoculation and increased significantly in the untreated samples from day 3 to 13 (Fig. 2B). The Hg-treated microcosms did not increase accordingly, and at days 6, 13, and 21 Hg significantly reduced the CFU culturability.
K. Johnsen et al.: Mercury Decreases Culturability of P. frederiksbergensis
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Fig. 2. P. frederiksbergensis JAJ28 as measured by different methods. (A) AODC. (B) Numbers of CFU on 1/10 TSA per AODC. (C) Numbers of mCFU on 1/10 TSA per AODC. (D) Headspace CO2 content per AODC-stained cell. Error bars represent standard error of the mean. There was no effect of protozoa on AODC, CFU/AODC, or mCFU/AODC, so treatments with and without protozoa are pooled.
The number of mCFUs was lower than the number of CFUs in samples without Hg at days 3, 6, and 28. In Hg-treated microcosms, there were no significant differences. The Hg effects on mCFU culturability results were even clearer (Fig. 2C) than the effects on CFU culturability. The increase in mCFU culturability with time after inoculation in untreated samples was more prominent than for CFU culturability, and values at days 13 and 21 were significantly above those on the other days. The Hg-treated samples remained at constant mCFU culturability throughout the experiment. Thus, there was a significant reduction in mCFU culturability with added mercury at days 9, 13, and 21. The headspace CO2 content per AODC-stained cell was significantly increased by Hg when looking at all samplings as a whole, but not at any specific sampling times. The tendency was most pronounced in the first half of the experiment. The protozoa-amended samples had an increased headspace CO2 content per AODCstained cell on days 21 and 28 (Fig. 2D).
Discussion The lower number of mCFUs compared with CFUs (Fig. 2B and 2C) is in contrast to earlier studies of specific strains [4] or communities [12, 19]. There are several possible explanations for this: (i) the difference is strainspecific; (ii) the mechanical stress caused by filtration decreased culturability; or (iii) the water potential was lower on the polycarbonate filter than on agar plates. On the other hand, Normander et al. [12] used agar plates and found higher mCFU numbers than CFU numbers, though using a different agar medium and strain. The delayed effect of Hg on mCFU compared with CFU may reflect that the mCFU cells are more resistant to the effects of Hg. After 28 days, mCFU culturability of untreated samples had fallen to the level of the treated samples. This indicates the introduction of a new stress factor, perhaps from the depletion of nutrients. The increased headspace CO2 content was also most likely a sign of stress. Previous results have suggested that in-
128 creased microbial production of CO2 after exposure to heavy metals may be attributable to the need of a higher respiration to survive [10]. The increased CO2 production is consistent with the suggestion that the presence of protozoa stimulates the production of CO2 by the prey bacteria [6]. In conclusion, our results demonstrated clear effects of Hg on P. frederiksbergensis JAJ 28 culturability; this factor must be taken into account when studying the effects of heavy metals on bacteria.
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11. ACKNOWLEDGMENT This work was supported by the Centre for Biological Processes in Contaminated Soil and Sediment (www.biopro.dk).
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