Oct 29, 1993 - 1994 Springer-Verlag New York Inc. Fate of Pseudomonas putida After Release into Lake Water. Mesocosms: Different Survival Mechanisms in ...
Microb Ecol (1994) 27:99-122
MICROBIAL ECOLOGY © 1994Springer-VerlagNewYorkInc.
Fate of Pseudomonas putida After Release into Lake Water Mesocosms: Different Survival Mechanisms in Response to Environmental Conditions I. Brettar, 1'* M.I. Ramos-Gonzalez, 2 J.L. Ramos, 2 M.G. H6fle l'* 1Max-Planck-Institut for Limnologie, Postfach 165, D-2320 P16n, Germany ZConsejo Superior de Investigaciones Cientificas, Estacion Experimental del Zaidin, 18080 Granada, Spain Received: 6 July 1993; Revised: 29 October 1993
Abstract. To study the fate of Pseudomonas putida DSM 3931 in an aquatic environment, cultures of the strain were released into lake water mesocosms. P. putida, bearing the TOL-plasmid, was released as a representative xenobiotic-degrading microorganism. The release was carried out in mesocosms with unamended lake water and in lake water with added culture medium to compare the survival of the strain due to the influence of different organic load. As a comparison, the survival of P. putida was followed in microcosms with sterile lake water. Survival and fate of the strain were determined by means of immunofluorescence with highly specific monoclonal antibodies and growth on selective agar medium for up to ten weeks after release. Addition of medium had a pronounced influence on survival in mesocosms. In mesocosms without added medium, the number ofP. putida cells decreased within ten days by over 2 orders of magnitude. In mesocosms with medium, cell numbers increased in the first two days by an order of magnitude and were, after ten days, in the same range as at the time of introduction. Over time, cell numbers decreased but remained detectable in both types of mesocosms for up to ten weeks after release. In mesocosms with unamended lake water, the major fraction of the cells was attached to particles after two days. In mesocosms with medium, large aggregates of P. putida cells formed which included algae. The observed decrease in cell numbers in mesocosms was attributed mainly to grazing. Sedimentation was an additional factor contributing to loss of cells out of the water column, which especially affected aggregate-forming ceils in
*Present address: Institut fOr Agrartechnik Bornim e. V., Abt. Bioverfahrenstechnik, Max-Eyth-Allee
1, D-14469 Potsdam, Germany. tPresent address: Gesellschaft far Biotechnologische Forschung, Abt. Mikrobiologie, Mascheroder
Weg 1, D-38124 Braunschweig, Germany. Correspondence to: M.G. Hrfle
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mesocosms with medium in the long run (beyond two weeks). These studies demonstrate that experimental tools on a mesoscale are crucial in order to understand the complex processes microorganisms are subjected to after release into a natural environment, and that single cell detection, such as immunofluorescence, is essential to understand mechanisms of survival and elimination.
Introduction
There is growing public concern about the release of genetically engineered microorganisms (GEMs) into the environment [32]. This led us to investigate basic features of release scenarios, i.e., (1) survival of released bacteria, (2) interaction of components of the ecosystem with the released bacteria, and (3) impact of the released bacteria on the ecosystem. In this paper, we emphasize the first two aspects. Data on the impact on the ecosystem are provided as background information, but are described in more detail elsewhere [ 12, 13, 14]. Aquatic environments seem most sensitive to release, due to the potential for uncontrolled distribution of the GEMs. Extensive knowledge about the ecosystem concerned is necessary to understand changes caused by the released bacteria. For this reason a eutrophic, well-studied lake was chosen as the ecosystem for release [11]. A major goal of the study was to gain insight into how natural ecosystems may interact with the released bacteria and thus influence their survival. Therefore, the creation of near-natural conditions was a basic challenge. Numerous studies on the survival of bacterial strains, wild type as well as engineered derivatives, have been performed in a variety of microcosms including some with freshwater [ 1, 8, 21, 34, 35]. These studies, well suited for the analysis of single factors, have only limited predictive power for effects on whole ecosystems [21, 35]. To fill the gap between laboratory microcosm studies and field release, mesocosm studies are appropriate for understanding the interaction of released bacteria and the ecosystem. Mesocosms are well-established experimental tools whose value for studying phenomena in aquatic ecology has been extensively demonstrated [7, 19, 24]. Pseudomonas putida DSM 3931 was released to gather basic knowledge about the survival of nonindigenous bacteria able to degrade xenobiotics. P. putida DSM 3931 is the source strain for a variety of genetically engineered derivatives whose degradative abilities have been strongly improved [26]. The major criterion for the choice of the strain was that it had features similar to strains introduced into sewage treatment plants for improvement of xenobiotic degradation. To avoid the release of a genetically engineered strain in open ecosystems like mesocosms installed in a lake, a nonengineered strain was released. Several studies in aquatic microcosms have shown that the survival of the source strain and derivatives with additional degradation features was comparable [1]. Large numbers of P. putida cells were introduced; the biomass of the introduced bacteria exceeded the indigenous microflora by a factor of 3. The influence of addition of organic nutrient provided as culture medium was also studied in single as well as in combined effect experiments. The medium addition was also intended to mimic a major disturbance of the system and should provide some insight about survival of the released strain under conditions of different organic loads.
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In this research, we followed the survival of P. putida by culture techniques as well as directly by immunofluorescence. This was done to overcome the problem of viable but nonculturable bacteria [28] and to gain insight into survival strategies and niches for survival at the single cell level. To understand interactions among the massive release, the nutrient input, and major components of the aquatic ecosystem, parameters for major processes like mineralization of the added medium and phytoplankton production were followed. Materials and Methods
Bacterial Strain and Culture Conditions The strain used in the experiment was Pseudomonas putida DSM 3931 (purchased from DSM, Braunschweig, Germany). P. putida DSM 3931 is identical to the earlier described P. putida mt-2 PaWl [23], a wild type strain carrying the naturally occurring TOL plasmid pWWO, and the source strain KT 2440 host for the genetic engineering of the plasmid [26]. The plasmid enables the bacterium to perform meta-cleavage of the aromatic ring [6]. Mass cultivation ofP. putida was performed in aerated, stirred 25-liter glass bottles at 28°C. Culture medium was Nutrient Broth (Difco Cow.) at a concentration of 4 g/1 amended with 0.8 g/1 glucose. For generation of the inoculum for the mesoscosm experiments, cells in the late logarithmic phase were harvested by flow-through centrifugation at 6000 g (Contifuge, Heraeus Corp.). Pellets were resuspended in 1 liter of filtered (0.2 Ixm pore size) and autoclaved lake water and kept stirred at 18°C. Cultivation of the strain and generation of the inoculum were described in more detail by Brettar and H6fle [3].
Design of the Mesocosm and Microcosm Experiment The mesocosms were installed in the central part of lake Plul3see, a small (0.14 krn2) eutrophic lake in northern Germany. Eight mesocosms (polyethlene bags of 0.95 m diameter and 2.4 m length that were closed at the bottom) were mounted on free-floating racks (basic design of Lampert et al. [19]). The period of intensive investigation was from 15 May to 31 May 1990. Weekly and biweekly sampling continued up to ten weeks after release. At the beginning of the experiment, the lake had just reached the clear water phase, i.e., macrozooplankton (mainly calanoid copepods) was completely controlling phytoplankton biomass (chlorophyll a content < 2 Ixg/1). The mesocosms were filled on 14 May 1990 with 1.7 m 3 of lake water each (water from epilimnion of lake Plu[3see, 1-2 m depth). The experiment was started the next morning (9-10am) by adding 3.0 liter of a suspension of (1) washed bacterial cells, (2) bacterial cells suspended in the culture medium, or (3) culture medium alone. Each experimental treatment was in duplicate, including unamended controls. This resulted in the following four different batches: (1) control, (2) medium alone, (3) P. putida plus medium, (4) P. putida alone. After thorough mixing of the water in the mesocosms by four strokes from bottom to surface with a perforated plastic disc (65 cm diameter) samples were taken from a depth of 1 m every morning with 2-liter Ruttner samples. The added culture medium increased the indigenous concentration of dissolved organic carbon (DOC) of the lake water of 8.5 mg/1 by 2.8 mg/1 C. The water column was mixed for 13 days. Previous studies revealed a good comparability of conditions in mixed mesocosms with natural epilimnic environments for up to two weeks [19]. After the mixing period (and for comparison also in the final phase of the mixed period), the unmixed water column (1 m) and the bottom water (2.4 m) were sampled separately. The experimental setup of the mesocosms and the observation of physical and chemical parameters are described in more detail elsewhere [3]. Filtered (0.2 ixm Nucleopore filter) and autoclaved lake water was used in l-liter Erlenmeyer flasks to test the survival of P. putida in an axenic environment (microcosm). P. putida was precultured as
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described above and, after inoculation into 400 ml of sterile lake water, microcosms were incubated at 18°C in the dark on a rotary shaker (20 rpm).
Agar Media for Detection of the Released Strains and Phage Susceptibility P, putida was grown on casitone-peptone-starch agar (CPS) and on a mineral medium with 1 g/1 4-methylbenzoate (4-MB). CPS-agar consisted of Bacto-Casitone (Difco) (0.5 g/l), Bacto-Peptone (0.5 g/l), glycerol (1 ml/1), starch (0.5 g/l), K2HPO 4 (0.2 g/l), MgSO 4 • 7H20 (0.05 g/l), (4 drops of a 0.01% FeC13 • 6H20 solution/1), and Difco-agar (14 g/l). 4-MB-agar consisted of 4-methylbenzoate (Merck) (1 g/l), NHaC1 1 g/l, K2HPO4 (0.5 g/l), MgSO4 • 7H20 (0.2 g/l), 2 drops of a 0.1% FeC12 solution/l), and Difco-agar (22 g/I). Both media were adjusted after autoclaving to a final pH of 7.3 + / - 0.1. For counting colony-forming units (CFUs) of P. putida, samples were plated after adequate diluting in three replicates on 4-MB-agar and CPS-agar plates. After one week of incubation at 35°C in the dark, the total number of colonies growing on the medium (t-CFU) was counted. Then plates were sprayed with a 1% catechol solution in water [22]. Catechol is degraded by the plasmid-encoded catechol 2,3-dioxygenase to 2-hydroxy-muconic semialdehyde, a substance with a bright yellow color. Colonies producing this enzyme turned bright yellow after spraying, and were counted separately as catecholase positive colonies (c-CFU). Plating of pure cultures on the respective media resulted in complete recovery, compared to direct microscopic counting after staining with the fluorochrome 4,6-diamidino-2-phenylindole (DAPI [25]). For samples from mesocosms, the coefficient of variance between the three replicate plates was an average of 18%. The presence of phages infectious for P. putida cells in the mesocosms was tested for every sample by the standard soft agar overlay technique (detection limit >/5 pfu/ml [30]) with Nutrient Agar (for details see [3]).
Fluorescent Antibody and DAPI Staining of Pure Cultures and Environmental Samples Techniques for the staining of cells and microscopic analysis were adapted from the descriptions by Harlow and Lane [10] and Enger et al. [5]. Bacterial cells were fixed with formaldehyde (2% final concentration) after sampling. Cells were concomitantly stained with DAPI and fluorescent antibodies as previously described [3]. Strain-specific detection of P. putida DSM 3931 was achieved with monoclonal mouse IgM antibodies against surface lipopolysaccharides (LPS). Production and testing of these antibodies are described in detail by Ramos-Gonzalez et al. [27]. Secondary fluoresceinisothiocyanate (FITC)-labeled antibodies (anti-mouse IgM, ix-chain-specific) were purchased from Sigma Chemical Co. (St. Louis, Mo., USA). For staining on filters, 0.5 ml of the primary antibody was applied for 1 h in a dilution of 1:100 in PBS + 0.5% BSA. The FITC-conjugate (0.5 ml) was applied twice for 15 min in a dilution of 1:120. The stained samples were analyzed with a Zeiss epifluorescence microscope using different filter sets that allowed concomittant observation of DAPI and FITC fluorescence and algal fluorescence [3]. The standard error for the method was better than 20% of the mean, when cell numbers exceeded 8000 cells/ml. The detection limit for the FITC-stained P. putida cells was 20 cells/mi, based upon examination of 110 fields of a filter containing particles from 30 ml of lake water. The limiting factor for lowering the detection limit was the particle content of the sample. The higher the particle content the lower the volume that could be used for one filter in order to guarantee a good screening of the whole sample material for the P. putida cells. If the detection limit exceeded 20 cells/ml, it will be indicated below. In lake water samples, no cells of the autochthonous microflora were detected that were stained by the immunofluorescence staining procedure. Length and width of the P. putida cells were measured on the projection of slides of the DAPI- and FITC-stained cells. After adjusting the measurement procedures for both staining techniques comparable values were obtained. Measurements were calibrated against 10 ixm-scales. Coefficient of variance
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(CV) of the method was always better than 10%, with the highest CV for the smallest cell dimensions of 0.5 I~m, and decreasingCV with increasing cell dimensions.
Parameters of Ecosystem Changes As a tracerfor mineralizationof the added culturemedium, the productionof ammoniumwas monitored. Ammoniumwas measuredby the indophenol-bluemethodwith an autoanalyzer[18]. Particulatechlorophyll a was used as a parameterto monitormajorecosystemresponses,i.e., changesof the algal biomass. Chlorophylla was measuredspectrophotometficallyafterhot ethanolextraction[20].
Results
Survival of the Released Strain in the Mesocosms Determined by Immunofluorescence Microscopy Cell numbers of P. putida in mesocosms with and without medium developed in different ways. During the first ten days, cell numbers decreased by over 2 orders of magnitude in the mesocosm without medium. In the mesocosm with medium, cell numbers increased in the first two days by an order of magnitude, and decreased within the following two days. However, after ten days, the cell numbers were still around 50% of the value at the start of the experiment (Fig. 1). Sampling from the 16th day onward was done without mixing of the water, and cell concentrations in the water column (1 m) and the bottom water (ca. 2.4 m) were followed separately (see Fig. 1). In the mesocosm without medium, cell numbers in the unmixed water column increased on the 22nd day and remained detectable up to the last sampling day ten weeks (70 days) after release (Fig. 1A). In mesocosms with medium added, cells were below the detection limit ( 40 Ixm) seemed to provide good shelter against grazing. Lampert et al. [19] showed the selection for larger algae at a given high grazing pressure by daphnids. Algae exceeding the ingestable size of daphnids (> 35 lxm) escaped grazing. Improved shelter provided by attachment to larger particles can therefore be assumed for our study.
Adhesion to Surfaces and Vectorial Transport in Mesocosms P. putida tended to adhere to surfaces, such as zooplankton, algae, and detritus. Sticking to biological surfaces was most pronounced for aggregate-forming cells in mesocosms with medium added. Cells in mesocosms without medium were observed mainly on detritus after the second day. We assume that the coccoid aggregate-forming cells were more adhesive than the rod-shaped single cells, as (1) the former were sticking together, thereby forming huge aggregates, and (2) in times of high density of these aggregates in the water, a major fraction of the particulate matter was colonized by these aggregates. The P. putida-algae aggregates could have even been caused by the stickiness of the P. putida cells that adhered to each other and to algae, leading in this way to the formation of huge P. putida-algae aggregates. Aggregate formation in mesocosms with medium led to an increased sedimentation of the cells out of the water column. This could be shown by cell counts in samples taken before and after mixing (during the mixed period of the first 13 days), and comparison of water from 1 m and bottom water from unmixed mesocosms. Cells were enriched in the bottom water before mixing, and cell numbers increased greatly in the water (1 m) after mixing from the tenth day onward. Thus, a major fraction of the cells settled down to the bottom of the mesocosms and was mixed back into the water column as long as there was mixing. In the unmixed period, cells lying on the bottom of the mesocosms were not likely to have been reached completely by our bottom-water sampling procedure. The sudden decrease in cell numbers after mixing was stopped may be partly explained by this missing cell fraction. In general, the cessasion of mixing of the mesocosms, intended as an experimental simulation of the transition from the well-mixed epilimnion to the physically stable metalimnion, demonstrated how important physical processes are if released bacteria form aggregates. In mesocosms without added medium, cell enrichment in the bottom water was much less and cells were detectable in 1 m for the whole observation period at only slightly lower concentrations than in the bottom water. This indicates that cells had only a very weak tendency to settle. This may be explained by the following: (1) detritus particles to which most of the cells were attached had a much weaker tendency to settle than the aggregates formed in mesocosms with medium, (2) sticking of cells to detritus was a reversible process and cells may detach, (3) grazing of particles may have liberated P. putida cells from particles by mechanical stress during the process of grazing or gut passage, and (4) single rod-shaped cells remained mobile while the small coccoid cells in aggregates did not.
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While attachment to particles or formation of aggregates increases the tendency for transport down to the bottom, attachment to surfaces of living animals may result in movements in different directions, i.e., all directions the animal goes. Even cells once settled down can in this way return to the free water. Adhesion to animals (in our study mainly zooplankton) is very crucial for risk assessment, as bacteria can be transported in all directions, even in unexpected ways such as upstream in river ecosystems. The importance of transportation via zooplankton has been shown for Vibrio cholerae by Tamplin and coworkers [33].
Comparison of the Survival Behavior of P. putida DSM 3931 with Other Released Strains Only a few studies have examined bacteria released in aquatic environments similar to nature [1, 34]. Most comparable to our study is the fate of an E. coli K12 strain that was released in mesocosms in the same experimental set-up as P. putida [3]. Conditions for the release were identical to the release ofP. putida. Cell numbers of E. coli decreased much more rapidly. Attachment of the major fraction of the E. coli cells to particles was observed a few days after release for mesocosms with and without medium. After ten weeks E. coli was detectable only in mesocosms with medium added. While survival of P. putida was quite different in response to the addition of organic nutrients, survival of E. coli was similar in the mesocosms of both treatments. Addition of medium did not change the general survival mechanisms of E. coli but only improved survival in the long run. Awong et al. [1] examined with culture techniques survival in microcosms for wild-type and genetically-engineered P. putida and E. coli strains. According to their results, survival of the genetically-engineered derivatives was never lower than that of the wild type strains. In the presence of indigenous microflora, E. coli was eliminated after 20 days, while P. putida survived. Survival of plasmidbearing strains of P. putida, P. fluorescens, and Klebsiella aerogenes was examined by van Overbeek and coworkers in microcosm studies with agricultural drainage water over one year [34]. Both Pseudomonas strains were still detectable in sterile as well as in nonsterile water after one year, while K. aerogenes survived only in sterile water. In sterile lake water, P. putida cell numbers did not decrease compared to the introduced number. Both studies are consistent with our findings of good survival of P. putida in nonsterile water. In the work of van Overbeek et al. [34], P.fluorescens numbers were determined by immunofluorescence and plate counts. Immunofluorescence gave higher numbers by up to 4 orders of magnitude, and samples where no colonies were detected displayed high numbers of immunocounts. We observed similar results here and in our release study with E. coli [3]. This indicates that studies with only culture techniques must be interpreted with care; culture techniques can be taken as minimum values but they never guarantee full recovery.
Conclusions P. putida can be an example of a released strain that is well adapted to survival in sterile lake water, as indicated by the microcosm experiment. In mesocosms, P.
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putida was able to survive for a long time (>70 days) with natural and increased concentrations of organic nutrients. However, cell numbers decreased in mesocosms of both treatments by several orders of magnitude, while in microcosms no reduction occurred. Reduction of cell numbers in mesocosms was attributed during the mixed period of the first ten days largely to grazing. Rotifers, daphnids, and flagellates may have played a role as potential grazers. During the unmixed period from the 16th to the 70th day, grazing may have had some further impact in mesocosms of both treatments. In mesocosms with organic nutrients added, major losses of cells out of the water column must also be attributed to sedimentation of aggregated cells. In mesocosms, survival varied in response to organic nutrient (medium) addition. With medium, cells showed high dividing activity thereby forming large aggregates of coccoid cells, which did not occur in unamended lake water. Rapid and effective uptake of organic nutrients by the P. putida cells, followed by multiplication, is probably responsible for this phenomenon. In both types of mesocosms, particles are assumed to have provided shelter from grazing and been a site of enhanced growth. Following P. putida by immunofluorescence in mesocosms provided more reliable results than plate counts. Though results of both methods agreed very well in microcosms with sterile lake water, plate counts failed in some respects in mesocosms, especially for long-term exposure. Particle attachment and nonculturabilty are considered to be major reasons for the failure of culture media to detect bacterial cells. Immunofluorescence microscopy was essential for insight into the survival and elimination mechanisms of the released bacteria as attachment to living and nonliving surfaces and aggregate formation. The complexity of the release process caused by the variety of the reactions of the released bacterium and its interactions with the components of the ecosystem demonstrate the necessity of experimental tools on a mesoscale. The results of the release experiment of P. putida show that survival mechanisms can strongly depend on the conditions in the ecosystem and its interaction with single ecosystem components. Furthermore, comparison of the survival mechanisms of P. putida with that of E. coli [3] under the same conditions indicates that survival depends strongly on special features of the respective strains.
Acknowledgments. This project was financed by the Bundesministerium ffir Forschung und Technologie. We acknowledge excellent technical assistance by R. Handels, S. Hamann, M. Keskin, and D. Albrecht. For cooperation on the project, we thank C. Krambeck, W. Hofmann, S. Grein, and U. Miinster. Special thanks to B. Albrecht for excellent photographic assistance and A. Konopka for helpful criticism on the manuscript. We acknowledge the valuable comments of two anonymous reviewers.
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