FACTORS CONTROLLING THE POPULATION DYNAMICS OF INTRODUCED BACTERIA IN STREAMS: A TIERED APPROACH TO THEIR STUDY

Thomas L. Bott and Louis A. Kaplan

Stroud Water Research Center, Academy of Natural Sciences, Avondale, PA 19311

SUMMARY

Studies are underway to characterize several autecological properties of bacterial isolates capable of 3-chlorobenzoate (3-CB) degradation. Isolates are being characterized with regard to: 3-CB degradation rate, growth in 3-CB, metabolism of other individual carbon sources, growth rates in tryptone yeast extract and freeze dried algal lysate, temperature optimum, ability to withstand starvation, ingestion by protozoa, and attachment to substrata. Data obtained thus far indicate that the isolates degrade 3-CB at rates spanning nearly three orders of magnitude, are capable of growth on other carbon sources, have temperature optima above stream water temperatures, and are differentially ingested by protozoa. Fluorescent antibodies are being prepared against four isolates to allow their population dynamics to be followed in microcosm and mesocosm experiments in which conditions are controlled to favor particular isolates to enable us to relate observed responses to autecological properties.

Key words: Population dynamics, 3-chlorobenzoate degraders, bacteria, autecology, streams

INTRODUCTION

The potential for problems resulting from the deliberate or accidental release of a genetically engineered microorganism (GEM) into the environment will be related to the combined probabilities of release, survival, growth, ability to transfer genetic material, dispersal to new habitats, and capacity to do harm (Alexander, 1985). The research discussed here is based on the premise that in addition to genetic and metabolic capabilities, the ecologically significant traits of a microorganism should be explicitly considered when making selections for genetic manipulation or applications for the purpose of bioremediation. Organismal traits such as nutrient utilization capabilities, growth rate, motility, attachment capability and temperature optimum interact with environmental conditions, e.g., available substrata and refugia, environmental concentrations of metabolic substrates, time of introduction, densities of competitors and predators, to affect the population dynamics of an introduced organism (Table 1). Microbial population ecology is presently largely descriptive and there is a need to identify factors that control the dynamics of introduced bacterial populations in nature (Tiedje et al., 1989).

There have been a few comparative studies of parent strains and genetically engineered derivatives. The population dynamics of a wild type parent were remarkably similar to those of genetically engineered derivative strains of Pseudomonas fluorescens when competition studies were conducted in sterile and non-sterile soils (van Elsas et al., 1991). Other studies with wild-type parent strains and GEMs in soils (Short et al., 1991) and aquatic microcosms (Scanferlato et al., 1989) provided similar conclusions. Chao and Feng (1990) considered that the extent of survival of GEMs in soil and water systems was "mainly determined by the host organism itself, with plasmids playing a minor part". McClure et al. (1991) reported that bacteria isolated from a particular activated sludge system survived better in that system than engineered strains from other sources, which apparently were out-competed by autochthonous organisms.

We are examining these relationships using wild type bacteria and GEMs capable of 3-chlorobenzoate (3-CB) degradation. Work thus far has been focused on the laboratory characterization of autecological properties. We will soon begin studies of the isolates in microcosm, mesocosm and field experiments designed to relate population dynamics to bacterial traits. We will use wild type bacteria to directly compare phenomena observed in microcosms and mesocosms with those in a stream and assume that the relationships established between wild type bacteria and GEMs in microcosms and mesocosms will hold in nature. Thus, our studies address another important question, namely, the utility of data gathered in laboratory cultures, microcosms and mesocosms for predicting the fates of bacteria in a natural habitat. Our study stream is the east branch of White Clay Creek (WCC, Chester Co., PA). Streams provide an important dispersion mechanism for any GEM which might be introduced from either a point (e.g., from failure of a containment system) or non-point (e.g., from application for bioremediation of nearby soils) source.

MATERIALS AND METHODS

Study Organisms. We acquired the following cultures for study: Pseudomonas putida P111, Pseudomonas alcaligenes CO and Pseudomonas acidovorans M3GY from D. D. Focht (Univ. California, Riverside); P. putida RC-4 and RC-4(pSI30) from F. Genthner (U.S. EPA, Gulf Breeze, FL); P. putida PPO300 and PPO301(pRO103) from R. Olsen (Univ. of Michigan Medical School, Ann Arbor) and Alcaligenes sp. BR60 from C. Wyndham (Carleton Univ., Ottawa, ONT).

Degradation of 3-chlorobenzoate. [¹⁴C]3-chlorobenzoate (sp. act. 19.07 mCi/mmol) was purchased from Sigma Radiochemicals (St. Louis, MO). Isolates were cultured in Cl^- -free basal salts solution (BSS, Hernandez et al., 1991) containing 500 mg/l of 3-CB (3.2 mM). 3-CB degradation rates were measured using cells harvested in mid- to late-log phase (determined by companion growth measurements) by centrifugation (6,423 x g, 10 min, 4C). Cells were washed, resuspended in BSS without 3-CB, and aliquots were transferred to shell vials. Controls for abiotic sorption of isotope were killed with formalin (2% final concentration). [¹⁴C]3-CB was added at a single concentration to all vials; additions varied between 0.079 and 0.1 µCi/vial between experiments. Vials were sealed with serum stoppers from which was suspended a CO₂trap (glass fiber filter in a small holder). The final volume in all vials was 5 ml. Experiments were performed as time courses, with 5 replicate live samples and 2 controls incubated for times between 0.25 and 4 h at 30C with agitation. Incubation was terminated by adding 0.2 ml 2N H₂SO4 which killed the cells and released [¹⁴C]CO₂. CO₂ was trapped by adding phenethylamine to the glass fiber wick in the trap and placing the samples on ice in a water bath for 2 h with agitation. CO₂ traps were transferred to liquid scintillation vials containing Cytoscint (ICN, Irvine., CA). Cells were recovered on 0.2 µm pore size polycarbonate filters, rinsed with 10 ml of water, and the filters were transferred to vials containing Ecolite (ICN). Radioactivity recovered in biomass and as CO₂was determined by liquid scintillation counting (Beckman Model 3800). The rate of 3-CB degradation was assessed by regressing the total DPM metabolized as a function of time, optimizing the regression by stepwise removal of sampling times from the latest time backward, and using the consecutive times from time 0 forward that gave the model with the highest r² value. Triplicate samples of the cell suspension were taken for epifluorescence microscopic counts (EMC) of bacterial densities determined by filtering samples onto 0.2 µm pore size membrane filters previously stained with irgalan black, staining with propidium iodide (PI, 2 µg/ml final concentration) and counting under a Zeiss epifluorescence microscope.

In a second approach, duplicate flasks of 3-CB (500 mg/l; 3.2 mM) in BSS were inoculated with an isolate and degradation was assessed from dissociation of the Cl^- ion. Growth rate was determined from absorbances at 650 nm over time. Cultures were incubated at 30C until maximum turbidity was achieved after which the culture was centrifuged. The supernatant fluid was analyzed for the concentration of Cl^- by argentometric titration (Greenberg et al., 1985). Samples were taken for EMCs to allow computation of cell specific rates of Cl^- dissociation. The weight of the pellet obtained on centrifugation was determined after drying at 60C overnight and biomass carbon was assumed to be 50% of the dry weight. Because we did not measure changes in DOC in the culture medium, cell yields for these studies were determined as follows. Since 60-80% (90% in one experiment) of the 3-CB metabolized was mineralized in experiments with [¹⁴C]3-CB, we assumed that secondary organic metabolites would not be excreted into the medium and that the fate of 3-CB carbon would be biomass or CO₂. Yield (biomass carbon produced/change in substrate carbon) was calculated using [mg 3-CB carbon x proportion Cl^-dissociated] in the denominator.

Use of other carbon sources. Two approaches were employed to test the ability of the isolates to use carbon sources other than 3-CB. In one, the isolate was grown on 3-CB in BSS until the 1 L culture was turbid. Cells were harvested by centrifugation, washed, and resuspended in 20-30 ml of groundwater. Aliquots (0.1 ml) were added to 4.8 ml sterile groundwater in sterile vials (controls received 4.7 ml plus 0.1 ml 37% formaldehyde). U-[¹⁴C] glucose (sp. act. 242 mCi/mmol, ICN), U-[¹⁴C] leucine (sp. act. 348 mCi/mmol, Amersham, Arlington Heights, IL), or [¹⁴C] 3-CB was added to 5 experimental vials and 2 killed- cell controls, and the vials were sealed. After incubation for 1 h, respired ¹⁴CO₂ was collected as described above for 3-CB degradation experiments. Cells were recovered by filtration and washed with sterile water. All samples were transferred to liquid scintillation vials with appropriate cocktail and counted.

In the other approach, cells grown on 3-CB in basal medium were harvested, washed, and transferred to either freeze dried algal extract (FDAE) or tryptone-yeast extract (TYE) in deionized water, and temporal changes in absorbance measured. To prepare FDAE White Clay Creek algae (a mix of diatoms and cyanobacteria) were homogenized in a blender, extracted in cold water for 16 h, centrifuged to remove particulates, and lyophilized. Initial carbon concentrations measured on filtered samples using an OI TOC analyzer were: FDAE, 269.7 ± 2.2 mg/l and TYE, 325.9 ± 15.7 mg/l (x ± SD, n=3). Triplicate 125 ml Nephlo flasks of each medium (25 ml) were inoculated with a suspension of the 3-CB grown cells and incubated at room temperature (20-22C) with shaking. Absorbances at 650 nm were measured for 48 h.

Temperature optima. The temperature optimum of each isolate was determined by measuring [¹⁴C]acetate metabolism over a range of temperatures from 5 to 44 C (Bott, 1975). Cells were grown overnight in 0.1% TYE broth at 30C, harvested by centrifugation, washed, and resuspended in sterile BSS. Aliquots were added to shell vials containing sterile BSS, transferred to temperatures over the range of 5-44C for a 15 min pre-incubation, after which 0.2 µCi of 2-[¹⁴C]acetate (sp. act. 55 mCi/mmol, New England Nuclear, Boston, MA) was added as a sole carbon source. Five replicates and three formalin killed-cell controls were used at each temperature. After a 0.5 h incubation metabolism was terminated by the addition of formalin. Incorporation and respiration of radioactivity was determined as described for the [¹⁴C]3-CB degradation experiments.

Protozoan Predation. Mixed cultures of predominantly ciliates or microflagellates from White Clay Creek were grown in Cerophyll medium bacterized with Enterobacter aerogenes (Trial 1 with ciliates) or a mixed inoculum of White Clay Creek bacteria (other trials). After suitable protozoan densities were reached, the 110 ml of the culture was transferred to seven 1 L Erlenmeyer flasks. Isolates were grown in 0.1% TYE broth, checked for purity microscopically, harvested by centrifugation (6,423 x g, 4C, 15 min), washed twice with sterile groundwater, pasteurized at 60 C for 2 h and stained with PI (0.2 mg/ml for 1 h). The fluorescently labeled bacteria (FLB) were washed 5 times with sterile groundwater (or until the supernatant fluid was clear). FLB of an isolate were added to a single flask to provide 10%-20% of the total densities of bacteria in the protozoan culture. Three replicate 2.5 ml samples were removed from the flask 5, 10, 20, 30 and 60 min. after addition, fixed with 5 µl Lugol's iodine solution followed by 63 µl of 37% formalin and 13 µl of sodium thiosulfate (3g/100 ml) to clear the iodine solution. Counts of ingested FLB were obtained for each aliquot. Grazing rates were calculated by regressing the number of ingested fluorescent prey per individual over time. The y-intercept was set to 0. We assumed the initial ingestion rate was linear, and based the ingestion rate on the portion of the curve for which the r² was maximum, determined using the criteria described above for the 3-CB regressions.

RESULTS

Degradation of 3-chlorobenzoate. Cell specific mineralization rates spanned three orders of magnitude. Alcaligenes sp. BR60 had the highest average cell specific rate (3.82 ± 2.61 x 10⁹µg/h, x ± SD, n = 3 trials). Activity for the other isolates ranged from 1.59 x 10^-11 µg/h to 9.95 x 10^-11 µg/h with P. alcaligenes CO having the lowest average mineralization rate (n = 2 or 3 trials for each isolate). From 60-86% of the 3-CB carbon was mineralized.

Growth rates in 3-CB varied considerably between isolates, between experiments and between replicates. Sometimes a culture grew quickly enough to become turbid within 48 h, but other times growth occurred only after a prolonged lag period. All of the isolates cleaved Cl^- from the 3-CB with similar cell specific activities. Thus, the amount of Cl^- dissociated appears to be dependent on the length of incubation and the amount of growth of the isolate. Generation times on 3-CB for four isolates ranged from 2.8 h for P. acidovorans M3GY to 13.0 ± 4.9 h (x ± SD, n = 3) for P. putida RC-4 (pSI30). The biomass produced varied widely. Yield estimates (cell C produced/substrate C utilized, as %) are considered approximate because the change in substrate C was estimated from Cl^- dissociation and was not measured directly in these experiments. The average yield (%) for the duplicate flasks was greatest for Alcaligenes sp. BR60 (26.0 ± 9.5) and least for P. putida RC-4(pSI30) (7.6 ± 3.4).

Use of Other Carbon Sources. The metabolism of 3-CB was compared with that of glucose and leucine. Leucine was metabolized to a greater extent than glucose by all the isolates and had the greatest retention in biomass of the isotopes tested. 3-CB was metabolized to a greater extent than glucose by Alcaligenes sp. BR60 and P. alcaligenes CO, and even more than leucine by Alcaligenes sp. BR60. Growth studies with P. alcaligenes CO in mixed carbon sources showed doubling times of 2.0-3.5 h in TYE and 3.5-4.3 h in FDAE with similar cell yields on each substrate. P. acidovorans M3GY also grew slightly faster in TYE than in FDAE (generation times 1.9 vs. 4.0-5.2 h, respectively) , although the maximum absorbance was similar in each medium. The isolates tested are clearly capable of metabolizing carbon sources other than 3-CB, implying survival in aquatic systems in the absence of 3-CB.

Temperature optima. All of the isolates had a temperature optimum in the range of 25 - 30C. P. putida PPO301 had a sharply defined optimum (30 C). Alcaligenes sp. BR60 and P. acidovoransM3GY had similar metabolic rates at 25 and 30C, and P. putida P111 had an even broader range with similar rates at 25, 30 and 37 C. Acetate was utilized differently than 3-CB by the isolates. Only 35-45% of the acetate was recovered as CO2 at the optimum temperature(s). All isolates will be at a slight disadvantage at ambient streamwater temperatures (0-20C) but the data suggest that Alcaligenes sp. BR60 and possibly P. putida PPO301 would be more active at streamwater temperatures than P. acidovorans M3GY, and possibly P. putida P111.

Protozoan Predation. Two trials were performed with ciliates and two with microflagellates. The isolates ranked practically identically in each trial with ciliates, with P. acidovorans M3GY being preferred and P. putida RC-4(pSI30) being least preferred in each trial. The results of experiments with microflagellates were more variable although P putida PPOP301 and P. acidovorans BR60 ranked high in both trials. Results of duplicate trials with P. putidaRC-4(pSI30) were anomalous. In contrast to ciliates, P. acidovorans M3GY may have been selected against by microflagellates.

DISCUSSION

These results are considered preliminary but they are encouraging because they indicate that we have isolates with a wide range of potential for 3-CB degradation in nature, different rates of growth on the compound, and possibly different thermal characteristics. We are continuing to study the isolates to obtain temperature optima for a few remaining isolates, and to test them for growth on other carbon sources, attachment to natural substrates, and response to starvation. Thus far we have data for ability to withstand starvation only for P. alcaligenes CO, which survived for over 50 days.

We will use fluorescent antibodies (FA) to identify the isolates in studies of population dynamics in microcosms, mesocosms and the field. We have used FA to document population dynamics in studies of the effects of introduced Cellulomonas species on the structural and functional characteristics of benthic communities and stream ecosystems (Bott and Kaplan, 1991; 1993), and to document densities of Bacillusthuringiensis var. israelensis (Bti) following spraying in the Susquehanna River (PA) (Jackson et al., 1994). Antigenic variability was not a problem during the 3 years of work with Cellulomonas, nor was it a problem in studies of methanogenic bacteria from lake sediments over a 1.5 to 2.5 year period (Strayer and Tiedje, 1978), or in studies of Legionella pneumophila in pond water over 15 months (Fliermans et al., 1981). We can detect FA stained cells at approximately 0.01 % of the total bacterial community density and thus, the technique is suitable for studies that involve tracking cell densities.

We have selected Alcaligenes sp. BR60 (greatest 3-CB degradation rate, preferred ingestion by microflagellates, possible advantage at lower temperatures), P. acidovorans M3GY (preferential ingestion by ciliates, possible metabolic advantage at higher temperatures), P. alcaligenes CO (slow 3-CB degradation rate, withstood starvation), and P. putida RC-4(pSI30) (high yield on 3-CB, complete 3-CB dissociation, slow grower) for preparation of antibodies.

ACKNOWLEDGMENTS

This research was sponsored by the U. S. Environmental Protection Agency under Cooperative Agreement No. CR 822887-01-0, administered through the Duluth Environmental Research Laboratory; Dr. Richard Anderson, Project Officer.

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