Examining the Fate of Released Pseudomonas putida F1 in Rhizosphere Environments

X. Wu, L.C. Davis and L.E. Erickson¹

Department of Biochemistry, Department of Chemical Engineering¹, Kansas State University, Manhattan, KS 66502

Abstract

A soil microcosm study was conducted to see the fate of released bacterial strain Pseudomonas putida F1 in soil. Although the P. p F1 population died off to low levels within the experimental period, the presence of alfalfa and poplar trees helped the survival of P. p F1 in soil. The P. pF1 populations were significantly higher ( p=0.05) in soil samples from the poplar tree soil microcosms than from unplanted control soil microcosms. There was no significant difference observed between soil microcosms planted with alfalfa and unplanted control. The better survival of P. p F1 in planted soil is due to the " rhizosphere effect," and therefore , is dependent on the root density in soil. This study shows the beneficial effect of vegetation on the survival of a laboratory cultured strain under conditions close to field condition.

Keywords: microcosm, bioremediation, rhizosphere, pseudomonas, toluene dioxygenase

INTRODUCTION

Bioremediation , especially plant-based bioremediation, is receiving increasing attention because compared to traditional soil and groundwater remediation techniques, it is rapid, safe, and cost-effective. Through the evapotranspiration of plants, the contaminated water is drawn up to the root zone of plants which has been shown to have a large and active microbial community which might be able to transform the contaminants to safe products of metabolism. Also due to the evapotranspiration, the water content in the soil is reduced and the groundwater table is lowered, thereby enhancing oxygen diffusion through the gas phase. This is important for the aerobic degradation of some contaminants and particularly trichloroethylene, because some products of the anaerobic degradation of TCE are even more toxic than TCE. Furthermore, plant root zone, and more precisely, the rhizosphere, is a unique environment for the microflora compared to bulk soil because of the release of organic matter from plant roots. Microbial growth is stimulated in the rhizosphere and, due to plant-microbe interaction, the relative abundance of different microbial species could be different in the rhizosphere compared to bulk soil.

On the other hand, laboratory cultured microorganisms which are capable of carrying out desired degradation reactions are often released into the contaminated site to enhance the natural degradation process. However, this kind of practice has experienced many failures. A key factor involved is that the laboratory cultured microorganisms often die off quickly under field conditions due to predation by soil fauna, competition with indigenous microbes, and other factors. How to maintain sufficient activity of an introduced population over a prolonged period after release is of great concern.

The purpose of this research was to study the effect of vegetation on the fate of a released laboratory cultured strain. Pseudomonas putida F1 was used as the target strain. This strain is able to grow on toluene and degrade TCE through cometabolism. It carries a toluene dioxygenase responsible for the degradation of TCE and the first reaction of toluene oxidation (Zylstra, et al., 1988). Whether or not this strain can colonize and maintain in the rhizosphere, and the possible effect of different plant species, are also of concern.

Alfalfa and poplar trees were chosen in this study. Alfalfa is a legume which can grow under relatively poor nutrient conditions. Poplar trees are popular trees used in plant-based bioremediation due to their rapid growth, deep rooting , and easy propagation (Jordall, et al., 1996).

MATERIALS AND METHODS

Bacterial strain and growth medium

Bacterial strain Pseudomonas putida F1 ( P.p F1 ) used for all studies was a gift from Dr. Nancy Duteau, Iowa State University. The microorganism was stored in LB broth with 50% glycerol at -70°C. The inoculum for introduction to soil was prepared by adding a loopful of P. p F1 to 3 ml minimal salts medium supplied with 0.5% glucose. The culture was grown overnight and a small amount of the culture was then transferred to 200 ml of the minimal salts medium with 0.5% glucose, and grown overnight at 37°C. Prior to inoculation, the culture was centrifuged at 4°C and 5,000´g for 20 min to pellet the cells. The cell pellets were then resuspended in 0.85% NaCl solution to the desired density. The ingredients of the minimal salts medium ( 1L ) are 6.9 g Na₂HPO₄×12 H₂O, 2.4 g KH₂PO₄, 0.5g ( NH₄)SO₄, 0.2g MgSO₄× 7H₂O, 30 mg yeast extract, 5 ml trace element solution formulated as 530 mg CaCl_2, 200 mg FeSO₄× 7H₂O, 10 mg ZnSO₄× 7H₂O, 10 mg H₃BO₃ , 10 mg CoCl₂×6 H₂O, 4 mg MnSO₄ ×5H₂O, 3 mg Na₂MoO₄×2H₂O, and 2 mg NiCl₂× 6H₂O per liter.

Soil

Soil used in all studies was collected from an area adjacent to the Riley County landfill near Manhattan, Kansas. No microorganism able to convert indole to indigo and form a dark blue color was found in this soil; therefore, the background of P. p F1 in this soil was regarded as blank .

Construction of soil microcosms

Two types of soil microcosms were constructed. In Type I microcosms, four groups of alfalfas were planted and formed a square with a side length of 4cm in soil columns 16 cm diameter and 46 cm high . Type II microcosms, about 10 cm diameter and 20 cm tall, each contained 2500g air dried soil. Each one was planted with one poplar tree in the middle of the container. P. p F1 cell suspensions with density of 5.6´10⁷ CFU/ g dry soil were inoculated to each soil microcosm. The studies on the two types of microcosms were conducted at different times. The inoculated soil microcosms were grown at 25±2°C with continuing fluorescent lighting. Soil moisture was maintained close to field capacity. Control soil microcosms without plants were inoculated with P.p F1 cell suspensions of the same density and the growth condition was maintained the same as the planted soil microcosms.

Sampling procedures and enumeration methods

The planted bulk soil samples were taken about 2 cm away from the plant stems almost every week using a brass coring apparatus with a diameter of 0.9 cm and a length of 9 cm. The unplanted bulk soil samples were taken randomly from the control microcosms. Each sample contained about 8 g ( dry wt ) soil. Rhizosphere soil samples consisted of the plant roots as well as the soil closely associated with the roots. Each rhizosphere soil sample was about 0.1-0.5 g (dry wt ).

The plate counting method was used to analysis the remaining P. p F1 population in the soil samples after release. After removal from microcosms, soil samples were put in 45 ml sterile 0.85% NaCl solution immediately and shaken with 15-20 glass beads, with a diameter of 4mm, on a shaker at 50 RPM for 30 min to release microorganisms from soil particles. The resulting soil slurry was then subject to serial dilution using 0.85% NaCl solution and spread out on P. p F1 culture medium (Wollum, 1982). The detection limit was 100 CFU/g dry soil.

Two kinds of media were used to culture P. p F1. A selective medium contains solid minimal salts medium supplied with 0.5mM toluene vapor in the gas phase as the sole carbon source. This concentration of toluene was tested to be the optimum concentration for P.p F1 pure culture. After one and half days of incubation at 25°C, the colonies that appeared on the plates with a similar morphology as P. p F1 were counted as P. p F1. The identity of these colonies was occasionally confirmed with random primer PCR (Wu and Davis, 1996). Otherwise, LB medium added with 1mM indole was used. After incubation at 37°C for one day, followed by incubation at 25°C for another day, colonies with dark blue color were counted as P.p F1. The identity of these colonies was also confirmed by random primered PCR .

The P. p F1 population measured from planted-soil microcosms was compared to those from control soil samples. SAS program was used to determine whether differences were statistically significant.

RESULTS AND DISCUSSION

The presence of alfalfa, and presumably the presence of alfalfa roots, helped the survival of P. p F1 compared to unplanted soil, although the difference between the two treatments is not statistically significant at 5% level ( Fig. 1). The explanation of this observation is " rhizosphere effect"-- the stimulation of microbial growth in the adjacent soil affected by roots. The rhizosphere effect is characterized as a phenomenon in which the indigenous microbial growth is stimulated due to the effect of plant roots (Foster, 1986). Notably, the rhizosphere effect helped to maintain the introduced microorganisms in the rhizosphere. On day 79, when the P. p F1 populations were below detection limit ( 100 CFU/ g dry soil) in both planted and unplanted bulk soil samples, there was still a considerable amount of P. p F1 in the plant rhizosphere sample ( 6300 ± 300 CFU/ g dry soil ), which shows that the laboratory-cultured strain colonized and remained active in the rhizosphere. In a study concerning the fate of a genetically altered Pseudomonas sp. (Bolton, 1991), the laboratory-cultured organisms introduced to the rhizosphere competed favorably with the native soil microflora, and the extent to which they colonized and survived was dependent on the stage of plant growth (Bolton, 1991; 1991b).

According to model prediction and experimental data , the rhizosphere effect only exists within a mm range from the root due to the limit of materials released from plants through diffusion (Bowen, 1980; Newman and Watson, 1977). Therefore, soil samples that contained more roots would be expected to have a higher P. p F1 population. The P.p F1 populations in soil samples taken right beside alfalfa were 9.2´10³± 1.8´10³ CFU/ g dry soil , compared to 3.3´10³± 1.2´10³ CFU/ g dry soil, P. p F1 populations in soil samples taken 2 cm away from alfalfa. The difference between P. p F1 populations was statistically significant at 5% level.

The presence of poplar trees also helped the survival of P. p F1 in soil ( Fig. 2 ), and the difference of the two treatments is statistically significant at the 5% level. Although the alfalfa and poplar soil microcosm studies were conducted at different times and therefore are incomparable in some sense, the higher root density in the poplar soil microcosms than in the alfalfa soil microcosms could be the reason why a significant difference was observed between the poplar soil microcosm and unplanted control; but there is no significant difference in the case of alfalfa soil microcosm and unplanted control. Furthermore, the bigger the plants are, the better the P.p F1 survived. In soil microcosms planted with small poplar cuttings, P.p F1 cells died off as quickly as they did in unplanted soil, probably because the root amount of cuttings was too small to have rhizosphere effect on the soil taken 2cm away from the plant stem ( data not shown). Interestingly, the presence of a large, dead poplar tree also helped the survival of P. p F1 to some extent ( Fig.3 ). An unplanted microcosm with subsoil which contains less organic matter than the top soil used in most experiments was also inoculated with the same amount of P. p F1 and treated under the same conditions. Interestingly, the P. p F1 population in this microcosm sustained the longest time, even longer than in the planted microcosm. The total heterotroph population observed was ten times less than that in the top soil microcosms ( Table 1 ), which reveals the important effect of competition between indigenous microbial community and introduced strain on the survival of the introduced strain.

In both microcosm studies, a three-stage decline of culturable P. p F1 population was observed. The first two weeks after inoculation were the fastest dropping stage, followed by a relative stable stage in the next three weeks; the last stage is a fast decline stage which leads the P. p F1 population as undetectable using the previously described plate counting method.

In conclusion, vegetation benefits the survival and activity maintainance of a laboratory-cultured bacterial strain P. p F1. This is due to the rhizosphere effect of plant roots. Considerable amounts of P. p F1 were observed colonizing and remaining active in the rhizosphere.

REFERENCE

Bolton, H., Jr., J.K. Fredrickson, S.A. Bentjen, D.J. Workman, S.W. Li, and J.M. Thomas. 1991a. Field calibration of soil-core microcosms: Fate of a genetic-altered rhizobacterium. Microb. Ecol. 21: 163-173.

Bolton, H, Jr., J.K. Fredrickson, J.M. Thomas, S.W. Li, D.J. Workman, S.A. Bentjen, and J.L. Smith. 1991b. Field calibration of soil-core microcosms: Ecosystem structural and functional comparisons. Microb. Ecol. 21: 175-189

Bowen, G.D. 1980. Misconceptions, concepts, and approaches in rhizosphere biology. In Contemporary Microbial Ecology, D.C. Ellwood, J.W. Hedger, M.J. Latham, J.M. Lynch, and J.H. Slater. Academic Press.

Foster, R.C. 1986. The ultrastructure of the rhizoplane and rhizosphere. Annu. Rev. Phytopathol. 24: 211-234.

Jordall, J.L., L. Foster, J.L. Schnoor, and P.J.J. Alvarez. 1996. Effect of hybrid poplar trees on microbial populations important to hazardous waste remediation. Environmental Toxicology and Chemistry, 16: 1318-1321.

Newman, E.I. and A. Watson. 1977. Microbial abundance in the rhizosphere. A computer model. Plant and soil. 48: 17-56.

Wollum II, A.G. 1982. Cultural methods for soil microorganisms. In Methods of Soil Analysis, Part 2. Chemical and Microbiological Properties- Agronomy Monograph no. 9 (2nd Edition).

Wu, X. and L.C. Davis. 1996. Detection of specific microorganisms using the arbitrarily primed PCR in bacterial communities of vegetated soil. In Proceedings of the Twenty-Six Annual Biochemical Engineering Symposium, L.E. Erickson, Department of Chemical Engineering, Kansas State University.

Zylstra, G.J. and Gibson, D.T. 1988. Toluene degradation by Pseudomonas putida F1. Journal of Biological Chemistry. 264: 14940-14946.

Table 1. P. p F1 and total heterotroph populations on day 68 in unplanted top soil, plant top soil, and unplanted sub soil microcosms.

	P. p F1 population on day 68	Total heterotrophs on day 68
unplanted top soil	< 100 CFU/ g dry soil	1.7´104
planted top soil	< 100 CFU/ g dry soil	1.6´105
unplanted sub soil	490 CFU/ g dry soil	9.3´104

Figure 1. P. p F1 populations in soil microcosms planted with alfalfa and unplanted microcosms after inoculation. Two planted and two unplanted bulk soil samples were taken at each time. Solid and broken lines show the log of the mean value.

Figure 2. The P. p F1 populations in soil microcosms planted with poplar and unplanted soil microcosms after inoculation. One planted bulk soil sample was taken from each of two or four planted soil microcosms, and one unplanted bulk sample was taken from each of two unplanted soil microcosms at each time. The lines show the log of the mean value.

Figure 3. The P. p F1 populations in soil microcosm with a dead poplar tree and unplanted soil microcosms. One soil sample was taken from the soil microcosm with a dead plant. The P. p F1 population of unplanted bulk soil was the average of two samples taken from two unplanted soil microcosms.