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ISSN 0096-3925, Moscow University Biological Sciences Bulletin, 2008, Vol. 63, No. 3, pp. 113117. © Allerton Press, Inc., 2008. Original Russian Text © P.V. Fursova, E.S. Mil'ko, A.P. Levich, 2008, published in Vestnik Moskovskogo Universiteta. Biologiya, 2008, No. 3, pp. 2529.

Carbon and Nitrogen As Resources Limiting the Growth of Mono- and Mixed Cultures of Pseudomonas aeruginosa Dissociants
P. V. Fursovaa, E. S. Mil'kob, and A. P. Levichc
c

Department of Biophysics Department of Microbiology Department of General Ecology, Moscow State University, Moscow, 119991 Russia e-mail: fursova@biophys.msu.ru
b

a

Received December 26, 2006

Abstract--New experiments for detection of resources limiting the growth of mono- and mixed cultures of Pseudomonas aeruginosa dissociants were carried out. The results were analyzed on the basis of the consumption and growth variational model in accordance with the data on the dissociant metabolism special traits. In 83% of cases, the theoretical calculation was confirmed by the experimental results. DOI: 10.3103/S0096392508030048

The work is the continuation of the study aimed at detection of resources limiting the growth of Pseudomonas aeruginosa dissociant cultures. The previous publication (Fursova et al., 2004a) showed the theoretical possibilities of predicting limiting substances on the basis of the consumption and growth variational model for biocenoses (Levich, 1980; Levich et al., 1997; Levich, 2000). Experiments with additives aimed at detection of limiting resources confirmed the model calculations in most cases. However, new data on the special traits of the metabolism of P. aeruginosa dissociants made it necessary to reconsider the obtained results. MATERIAL AND METHODS Mono- and mixed cultures of R, S, and M dissociants of bacterial strain Pseudomonas aeruginosa K-2 were cultivated, without replenishment of resources, on media with various initial concentrations of glucose,

nitrates, and phosphates. The total number of experiments neared 200. The bacteria were cultivated in 50-ml tubes on 10 ml of the media on a shaker (180 rpm) at a temperature of 28°C until they reached the stationary growth phase (Tables 13). One-day cultures of pseudomonade dissociants cultivated on solid medium containing meatpeptone broth and wort in the ratio of 1 : 1 (BSA) were used for inoculation. The bacteria from agar slant were transferred with a loop to a tube with physiological solution. The inoculum density of each dissociant in all experiments was adjusted with the help of a nephelometer or according to opacity standards until the cell concentration was 109 (for mixed cultures, 107) in 1 ml. The inoculum was administered at a rate of 3% of the total volume. The bacterial growth was estimated on the basis of culture density using the nephelometer. The dissociant correlation in a population was determined by sifting on BSA on the basis of colony morphology.

Table 1. Initial concentration of carbon, nitrogen, and phosphorus in media for monocultures (mg/ml) (Fursova et al., 2004a) Medium number 1 2 3 4 5 6 R dissociant carbon 0.78 3.18 3.18 0.282 1.6 1.6 nitrogen 0.4 0.1 0.4 0.1 0.03 0.1 phosphorus 0.028 0.028 0.007 0.008 0.008 0.002 113 carbon 0.78 3.18 3.18 0.282 0.78 0.78 S and M dissociants nitrogen 0.4 0.1 0.4 0.1 0.03 0.1 phosphorus 0.028 0.028 0.007 0.008 0.008 0.002

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FURSOVA et al. Table 3. Initial concentration of carbon, nitrogen, and phosphorus in media for mono- and mixed cultures under specified limitation conditions (mg/ml) (Fursova et al., 2007) Medium number 1 2 3 4 5 6 7 Carbon 0.9 0.76 2.4 1.6 1.6 4.8 0.76 Nitrogen 0.05 0.165 0.04 0.04 0.08 0.25 0.04 Phosphorus 0.008 0.02 0.02 0.006 0.006 0.006 0.006

Table 2. Initial concentration of carbon, nitrogen, and phosphorus in media for mono- and mixed cultures (mg/ml) (Fursova et al., 2004a) Medium number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Carbon 0.4 1.62 0.4 1.6 0.12 0.48 0.12 0.48 0.78 3.24 1.6 6 1.2 4.8 Nitrogen 0.035 0.14 0.015 0.06 0.035 0.14 0.015 0.06 0.1 0.4 0.2 0.8 0.035 0.14 Phosphorus 0.01 0.04 0.01 0.04 0.01 0.04 0.01 0.04 0.01 0.04 0.01 0.04 0.01 0.04

content (the range of determined concentrations is 10 2550100500 mg/l). The technique of the experiments with additives aimed at detection of limiting resources was improved. At the estimated time of entering the stationary phase, the culture was divided into seven tubes. Six of them were filled with additives (glucose, nitrate or phosphate, their paired combinations, and all three substances) and one was left unchanged (control). The amount of the additives was equal to their initial concentration in the medium. The culture was left for 12 more hours to grow, and then the optical density was measured. If the added nutritional component caused the renewal of cell division, the resource (or combination of resources) was considered to be limiting. If administering of substances did not lead to culture growth, it was assumed that the respective factors did not limit the community development. As compared to the previously conducted experiments (Fursova et al., 2004a), the time of culture exposure to the medium with additives was increased, thus excluding the lag phase effect. Moreover, using combinations of resources, as opposed to addition of separate nutritional components, enabled joint limitaCarbon, mg/ml 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2

For pH measurements of the medium, a Checker micropotentiometer (HANNA Instruments) was used. Glucose concentration was determined with the help of triphenyltetrazolium chloride (Chemistry of Carbohydrates, 1967), nitrogen was determined with sulfophenol reagent (Polyakov, 1950), and phosphorus was determined by the Panush method (Practical Course of Biochemistry, 1979). In subsequent experiments, express methods of substance determination were used: Diagluc test strips for semiquantitative determination of glucose in blood (the range of determined concentrations is 0.01000 mg% (0.055.5 mM)); Merck photometric phosphate test (concentration determination is possible within the range from 0.010 to 5.00 mg/l); Merck analytic test strips for determination of nitrate
L1

III I II L2
Fig. 1. Spatial stratification of two consumed resources. In Area I, both factors are limiting; in Area II, L1; in Area III, L2.

0

0.02

0.04

0.06

0.08

0.10 0.12 Nitrogen, mg/ml

Fig. 2. Spatial stratification of two resources for the mixture of R, S, and M dissociants of P. aeruginosa. Vol. 63 No. 3 2008

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tion of bacterial growth by several substances to be fixed. According to the results of studying the consumption and growth mathematical model (Levich et al., 1904; Fursova, 2003), the space of initial resource stocks falls into areas where one of the possible combinations of consumed substances (including each individual substance) is limiting. The boundaries of these areas are calculated on the basis of the experimentally determined requirements of organisms in environmental resources (Fursova et al., 2004b). RESULTS AND DISCUSSION In the cited work of Fursova et al. (2004a), the analysis of the experimental results allowed one to suppose that the main resources that can limit the growth of mono- and mixed cultures of P. aeruginosa dissociants are carbon, nitrogen, and phosphorus. It was shown that the resource vectors defining the experimental media at the given stage of research (Tables 1, 2) belong to strata with single-factor limitation. Limitation of the culture growth by the model-predicted substance was confirmed in 75% of the analyzed experiments. Next, a series of experiments with preset limitation conditions was conducted. Seven media (Table 3) were chosen so that they would be limited by various resources: a balanced medium (no. 1) where the concentration of each resource on average is proportional to the respective requirements of all dissociants (three monocultures were cultivated on this medium); a medium limited by carbon for all possible combinations of mono- and mixed cultures (no. 2); a medium limited by nitrogen for monocultures (no. 3) and mixed cultures (no. 4); a medium limited by phosphorus for all possible combinations of mono- and mixed cultures (no. 5); for monocultures and a mixture of three dissociants (no. 6). The composition of medium no. 7 was balanced for the monoculture of R dissociant (the concentration of resources was proportional to the requirements of the dissociant) and limited by phosphorus for mixed cultures and S and M dissociant monocultures. The analysis of the data obtained in the above-mentioned series of experiments resulted in the assumption that phosphorus is repeatedly consumed by P. aeruginosa dissociants (Fursova et al., 2007). If we accept the hypothesis about cyclic consumption of phosphorus during culture growth, it is necessary to reconsider the results of the experimental analysis. Let us assume that the growth of P. aeruginosa dissociants can actually be limited only by carbon and nitrogen, and all other substances necessary for their growth, including phosphorus, are present in sufficient amount in the media. In this case, the boundaries of limitation areas are defined by two rays coming from the center of coordinates and positive semiaxes of the Cartesian plane (Fig. 1) (Levich et al., 1994). These rays coincide for monocultures. Stratification for the

Table 4. Reaction to additives in monocultures (%) (Fursova and Levich, 2007) (see explanations in the text) ModelExperipredicted Additive ment limiting number resource 1 2 3 4 5 6 7 8 9 10 11 12 13 14 C N C C C N C N C C C C C C C N C N C N C N C N C N C N C N C N C N C N C N C N C N CN C1 N1 CN1 C N CN C1 N1 CN1 C N CN C1 N1 CN1 C N CN C1 N1 CN1
2008

R 121 91

S 220 102

M 110 96 101 131 117 119 217 74

220 97

140 80 227 73

93 93 94 95 95 143

62 100 100 112 122 100

212 162 129 86 131 76

129 100 101 68

246 103

171 76

100 68 90 100 160 90 97 66 143 86 119 81 112 91 184 92

15

C

16

CN

17

CN

195 213 104 204 223 100 223 105 105 86 104 82 168 135 91 214 135 100 207

140 213 100 178 209 120 219

163 222 110 208 212 120 194

149 102 202 153 104 196

178 104 216 158 92 218

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Table 5. Additives in mixed cultures (%) (Fursova and Levich, 2007) (see explanations in the text) ModelExperipredicted Additive ment limiting number resource 1 2 3 4 5 6 7 8 C N C C C C C C C N C N C N C N C N C N C N C N CN C1 N1 CN1 C N CN C1 N1 CN1 C N CN C1 N1 CN1 C N CN C1 N1 CN1 C N CN C1 N1 CN1 RS RM 82 59 96 104 160 100 SM 89 84 104 116 160 90 121 79 RSM 87 98 96 128 150 71

92 104 142 75

120 100 103 81 103 88 220 98 202 204 110 212

9

C

120 93 143 102 109 81 200 94 198 227 114 227 233 102 246 200 173

113 75

194 98 214 276 94 270

129 91 254 114 237 254 116 237

mixed culture of three P. aeruginosa dissociants is shown in Fig. 2 (for double mixtures, the stratification patterns are the same). According to such division of the resource space, the limiting factor remained the same for all media that were limited by carbon or nitrogen (media nos. 1, 2, 4, 5 in Table 1; all media except nos. 11 and 12 in Table 2; media nos. 14 in Table 3). Media nos. 3 and 6 from Table 1 and nos. 11 and 12 from Table 2, initially limited by phosphorus, became limited by carbon according to the new conditions. Cultivation conditions on media nos. 57 from Table 3 set combined limitation by carbon and nitrogen. The results of the experiments with additives are summarized in Tables 4 and 5. The optical density of the culture without additives was taken to be 100% (the presence of phosphorus in the added resources is marked by 1). The characteristics pointing to the growth renewal as a result of introduction of the modelpredicted limiting nutrient are given in bold. Thus, in 83% of cases, the theoretical and experimental data coincide. It should be noted that the corrections to the experimental procedure (see Materials and Methods) made on the basis of the work by Fursova et al. (2004a) allowed the number of experiments to be increased substantially, the results of which are suitable for analysis in terms of a variational model. Account of the metabolic peculiarities of P. aeruginosa dissociants in the model approach and improvement of the experimental techniques made it possible to describe the experimental results more precisely. The previous model predictions were confirmed in 75% of cases. ACKNOWLEDGMENTS

10

C

11

CN

12

CN

102 116 116 111 96 113 100 90 104 90 92 170

99 81 157 104 68 157 108 92 125 98 92 163

77 87 107 103 91 125 116 110 127 112 102 163

222 100 222 256 113 220 126 93 137 86 89 200 142 102 205 142 98 193

The work was supported by the Russian Foundation for Basic Research (grant nos. 05-04-49238 and 05-0680062). REFERENCES
Fursova, P.V., Computation Algorithms for Biocenosis Variational Model, Mat. Modelirovaniye, 2003, vol. 15, no. 5, pp. 115128. Fursova, P.V. and Levich, A.P., Quantitative Regularities of Development of Pseudomonas Aeruginosa Dissociant Community, Izv. Akad. Nauk, 2007, Ser.: Biol., no. 1, pp. 110. Fursova, P.V., Mil'ko, E.S., Il'inykh, I.A., and Levich, A.P., Detection of Nutrition Components Limiting the Growth of Mono- and Mixed Cultures of Pseudomonas Aeruginosa Dissociants, Vestn. Mosk. Univ., 2004a, Ser.: Biol., no. 1, pp. 1923. Fursova, P.V., Mil'ko, E.S., Il'inykh, I.A., Maksimov, V.N., and Levich, A.P., Determination of Requirements of Pseudomonas Aeruginosa Dissociants in CarVol. 63 No. 3 2008

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CARBON AND NITROGEN AS RESOURCES bon, Nitrogen and Phosphorus, Mikrobiologiya, 2004b, vol. 73, no. 1, pp. 4550. Fursova, P.V., Mil'ko, E.S., and Levich, A.P., Cultivation of Pseudomonas aeruginosa Dissociants under Specified Limitation Conditions, Mikrobiologiya, 2008, vol. 77, no. 2, pp. 226231. Khimiya uglevodov (Chemistry of Carbohydrates), Kochetkov N.K. et al., Ed., Moscow, 1967 [in Russian]. Levich, A.P., Struktura biologicheskikh soobshchestv (Structure of Biocenoses), Moscow: 1980 [in Russian]. Levich, A.P., Variational Modeling Theorems and Algocenoses Functioning Principles, Ecological Modeling, 2000, vol. 131, pp. 207227. Levich, A.P., Alekseev, V.L., and Nikulin, V.A., Mathematical Aspects of Variational Modeling in Community

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Ecology, Mat. Modelirovaniye, 1994, vol. 6, no. 5, pp. 5576. Levich, A.P., Maksimov, V.N., and Bulgakov, N.G., Teoreticheskaya i eksperimentalnaya ekologiya fitoplanktona. Upravleniye strukturoi i funktsiyami soobshchestv (Theoretical and Experimental Phytoplankton Ecology. Management of Community Structure and Functions), Moscow, 1997. Malyi praktikum po biokhimii (Practical Course of Biochemistry), Yurkevich, V.V., Ed., Moscow, 1979 [in Russian]. Polyakov, G., Posobiye po gidrokhimii dlya rybovodov (Hydrochemistry Textbook for Fish Breeders), Moscow, 1950 [in Russian].

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