Effect of carbon, nitrogen and phosphorus nutrition on the R, S, and M dissociants of Pseudomonas aeruginosa in mixed cultures

The effect of glucose, nitrate, and phosphate on the stationary-phase growth characteristics of R, S, and M dissociants of the hydrocarbon-oxidizing strain P. aeruginosa K-2 was studied. The optimal concentrations of glucose and phosphate providing for at least 90% of the maximal culture density were found to be 2-7% glucose and 0.02-0.12% phosphate. The main factor that determined the proportion of dissociants in bacterial populations was the initial concentration of phosphate. The fraction of R dissociant in populations increased linearly with the concentration of glucose and varied nonlinearly with the concentration of phosphate in the growth medium. The fraction of M dissociant depended solely on the concentration of phosphate in a manner inverse to that typical of R dissociant. In glucose-deficient media containing sufficient amounts of phosphorus, S dissociant prevailed over R dissociant.     

Effect of major nutrient elements on the growth and population homogeneity of the R, S and M dissociants of Pseudomonas aeruginosa and the glucose oxidation and fermentation pathways

The population homogeneity of the stationary-phase monocultures of Pseudomonas aeruginosa dissociants was studied as a function of the initial content of major nutrient elements (C, N, and P) in the cultivation medium. The monocultures of the dissociants remained homogeneous during cultivation if the initial concentrations of the major nutrient elements were either sufficiently high or, conversely, very low, but became heterogeneous during cultivation in unbalanced (with respect to the major nutrient elements) media. At the initial concentration of nitrate in the medium equal to 0.07% or phosphate equal to 0.004-0.014%, the initially homogeneous population of R dissociant cultivated to the stationary growth phase turned out to contain 30-40% of S-type cells, whereas the initially homogeneous population of S dissociant was found to contain 50-80% of M-type cells. The population of M dissociant remained homogeneous throughout the cultivation period. R dissociant grew better at sufficiently high concentrations of glucose, nitrate, and phosphate in the medium, whereas M dissociant grew better when the initial concentrations of these nutrients were low. During the cultivation of R dissociant, the pH of the medium changed insignificantly, and the C/P ratio (the ratio of the carbon and phosphorus consumed during growth) was minimal (among the three dissociants), indicating that the R dissociant accomplishes the oxidative pathway of glucose metabolism. During the cultivation of the M dissociant, the pH of the medium dropped to 3.4-3.9, and the C/P ratio was maximal, indicating that this dissociant accomplishes the fermentative pathway of glucose metabolism. During the cultivation of the S dissociant, the pH of the medium and the C/P ratio exhibited variations, indicating that this dissociant triggers its pathways of glucose metabolism.     

The Effect of Major Nutrient Elements on the Growth and Population Homogeneity of the R,S, and M Dissociants of Pseudomonas aeruginosa and the Glucose Oxidation and Fermentation Pathways

The population homogeneity of the stationary-phase monocultures of Pseudomonas aeruginosa dissociants was studied as a function of the initial content of major nutrient elements (C, N, and P) in the cultivation medium. The monocultures of the dissociants remained homogeneous during cultivation if the initial concentrations of the major nutrient elements were either sufficiently high or, conversely, very low, but became heterogeneous during cultivation in unbalanced (with respect to the major nutrient elements) media. At an initial concentration of nitrate in the medium equal to 0.07% or phosphate equal to 0.004–0.014%, the initially homogeneous population of R dissociant cultivated to the stationary growth phase turned out to contain 30–40% of S-type cells, whereas the initially homogeneous population of S dissociant was found to contain 50–80% of M-type cells. The population of M dissociant remained homogeneous throughout the cultivation period. R dissociant grew better at sufficiently high concentrations of glucose, nitrate, and phosphate in the medium, whereas M dissociant grew better when the initial concentrations of these nutrients were low. During the cultivation of R dissociant, the pH of the medium changed insignificantly, and the C/P ratio (the ratio of the carbon and phosphorus consumed during growth) was minimal (among the three dissociants), indicating that the R dissociant accomplishes the oxidative pathway of glucose metabolism. During the cultivation of the M dissociant, the pH of the medium dropped to 3.4–3.9, and the C/P ratio was maximal, indicating that this dissociant accomplishes the fermentative pathway of glucose metabolism. During the cultivation of the S dissociant, the pH of the medium and the C/P ratio exhibited variations, indicating that this dissociant triggers its pathways of glucose metabolism.     

Effect of reduced concentrations of carbon, nitrogen, and phosphorus in a medium on growth of three dissociants of Pseudomonas aeruginosa]

The effect of lowered concentrations of carbon, nitrogen, and phosphorus sources in the medium on the specific growth rate mu of the R, S, and M dissociants of the hydrocarbon-oxidizing strain Pseudomonas aeruginosa K-2, culture pH, and the population composition was studied. Within the first 16 hours of cultivation in all of the four media tested, the R, S, and M dissociants had virtually identical mu. The maximal values of mu were reached by the 20th h of growth in the basal medium (R and S dissociants) and in the carbon-deficient medium containing 0.4% glucose (M dissociant). The R and M dissociants showed the most rapid decrease in mu in the nitrogen-deficient medium containing 0.55% NaNO3. By the end of cultivation in the basal medium, the pH of the R, S, and M cultures decreased to 6.3, 5.3, and 3.3, respectively. In the case of the carbon-deficient medium, the drop in the culture pH was lower. After a 2.5-day incubation of the S dissociant in the phosphorus-deficient medium containing 0.028% NaH2PO4.2H2O and of the M dissociant in the basal medium supplemented with chalk powder, these dissociants were completely displaced from the media.     

Characteristics of carbohydrate metabolism of R-, S- i M-dissociants of Pseudomonas aeruginosa]

R and S dissociants of the hydrocarbon-oxidizing strain Pseudomonas aeruginosa K-2 differed but little in their growth in a minimal defined medium with glucose as the source of carbon and energy. At the same time, the number of cells of M dissociant in the late exponential phase was five orders of magnitude less than that of R and S dissociants. The growth of M dissociant was accompanied by the accumulation of formate in the culture liquid and a concurrent decrease in pH. All three dissociants contained the key enzymes of the Entner-Doudoroff pathway of glucose oxidation; however, the activities of these enzymes, especially 6-phosphogluconate dehydrogenase, were low in M dissociant. Conversely, the activity of formate dehydrogenase in cells of M dissociant was higher than in other dissociants. The activity of 6-phosphogluconate dehydrogenase, a key enzyme of the pentosephosphate pathway of glucose oxidation, was detected only in S dissociant. The peculiarities of the carbohydrate metabolism of M dissociant are probably responsible for its poor growth on glucose and determine the more pronounced anaerobic type of its metabolism.     

The Effect of Lowered Concentrations of Carbon,Nitrogen and Phosphorus Sources on the Growth Dynamics of the R,S, and M Dissociants of Pseudomonas aeruginosa

The effect of lowered concentrations of carbon, nitrogen, and phosphorus sources in the medium on the specific growth rate of the R, S, and M dissociants of the hydrocarbon-oxidizing strain Pseudomonas aeruginosaK-2, culture pH, and the population composition was studied. Within the first 16 hours of cultivation in all of the four media tested, the R, S, and M dissociants have virtually identical . The maximal values of were reached by the 20th h of growth in the basal medium (R and S dissociants) and in the carbon-deficient medium containing 0.4% glucose (M dissociant). The R and M dissociants showed the most rapid decrease in in the nitrogen-deficient medium containing 0.55% NaNO₃. By the end of cultivation in the basal medium, the pH of the R, S, and M cultures decreased to 6.3, 5.3, and 3.3, respectively. In the case of the carbon-deficient medium, the drop in the culture pH was lower. After a 2.5-day incubation of the S dissociant in the phosphorus- deficient medium containing 0.028% NaH₂PO₄· 2H₂O and of the M dissociant in the basal medium supplemented with chalk powder, these dissociants were completely displaced from the media.     

The Requirements of Pseudomonas aeruginosa Dissociants for Carbon,Nitrogen, and Phosphorus

Quantitative data on the nutritional requirements of microorganisms are necessary to predict the behavior of bacterial populations and to control their cultivation. The requirements of the R, S, and M dissociants of P. aeruginosa for carbon, nitrogen, and phosphorus were derived from the results of 88 cultivation experiments. For each of the dissociants, we derived a coefficient that relates the optical density and the number of cells in the dissociant culture, determined the time when the cultures entered the stationary growth phase, studied cultural changes induced by transfer to the stationary phase, and determined what nutrients limit the growth of particular dissociants. The nutritional requirements of the dissociants are discussed in relation to our earlier data.     

The requirements of Pseudomonas aeruginosa dissociants for carbon, nitrogen, and phosphorus

Quantitative data on the nutritional requirements of microorganisms are necessary to predict the behavior of bacterial populations and to control their cultivation. The requirements of the R, S, and M dissociants of Pseudomonas aeruginosa for carbon, nitrogen, and phosphorus were derived from the results of 88 cultivation experiments. For each of the dissociants, we derived a coefficient that relates the optical density and the number of cells in the dissociant culture, determined the time when the cultures entered the stationary growth phase, studied cultural changes induced by transfer to the stationary phase, and determined what nutrients limit the growth of particular dissociants. The nutritional requirements of the dissociants are discussed in relation to our earlier data.     

Development and population structure of mixed (S + M) Pseudomonas aeruginosa cultures in the late stationary growth phase

Population growth, the ratio between dissociants, pH, and levels of reducing sugars in the medium were monitored during prolonged (375 h) batch cultivation of Pseudomonas aeruginosa S and M dissociants on mineral medium with glucose. During the stationary growth phase (100-375 h), two scenarios were possible. The first one included extensive cell autolysis coupled to alkalinization of the medium and an increased ratio of the M dissociant. In the second case, acidification of the medium was coupled to the oscillating secondary growth, mostly of the M dissociant; the dynamics of cell numbers of this dissociant correlated with the dynamics of the culture optical density. In this scenario, periodical appearance of reducing sugars in the medium was detected; it was in the opposite phase with the changes of the M dissociant cell numbers. The differences between scenarios of P. aeruginosa growth in the late stationary phase were probably due to the physiological and biochemical characteristics of the S and M dissociants, including different pathways of glucose utilization (respiration or fermentation), resistance to acidification, synthetic (proteolytic) activity, and productivity of autoinducers.     

Cultivation of Pseudomonas aeruginosa dissociants under specified limitation conditions

The possibility of controlling the development of microbial communities was investigated on the basis of experimentally determined requirements for basic nutrients in R, S, and M dissociants of Pseudomonas aeruginosa. On media with the limitation conditions preset on the basis of the predictions of a mathematical model, exhaustion of glucose was experimentally confirmed for all monocultures and mixed cultures, as well as balanced consumption of glucose, nitrogen, and phosphorus by the R dissociant at the corresponding initial medium composition. The experimentally determined composition of mixed cultures was found to conform to the ones calculated using the mathematical model. The data obtained suggest the possibility of cyclic consumption of phosphorus by P. aeruginosa.     

Development and population structure of mixed (S + M) <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> cultures in the late stationary growth phase

Population growth, the ratio between dissociants, pH, and levels of reducing sugars in the medium were monitored during prolonged (375 h) batch cultivation of Pseudomonas aeruginosa S and M dissociants on mineral medium with glucose. During the stationary growth phase (100–375 h), two scenarios were possible. The first one included extensive cell autolysis coupled to alkalinization of the medium and an increased ratio of the M dissociant. In the second case, acidification of the medium was coupled to the oscillating secondary growth, mostly of the M dissociant; the dynamics of cell numbers of this dissociant correlated with the dynamics of the culture optical density. In this scenario, periodical appearance of reducing sugars in the medium was detected; it was in the opposite phase with the changes of the M dissociant cell numbers. The differences between scenarios of P. aeruginosa growth in the late stationary phase were probably due to the physiological and biochemical characteristics of the S and M dissociants, including different pathways of glucose utilization (respiration or fermentation), resistance to acidification, as well as synthetic (proteolytic) activity and productivity of autoinducers.     

Cultivation of Pseudomonas aeruginosa dissociants under specified limitation conditions

The possibility of controlling the development of microbial communities was investigated on the basis of experimentally determined requirements for basic nutrients in R, S, and M dissociants of Pseudomonas aeruginosa. On media with the limitation conditions present on the basis of the predictions of a mathematical model, exhaustion of glucose was experimentally confirmed for all monocultures and mixed cultures, as well as balanced consumption of glucose, nitrogen, and phosphorus by the R dissociant at the corresponding initial medium composition. The experimentally determined composition of mixed cultures was found to conform to the ones calculated using the mathematical model. The data obtained suggest the possibility of cyclic consumption of phosphorus by P. aeruginosa.     

Simulation of a bacterial population structure in continuous cultivation by a dissociation process: An application to Rhodococcus rubropertinctus

An attempt is made to give a quantitative estimation of a possible contribution of the dissociation process in the formation of the heterogeneous population structure for the Rhodococcus rubropertinctus (Micobacterium lacticolum) in continuous cultivation. This estimation is realised with the help of a constructed mathematical model of the process. The model equations obtained describe particular biomass changes for R-, S- and M-dissociants and changes for substrate (glucose) concentration within the chemostate in continuous cultivation. Next, contributions to dissociant biomass changes were taken into account: (1) cell reproduction (each dissociant has its own specific growth rate depending on the substrate concentration), (2) cell outflow (in proportion to dissociant biomass) and (3) dissociation (dissociant genotype change during reproduction occurs for each dissociant with its own specific frequency). It is determined that if substrate growth constants are equal the rapidly growing dissociants (possessing maximal μ_max) outs the rest of them, and the greater the speed of the flow the less the time to reach a steady-state distribution of the dissociant biomasses. But, at the same time the slowly growing dissociant can become dominant in the population if it has a lower substrate growth constant.     

Dynamics of the growth and population composition of the mixed cultures of R, S, and M dissociants of Pseudomonas aeruginosa

The population composition of polycultures of Pseudomonas aeruginosa dissociants (R + M and R + S + M) developing on media with various contents and ratios of nitrogen and phosphorus has been studied. Irrespective of its proportion (10 to 90%) in the inoculum, the R variant accounted for 65 to 84% of the whole population of linear-phase and stationary-phase binary cultures of R and M dissociants, which differ in terms of energy metabolism and nutritional requirements. After prolonged cultivation, the population in the binary culture contained only R cells (100%), which are characterized by minimum requirements with respect to the main biogenic elements. These data agree with the predictive data of model studies and can be attributed to regulation of the population composition of bacterial cultures by trophic factors. It was established that the proportion of M cells, which are distinguished by maximum nutrient requirements and enhanced stability, increased during two developmental stages of the Ps. aeruginosa polycultures (R + M and R + S + M): the lag phase and the decay stage. This result cannot be due to the influence of trophic factors and presumably results from changes in the levels of autoregulatory factors (anabiosis autoinducers) involved in stress resistance and plausibly in the adaptive interconversion of dissociants upon transfer to a new medium (during the lag phase) and under starvation conditions (at the onset of the decay phase).     

Carbon, nitrogen, and phosphorus dynamics during leaf decay in nutrient-enriched stream microecosystems

We investigated the effects of long-term enrichment with nitrate, phosphate, and nitrate+phosphate on the first 5 weeks of leaf detritus processing in laboratory stream microecosystems. Enrichment with nitrate+phosphate accelerated leaf weight loss and increased rates of respiration associated with the leaves. However, whole-system respiration was little changed from that observed in the control stream since respiration in the water was greatly reduced. Enrichment with phosphate alone had little effect except to lower respiration associated with leaf discs. Enrichment with nitrate alone also decreased leaf-disc respiration but resulted in a greatly increased rate of respiration in the water. Net leaching and fragmentation of carbon from the leaves was also increased by nitrate enrichment. Nitrogen and phosphorus levels in leaf material were little affected by enrichment with nitrate or phosphorus alone. Leaves in those streams and in the control stream released nitrogen and phosphorus to the water. In contrast, percent nitrogen and phosphorus increased greatly in the leaves in the stream enriched with both nitrate and phosphate. The leaves in this system immobilized both nitrogen and phosphorus from the water. We also studied the importance of nitrogen fixation as a vector for nitrogen incorporation associated with leaf decomposition in streams. Somewhat surprisingly, fixation by microbes associated with the leaves and by microbes suspended in the water occurred under all three experimental enrichment treatments as well as in the control, casting doubt on the effectiveness of nitrate in inhibiting nitrogenase synthesis in nature. However, N₂-fixation is only a minor source of nitrogen for leaves decaying under the conditions studied.     

Dynamics of the Growth and Population Composition of Mixed Cultures of R,S, and M Dissociants of Pseudomonas aeruginosa

The population composition of polycultures of Pseudomonas aeruginosa dissociants (R + M and R + S + M) developing on media with various contents and ratios of nitrogen and phosphorus has been studied. Irrespective of its proportion (10 to 90%) in the inoculum, the R variant accounted for 65 to 84% of the whole population of linear-phase and stationary-phase binary cultures of R and M dissociants, which differ in terms of energy metabolism and nutritional requirements. After prolonged cultivation, the population in the binary culture contained only R cells (100%), which are characterized by minimum requirements with respect to the main biogenic elements. These data agree with the predictive data of model studies and can be attributed to regulation of the population composition of bacterial cultures by trophic factors. It was established that the proportion of M cells, which are distinguished by maximum nutrient requirements and enhanced stability, increased during two developmental stages of the Ps. aeruginosa polycultures (R + M and R + S + M): the lag phase and the decay stage. This result cannot be due to the influence of trophic factors and presumably results from changes in the levels of autoregulatory factors (anabiosis autoinducers) involved in stress resistance and plausibly in the adaptive interconversion of dissociants upon transfer to a new medium (during the lag phase) and under starvation conditions (at the onset of the decay phase).__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 475–482.Original Russian Text Copyright © 2005 by Mil’ko, Khabibullin, Nikolaev, Kozlova, El’-Registan.     

Carbon,nitrogen, phosphorus,and sulphur concentration and partitioning in beech ecotypes (Fagus sylvatica L.): phosphorus most affected by drought

Beech seedlings of different drought sensitivity originating from 11 German provenances were grown in pots and cultivated in a greenhouse. The present paper aims to give insights on uptake, transport and use of macronutrients, since the knowledge of drought effects on the nutrition of trees is low compared to water relations. Therefore, the elemental composition, the ratio of inorganic to total content, and the partitioning between roots and shoots of carbon, nitrogen, phosphorus, and sulphur were investigated as affected by provenance and drought treatment. Phosphorus and phosphate concentrations decreased in all tissues after a 3 week drought treatment simulating a summer drought period. In roots carbon increased and nitrate decreased, in stems nitrogen decreased but nitrate increased following drought. The observed effects on phosphorus and phosphate are discussed in terms of lower phosphate mobility in the substrate due to lower water availability. The decrease in the ratio of phosphate to phosphorus in the tissues suggests the use of vacuolar phosphate pools for maintaining organic phosphorus homeostasis. The partitioning of all macronutrients was not affected by drought, although phosphorus and phosphate were significantly lowered in tissues. In most of the parameters studied significant differences between provenances were found. The recently observed drought sensitivity of provenances was not reflected in the strength of concentration changes or partitioning of macronutrients by drought over provenances.     

Nitrogen and Phosphorus in a Stretch of the Guadalupe River,Texas, with Five Main-Stream Impoundments by

Nitrogen and phosphorus were studied in a 168-km stretch of the Guadalupe River that had five main-stream impoundments. Flow through the study area was controlled by releases from these five reservoirs and from Canyon Reservoir, a deep-storage reservoir, located 30 km upstream. Parameters measured monthly on a diel basis at 16 stations were nitrate nitrogen, nitrite nitrogen, ammonia nitrogen, Kjeldahl nitrogen, inorganic phosphate phosphorus, organic phosphate phosphorus, and total phosphate phosphorus.Inorganic nitrogen concentrations observed in this study were as high or higher than that previously reported for other bodies of water. Nitrate nitrogen entered the study area in relatively high concentrations from Comal Springs which was a major source of water for the Guadalupe River. Water from Canyon Reservoir, the other major source of water, was relatively low in nitrate nitrogen. The concentration of nitrate nitrogen was, therefore, dependent in part upon the portion of the total river flow originating from the two sources. Increased discharge from Canyon Reservoir and utilization by plants in areas of high chlorophyll a resulted in low nitrate-nitrogen levels. Retention of water in reservoirs reduced the concentration of nitrate nitrogen due to increased utilization by plants in areas of low flow. Nitrate nitrogen, in general, reached seasonal minima in summer and maxima in winter. Nitrite nitrogen showed considerable variation with no meaningful pattern except that higher concentrations occurred in association with high chlorophyll a and high Kjeldahl nitrogen, regions and periods of low river flow, and large phytoplankton populations. There was no increase in concentration of any form of nitrogen in the vicinity of sewage outfalls and no downstream accrual.Phosphorus levels in the study area were as high or higher than those reported in studies of other bodies of water. Sewage treatment plants at New Braunfels and Seguin, Texas, were major sources of phosphorus to the Guadelupe River. Total phosphate phosphorus was determined to be the most critical phosphate parameter in assessing eutrophication. Seasonally, it ranged from a winter high to a summer low. Concentrations were highest immediately below sewage outfalls and decreased as water progressed downstream. Inorganic-phosphate-phosphorus concentrations showed no clear seasonal trend but were clearly associated with sewage outfalls. Since large standing crops of phytoplankton were observed in areas of low inorganic phosphate phosphorus, it was not considered to limit photosynthesis. Total organic phosphate phosphorus varied seasonally, with high concentrations occurring during the spring and low concentrations in the fall. Total organic phosphate phosphorus showed no correlation with sewage outfalls, but was correlated to a degree with total Kjeldahl nitrogen and chlorophyll a. No consistent pattern of diel fluctuations was evident for any phosphorus or nitrogen compounds analyzed.     

Ultrastructural features of Rhodococcus rubropertinctus and Streptococcus lactis dissociants

Three Rhodococcus rubropertinctus dissociants (R, S and M) and two Streptococcus lactis dissociants (R and S) were compared using the electron microscopy method of negative contrasting. The cell wall of R.rubropertinctus dissociants had a thickness of 40 nm (R), 30 nm (S) and 20 nm (M). The cell wall of S. lactis dissociants was 35-55 nm (R) and 25-30 nm (S) thick. 1.5-2-fold variations in the thickness of cell walls could account for the different resistance of the dissociants against the action of such external factors as dehydration, antibiotics, UV, elevated NaCl concentrations, etc.     

牡丹根际溶磷放线菌的筛选及其溶磷特性

通过从牡丹根际土壤中分离筛选溶磷放线菌,得到一株具有较强溶磷能力的菌株PSPSA1,根据形态特征、生理生化特性以及16S rDNA序列分析对菌株进行鉴定,并研究其溶磷遗传稳定性及溶磷特性.菌株PSPSA1被鉴定为白网链霉菌,具有较好的溶磷遗传稳定性.在不同磷源培养液中溶磷量依次为磷酸钙(158.5 mg·L^-1)>磷酸铝(139.9 mg·L^-1)>磷酸铁(127.7 mg·L^-1)>卵磷脂(45.6 mg·L^-1),在无机磷培养液中的溶磷量均与pH呈现显著负相关性,在有机磷培养液中的溶磷量与pH没有显著相关性.在不同碳源条件下的溶磷量依次为乳糖>葡糖糖>麦芽糖>果糖>蔗糖>淀粉>纤维素,在不同氮源条件下的溶磷量依次为蛋白胨>硝酸铵>硫酸铵>硝酸钾>尿素,以葡萄糖为碳源、蛋白胨为氮源时,菌株的溶磷量最高可达202.6 mg·L^-1.土培60 d,单施菌株土壤有效磷含量比对照增加68.2%,菌株与有机肥混施土壤有效磷含量比单施有机肥增加76.7%.表明菌株PSPSA1能够溶解多种难溶磷,在土壤中溶磷效果显著,与有机肥混施其溶磷能力明显提高,有望成为高效生物磷肥的优良菌种.