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.     

Carbon and nitrogen as resources limiting the growth of mono- and mixed cultures of Pseudomonas aeruginosa dissociants

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.     

Quantitative patterns of development of community of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> dissociants

The stationary phase of batch culture of Pseudomonas aeruginosa dissociants has been described by a variational model of consumption and growth. The generalized entropy functional was used as the objective function. The model parameters include the requirements of the dissociants for the main nutrients: carbon, nitrogen, and phosphorus. The variational model was used to calculate the limiting regions and microbial community composition during stationary growth for different initial combinations of the resources as a function of the limiting resources. A correspondence between the experimental data and model calculations has been demonstrated. A possibility to control the community structure is discussed.     

Growth characteristics and growth modeling of <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> and <Emphasis Type="Italic">Planktothrix agardhii</Emphasis> under iron limitation

Although iron is a key nutrient for algal growth just as are nitrogen and phosphorus in aquatic systems, the effects of iron on algal growth are not well understood. The growth characteristics of two species of cyanobacteria, Microcystis aeruginosa and Planktothrix agardhii, in iron-limited continuous cultures were investigated. The relationships between dissolved iron concentration, cell quota of iron, and population growth rate were determined applying two equations, Monod’s and Droop’s equations. Both species produced hydroxamate-type siderophores, but neither species produced catechol-type siderophores. The cell quota of nitrogen for both M. aeruginosa and P. agardhii decreased with decreasing cell quota of iron. The cell quota of phosphorus for M. aeruginosa decreased with decreasing cell quota of iron, whereas those for P. agardhii did not decrease. Iron uptake rate was measured in ironlimited batch cultures under different degrees of iron starvation. The results of the iron uptake experiments suggest that iron uptake rates are independent of the cell quota of iron for M. aeruginosa and highly dependent on the cell quota for P. agardhii. A kinetic model under iron limitation was developed based on the growth characteristics determined in our study, and this model predicted accurately the algal population growth and iron consumption. The model simulation suggested that M. aeruginosa is a superior competitor under iron limitation. The differences in growth characteristics between the species would be important determinants of the dominance of these algal species.     

Quantitative studies on phosphorus transference occuring between <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> and its attached bacterium (<Emphasis Type="Italic">Pseudomonas</Emphasis> sp.)

Phosphorus release from Microcystis aeruginosa and attached bacterium (Pseudomonas sp.) isolated from Lake Taihu was examined using a phosphorus isotope tracer in order to investigate the phosphorus transference between the two species. Our results reveal that the amount of phosphorus released form ³²P-saturated M. aeruginosa is determined by its growth phase and most of phosphorus is assimilated by Pseudomonas finally while the amount of phosphorus released from ³²P-saturated Pseudomonas is also determined by the growth phase of M. aeruginosa and most of them are assimilated by M. aeruginosa. The results suggest that phosphorus transference occurs between M. aeruginosa and its attached Pseudomonas . This process makes microenvironment of mucilage of M. aeruginosa attached bacteria maintain relative high amounts of phosphorus. Attached bacteria may be a temporary phosphorus bank to the growth of M. aeruginosa, and assimilation of phosphorus by M. aeruginosa becomes easy when M. aeruginosa is in lag growth phase. Thus, the phosphorus exchange between M. aeruginosa and attached Pseudomonas in microenvironment may be important to microfood web and cyanobacteria bloom.     

The process of bacterial population splitting into dissociants and long-term batch cultivation of bacteria

The growth and composition of a population were studied during long-term (up to 50 days) batch cultivation of mono and mixed cultures of Pseudomonas aeruginosa S- and M-dissociants and Rhodobacter sphaeroides R- and M-dissociants without the addition of nutrients. During the cultivation of P. aeruginosa on a glucose-containing mineral medium, periodic lysis followed by polyculture growth resumption in the late stationary phase occurred on account of the M-dissociant: the change in its cell number corresponded to the change in the total cell number of the association. It was shown that the periodic occurrence of reducing sugars in the medium preceded the resumption of polyculture growth. Periodic secondary growth of the mixed culture of R. sphaeroides photosynthesizing bacteria occurred because of fast growing R-cells after the lysis of some part of the R-dissociant population. In the monoculture of the R. sphaeroides M-dissociant, R-cells were found during the whole period of cultivation, making up to 1–10% of the population irrespective of its size, which probably corresponded to the frequency of occurrence of this dissociant. In the R-dissociant monoculture, M-cells were found only after 26 days, and their number gradually decreased to half of population by the end of cultivation period. The joint growth of dissociants was characterized by the biomass increment and bacterial growth acceleration compared to monocultures, which is important for the fast development of new habitats under natural conditions. The cells of both bacterial species were lysed during long-term cultivation by exoproteinases secreted by the thin-wall cells of M-dissociants.     

Dynamics of water-extractable phosphorus during the degradation of Microcystis aeruginosa by four bacteria species

We carried out a laboratory experiment for studying the dynamics of water-extractable phosphorus release during the decomposition of dead Microcystis aeruginosa driven by the four bacteria strains. Our results showed that water-extractable phosphorus had significant correlation to alkaline phosphatase but without obvious correlation to acid phosphatase, suggesting that alkaline phosphatase was the primary enzyme decomposing organic phosphorus. Also, water-extractable phosphorus exhibited an obvious correlation to pH, indicating that the alkaline environment contributed to the release of water-extractable phosphorus. The quantity of bacteria hardly correlated to the release of water-extractable phosphorus for all species. This could result from decrease of bacterial quantity after consumption of substrate in closed system. Obvious variation in water-extractable phosphorus was observed across the strains in two water treatments. Sporolactobacillus sp. and Bacillus macerans had the strongest ability for decomposing organic phosphorus among five strain treatments in the lake water. In the distilled water, Bacillus sp. had the strongest ability to degrade organic phosphorus. In both water treatments, mixed species exhibited intermediate ability for decomposing organic phosphorus. It was true that water quality strongly affected the release of phosphorus by regulating phosphatase activity.     

Kinetic Study and Mathematical Modeling of Chromium(VI) Reduction and Microorganism Growth Under Mixed Culture

The kinetics of chromium(VI) reduction by Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli) was studied under both pure and mixed cultures. Initially, the study of kinetics was performed in pure culture. It was observed that the growth of the two bacteria was both inhibited in the presence of chromium(VI). The maximum specific growth rate (μ _m ) of P. aeruginosa decreased from 2.3942 h^?1 (without Cr(VI)) to 1.8551 h^?1 (with Cr(VI)). Under the mixed culture, the growth of E. coli was inhibited by P. aeruginosa. The maximum specific growth rate (μ _m ) of E. coli decreased form 0.871 h^?1 (in pure culture) to 0.153 h^?1 (in mixed culture). When the concentration of each bacterium was 4.5 × 10⁸ cells ml^?1, the half-velocity reduction rate constant (K _C) and the maximum specific reduction rate constant (v _max) of chromium(VI) were 80.05 mg chromium(VI) l^?1 and 3.674 mg chromium(VI) cells^?1 h^?1, respectively. The results showed that the simulation appeared in good agreement with the experimental data, supporting the series of mathematical models represented the bacteria growth and chromium(VI) reduction in both pure and mixed cultures usefully.     

Changes in the antibiotic production by co-culture of Rhizopus peka P8 and Bacillus subtilis IFO3335

The co-culture of Bacillus subtilis IFO 3335 with Rhizopus peka P8 or Rhizopus oligosporus P12 in liquid medium was found to increase production of antibiotic activity and to alter the spectrum of activity relative to the pure cultures. However, a mixed culture of Rhizopus arrhizus P7 and Rhizopus oryzae P17 did not produce antibiotic activity. The concentration, ratio, and time of addition of B. subtilis to the R. peka culture was found to influence antibiotic yields. Solid-state fermentations using mixed cultures of R. peka and B. subtilis were investigated. The growth of Escherichia coli IFO 3792 as a target bacterium was inhibited by the mixed culture. These results suggest the possibility of biopreservation of fermented foods by novel co-culture systems.     

SCP production by mixed culture of Rhodocyclus gelatinosus and Rhodobacter sphaeroides from Cassava Waste

The amylolytic enzymes produced by Rhodocyclus gelatinosus hydrolyzed cassava starch mainly to maltose and a small amount of glucose. The organism utilized maltose at the specific growth rate of 0.15 l/h, but in the presence of glucose, maltose consumption rate was retarded. Therefore, a series of mixed cultures was conducted with Rhodobacter sphaeroides P47, which showed a high growth rate of 0.2 l/h on glucose and contained 29.5 μg/g cell of vitamin B12 and 0.49 mg/g cell of carotenoid compared with the 18.4 μg/g cell and 0.23 mg/g cell respectively of Rc. gelatinosus. Mixed cultures with three different inoculum ratios of the two organisms based on cell number all gave higher growth yields and contents of vitamin B12 and carotenoid in the total cell mass than single cultures. When the inoculum ration of Rc. gelatinosus to Rb. sphaeroides P47 was over 1.0, the culture time was shortened due to the synergistic effect of sugar consumption. Therefore, it was suggested that the mixed culture of these two organisms would be practically profitable for more nutritive SCP production from cassava waste.     

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.     

Production of lactic acid from date juice extract with free cells of single and mixed cultures of Lactobacillus casei and Lactococcus lactis

The production of lactic acid from date juice by single and mixed cultures of Lactobacillus casei and Lactococcus lactis was investigated. In the present conditions, the highest concentration of lactic acid (60.3 g l⁻¹) was obtained in the mixed culture system while in single culture fermentations of Lactobacillus casei or Lactococcus lactis, the maximum concentration of lactic acid was 53 and 46 g l⁻¹, respectively. In the case of single Lactobacillus casei or Lactococcus lactis, the total percentage of glucose and fructose utilized were 82.2; 94.4% and 93.8; 60.3%, respectively, whereas in the case of mixed culture, the total percentage of glucose and fructose were 96 and 100%, respectively. These results showed that the mixed culture system gave better results than single cultures regarding lactic acid concentration, and sugar consumption.     

Improved ethanol tolerance of Saccharomyces cerevisiae in mixed cultures with Kluyveromyces lactis on high-sugar fermentation

The influence of non-Saccharomyces yeast, Kluyveromyces lactis, on metabolite formation and the ethanol tolerance of Saccharomyces cerevisiae in mixed cultures was examined on synthetic minimal medium containing 20% glucose. In the late stage of fermentation after the complete death of K. lactis, S. cerevisiae in mixed cultures was more ethanol-tolerant than that in pure culture. The chronological life span of S. cerevisiae was shorter in pure culture than mixed cultures. The yeast cells of the late stationary phase both in pure and mixed cultures had a low buoyant density with no significant difference in the non-quiescence state between both cultures. In mixed cultures, the glycerol contents increased and the alanine contents decreased when compared with the pure culture of S. cerevisiae. The distinctive intracellular amino acid pool concerning its amino acid concentrations and its amino acid composition was observed in yeast cells with different ethanol tolerance in the death phase. Co-cultivation of K. lactis seems to prompt S. cerevisiae to be ethanol tolerant by forming opportune metabolites such as glycerol and alanine and/or changing the intracellular amino acid pool.     

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.     

Light and Phosphate Competition Between Cylindrospermopsis raciborskii and Microcystis aeruginosa is Strain Dependent

The hypothesis that outcomes of phosphorus and light competition between Cylindrospermopsis raciborskii and Microcystis aeruginosa are strain dependent was tested experimentally. Critical requirements of phosphorus (P*) and of light (I*) of two strains of each species were determined through monoculture experiments, which indicated a trade-off between species and also between Microcystis strains. Competition experiments between species were performed using the weakest predicted competitors (with the highest values of P* and of I*) and with the strongest predicted competitors (with the lowest values of P* and of I*). Under light limitation, competition between the weakest competitors led C. raciborskii to dominate. Between the strongest competitors, the opposite was observed, M. aeruginosa displaced C. raciborskii, but both strains co-existed in equilibrium. Under phosphate limitation, competition between the weakest competitors led C. raciborskii to exclude M. aeruginosa, and between the strongest competitors, the opposite was observed, M. aeruginosa displaced C. raciborskii, but the system did not reach an equilibrium and both strains were washed out. Hence, outcomes of the competition depended on the pair of competing strains and not only on species or on type of limitation. We concluded that existence of different trade-offs among strains and between species underlie our results showing that C. raciborskii can either dominate or be displaced by M. aeruginosa when exposed to different conditions of light or phosphate limitation.     

Intraspecific variability in response to phosphorus depleted conditions in the cyanobacteria Microcystis aeruginosa and Raphidiopsis raciborskii

Phosphorus loading plays an important role in the occurrence of cyanobacterial blooms and understanding how this nutrient affects the physiology of cyanobacteria is imperative to manage these phenomena. Microcystis aeruginosa and Raphidiopsis raciborskii are cyanobacterial species that form potentially toxic blooms in freshwater ecosystems worldwide. Blooms comprise numerous strains with high trait variability, which can contribute to the widespread distribution of these species. Here, we explored the intraspecific variability in response to phosphorus depleted conditions (P-) testing five strains of each species. Strains could be differentiated by cell volume or genetic profiles except for those of the same species, sampling location and date, though these presented differences in their response to (P-). Although differently affected by (P-) over 10 days, all strains were able to grow and maintain photosynthetic activity. For most M. aeruginosa and R. raciborskii strains growth rates were not significantly different comparing (P+) and (P-) conditions. After ten days in (P-), only one M. aeruginosa strain and two R. raciborskii strains showed reduction in biovolume yield as compared to (P+) but in most strains chlorophyll-a concentrations were lower in (P-) than in (P+). Reduced photosystem II efficiency was found for only one R. raciborskii strain while all M. aeruginosa strains were affected. Only two M. aeruginosa and one R. raciborskii strain increased alkaline phosphatase activity under (P-) as compared to (P+). Variation in P-uptake was also observed but comparison among strains yielded homogeneous groups comprised of representatives of both species. Comparing the response of each species as a whole, the (P-) condition affected growth rate, biovolume yield and chlorophyll yield. However, these parameters revealed variation among strains of the same species to the extent that differences between M. aeruginosa and R. raciborskii were not significant. Taken together, these results do not support the idea that R. raciborskii, as a species, can withstand phosphorus limitation better than M. aeruginosa and also point that the level of intraspecific variation may preclude generalizations based on studies that use only one or few strains.     

A relationship between Pseudomonal growth behaviour and cystic fibrosis patient lung function identified in a metabolomic investigation

Chronic polymicrobial lung infections in adult cystic fibrosis patients are typically dominated by high levels of Pseudomonas aeruginosa. Determining the impact of P. aeruginosa growth on airway secretion composition is fundamental to understanding both the behaviour of this pathogen in vivo, and its relationship with other potential colonising species. We hypothesised that the marked differences in the phenotypes of clinical isolates would be reflected in the metabolite composition of spent culture media. ¹H NMR spectroscopy was used to characterise the impact of P. aeruginosa growth on a synthetic medium as part of an in vitro CF lower airways model system. Comparisons of 15 CF clinical isolates were made and four distinct metabolomic clusters identified. Highly significant relationships between P. aeruginosa isolate cluster membership and both patient lung function (FEV₁) and spent culture pH were identified. This link between clinical isolate growth behaviour and FEV₁ indicates characterisation of P. aeruginosa growth may find application in predicting patient lung function while the significant divergence in metabolite production and consumption observed between CF clinical isolates suggests dominant isolate characteristics have the potential to play both a selective role in microbiota composition and influence pseudomonal behaviour in vivo.     

Kinetic modeling of kefiran production in mixed culture of Lactobacillus kefiranofaciens and Saccharomyces cerevisiae

Growth and kefiran production rates of Lactobacillus kefiranofaciens were significantly enhanced in a mixed culture with Saccharomyces cerevisiae as compared with those in a pure culture. Because a positive effect on growth and kefiran production of L. kefiranofaciens in a mixed culture was observed, the elucidation of interaction between L. kefiranofaciens and S. cerevisiae may lead to higher productivity. Hence, microbial performance of each strain was investigated and analyzed by a mathematical model. The mathematical model for kefiran fermentation in a mixed culture of L. kefiranofaciens and S. cerevisiae was established, and the impact of S. cerevisiae on cell growth, kefiran formation, and substrate assimilation of L. kefiranofaciens were considered. The behavior of L. kefiranofaciens in a mixed culture was predicted using a developed mathematical model in this work, and the predictions were compared with the results from mixed culture experiments. The overall mathematical model is capable of describing the behavior of S. cerevisiae in a mixed culture as a lactic acid consumer, nitrogen source competitor and protective function inducer for L. kefiranofaciens. Furthermore, the constructed model described the phenomena in mixed cultures under aerobic and anaerobic conditions. Finally, the optimal inoculation ratios of S. cerevisiae to L. kefiranofaciens at 7-fold and 10-fold under aerobic and anaerobic conditions were obtained by applying the mixed culture model, respectively.     

Kinetic analysis of the uptake of glucose and corn oil used as carbon sources in batch cultures of <Emphasis Type="Italic">Gibberella fujikuroi</Emphasis>

This study determined the specific uptake rate of glucose and corn oil substrates used as carbon sources in batch cultures of Gibberella fujikuroi. We tested three biological models of growth rate: Monod, logistic and lag-exponential. With respect to the substrate consumption rate, we tested two models: constant cell yield (CCY) and law of mass action (LMA). The experimental data obtained from the culture with glucose as substrate correlated satisfactorily with the logistic/LMA model, indicating that the cell yield was variable. In the case of corn oil as carbon source, considering total residual lipids as substrate in the culture broth, the model with the best correlation was the lag-exp/CCY model. The quantification by GC of the three main fatty acids (linoleic, oleic and palmitic) in the culture medium showed a cumulative behavior, with a maximum concentration of each acid at 36 h. We established a more explicit mechanism of the consumption of corn oil, consisting of two stages: generation of fatty acids by hydrolysis and consumption by cellular uptake. The kinetic of hydrolysable lipids was of first order. We found that the hydrolysis rate of corn oil is not a limiting factor for the uptake of fatty acids by the microorganism. We also established, based on the analysis of the identical mathematical structure of consumption kinetics, that the uptake of fatty acids is faster than the uptake of glucose.