Phosphate Concentration In Medium Research Articles

#1288 Bioinformatic analysis of phosphate transporters in peritoneal cells and tissues and phosphate transport kinetics in vitro

Abstract Background and Aims Hyperphosphatemia is frequent in peritoneal dialysis (PD) patients due to limited dialytic clearance, and significantly increases cardiovascular risk. Molecular mechanisms of phosphate removal across the peritoneal membrane remain unclear, PD specific in vitro models for phosphate transport and modulator studies are not established. Method Phosphate transporter expression was assessed by RNAseq in mesothelial cells (HPMC, MeT5A) and human venous and microvascular endothelial cells (HUVEC, HCMEC). Transcriptome profiles of microdissected omental arterioles from 10 children with normal kidney function (NKF), 15 with chronic kidney disease (CKD5), and 10 children on PD were analyzed for phosphate transporter expression. Specific phosphate transporter localization and abundance in parietal peritoneum membranes and in the cell lines were assessed by immunostaining. In Transwell system, a phosphate transport model across polarized monolayers was established. Cell viability and integrity were verified by MTT assay and transepithelial resistance measurement (TER). Results Out of nine identified phosphate transporters, PiT1 (SLC20A1) and PiT2 (SLC20A2) exhibited high expression in all four cell lines, while SLC34A2 was specific in HPMC and SLC34A3 in MeT5A. In human arterioles, four phosphate transporters, SLC34A1, PiT1, PiT2, and SLC17A1, were detected. Arteriolar PiT1 was two-fold more abundant in PD patients treated with low glucose degradation product (GDP) fluids compared to those with CKD5 and NKF. In parietal peritoneal membranes, PiT1 was abundant in mesothelial and endothelial cells, with no difference between children with NKF, CKD5 and low GDP PD treatment. Concentration and time dependent phosphate kinetics across polarized mesothelial cells were studied in Transwells. Upon increased phosphate concentration in medium (1 mM and 2 mM vs. 0.1 mM), cells didn´t show signs of impaired viability, reduced TER or junction dysregulation (ZO-1 abundance) for up to 12 h. Independent of the added phosphate concentration, 60% of expected equilibrium was achieved after 12 h. Addition of PFA (SLC20/34 sodium-phosphate cotransporter inhibitor) alone or in combination with Tenapanor (an NHE3 blocker inhibiting paracellular phosphate transport) reduced phosphate transport across mesothelial cells by 10% and 20%, respectively. Conclusion We describe phosphate transporter expression and localization in peritoneal cells, in arterioles and parietal peritoneal tissues, and the impact of CKD5 and PD. The Transwell system allowed for quantification of phosphate transport across cell barriers and demonstrated the role of peritoneal sodium-phosphate cotransporter and of the paracellular route for phosphate transport across the mesothelial cell barrier. Further, yet unknown phosphate transport mechanisms should be active.

Molecular Characterization and Mineralizing Potential of Phosphorus Solubilizing Bacteria Colonizing Common Bean (Phaseolus vulgaris L.) Rhizosphere in Western Kenya.

Phosphorus solubilizing bacteria (PSB) are a category of microbes that transform insoluble phosphates in soil into soluble forms that crops can utilize. Phosphorus in natural soils is abundant but poorly soluble. Hence, introducing PSB is a safer way of improving its solubility. The aim of this study was to genetically characterize and determine the mineralization capability of selected PSB colonizing rhizospheres of common beans in Western Kenya. Seven potential phosphorus solubilizing bacteria (PSB) were isolated from various subregions of Western Kenya. 16S ribosomal RNA gene sequencing and National Center for Biotechnology Information (NCBI), Basic Local Alignment Search Tool (BLAST) identified the isolates. The phosphate solubilization potential of the isolates was evaluated under agar and broth medium of National Botanical Research Institute's phosphate (NBRIP) supplemented with tricalcium calcium phosphate (TCP). Identified isolates were as follows: KK3 as Enterobacter mori, B5 (KB5) as Pseudomonas kribbensis, KV1 as Enterobacter asburiae, KB3 as Enterobacter mori, KK1 as Enterobacter cloacae, KBU as Enterobacter tabaci, and KB2 as Enterobacter bugandensis. The strains B5 and KV1 were the most effective phosphorus solubilizers with 4.16 and 3.64 indices, respectively. The microbes converted total soluble phosphate concentration in broth medium which was 1395 and 1471 P μg/mL, respectively. The least performing isolate was KBU with a 2.34 solubility index. Significant (p ≤ 0.05) differences in plant biomass for Rose coco and Mwitemania bean varieties were observed under inoculation with isolates B5 and KV1. PSB isolates found in common bean rhizospheres exhibited molecular variations and isolates B5 and KV1 are the potential in solving the insufficiency of phosphorus for sustainable crop production.

EOS789, pan-phosphate transporter inhibitor, ameliorates the progression of kidney injury in anti-GBM-induced glomerulonephritis rats.

Hyperphosphatemia associated with chronic kidney disease (CKD) not only dysregulates mineral metabolism and bone diseases, but also strongly contributes to the progression of kidney disease itself. We have identified a novel drug for hyperphosphatemia, EOS789, that interacts with several sodium‐dependent phosphate transporters (NaPi‐IIb, PiT‐1, and PiT‐2) known to contribute to intestinal phosphate absorption. In this study, we investigated whether EOS789 could ameliorate kidney disease progression in glomerulonephritis rats. Anti‐glomerular basement membrane (GBM) nephritis was induced in rats by intravenously administering two types of anti‐rat GBM antibodies. We evaluated the effect of EOS789 administered in food admixture on hyperphosphatemia and kidney disease progression. In an anti‐GBM nephritis rats, which exhibit a significant increase in serum phosphate and a decline in renal function, EOS789 dose‐dependently improved hyperphosphatemia and EOS789 at 0.3% food admixture significantly ameliorated kidney dysfunction as shown in the decline of serum creatinine and BUN. Renal histopathology analysis showed that EOS789 significantly decreased crescent formation in glomeruli. To elucidate the mechanism underlying glomerular disease progression, human mesangial cells were used. High phosphate concentration in media significantly increased the expression of Collagen 1A1, 3A1, and αSMA mRNA in human mesangial cells and EOS789 dose‐dependently suppressed these fibrotic markers. These results indicate that EOS789 prevented glomerular crescent formation caused by mesangial fibrosis by ameliorating hyperphosphatemia. In conclusion, EOS789 would not only be useful against hyperphosphatemia but may also have the potential to relieve mesangial proliferative glomerulonephritis with crescent formation.

Low-cost media engineering for phosphate and IAA production by Kosakonia pseudosacchari TCPS-4 using Multi-objective Genetic Algorithm (MOGA) statistical tool.

The plant growth-promoting rhizobacteria (PGPR) can improve the biotic or abiotic stress condition by exploiting the productivity and plant growth of the plants under stressful conditions. This study examines the role of a rhizospheric bacterial isolate Kosakonia pseudosacchari TCPS-4 isolated from cluster bean plant (Cyamopsis tetragonoloba) under dryland condition. The low-cost media engineering was evaluated, and the phosphate-solubilizing and IAA-producing abilities of Kosakonia pseudosacchari TCPS-4 were improved using a hybrid statistical tool viz. Multi-objective Genetic Algorithm (MOGA). Further, the effect of carbon and nitrogen media constituents and their interactions on IAA production and phosphate solubilization were also confirmed by a single-factor experiment assay. This revealed that MOGA-based model depicted 47.5mg/L inorganic phosphate as the highest phosphate concentration in media containing 45g/L carbon source, 12g/L nitrogen source and 0.20g/L MgSO4. The highest IAA production was 18.74mg/L in media containing 45g/L carbon source, 12g/L nitrogen source and 0.2g/L MgSO4. These values were also confirmed and measured by the experiments with phosphate solubilization of 45.71mg/L and IAA production of 18.71mg/L with 1012cfu/mL. This concludes that effective media engineering using these statistical tools can enhance the phosphate and IAA production by each model. A good correlation between measured and predicted values of each model confirms the validity of both responses. The present study gives an insight on media engineering for phosphate and IAA production by Kosakonia pseudosacchari TCPS-4.

Optimizing Medium Components to Enhance High Cell Mass Production of Biotherapeutic Strai <em>Lactobacillus reuteri</em> DSM 20016T by Statistical Method

Probiotics referred to a group of living microorganisms which highly influence the human health. A number of studies have highlighted on the bio-therapeutic potential of Lactobacillus reuteri strains, especially in treating eradication of Helicobacter pylori . Therefore, in present investigation, statistical methods were applied to optimize medium composition for high cell mass production of L. reuteri strain DSM 20016 T . Most influencing medium components were screened by using Plackett-Burman and optimized using Box-Bhenken experimental design. The concentration of lactose, yeast extract and phosphate in cultivation medium has shown significant effect on the cell mass production. The highest cell mass obtained after 48h incubation was 3.96 ± 0.02 gL −1 in RSM-optimized medium compared to 1.76 ± 0.17 gL −1 in un-optimized medium.

Enzymatic characterization and regulation of gene expression of PhoK alkaline phosphatase in Sphingobium sp. strain TCM1.

Sphingobium sp. strain TCM1 can significantly degrade chlorinated organophosphorus flame retardants, such as tris(2-chloroethyl) phosphate. The PhoK of strain TCM1 (Sb-PhoK) is the main alkaline phosphatase (APase) that catalyzes the last step in the degradation pathway. Here, we purified and characterized Sb-PhoK produced in E. coli, and analyzed the regulation of Sb-phoK gene expression in strain TCM1. The recombinant Sb-PhoK was produced in the mature form, lacking a putative signal peptide, and formed a homodimer. Purified Sb-PhoK exhibited 384U/mg of specific activity at 37°C. The optimum temperature was 50°C, and Sb-PhoK was completely inactivated when incubated at 60°C for 10min. The optimum pH was 10, with stability observed at pH6.0-10.5. Sb-PhoK was suggested to contain two Ca2+ and one Zn2+ per subunit, but excess addition of Zn2+ into the reaction mixture markedly inhibited the enzyme activity. Sb-PhoK showed phosphatase activity against various phosphorylated compounds, except for bis(p-nitrophenyl) phosphate, indicating that it is a phosphomonoesterase with broad substrate specificity. The Km and kcat for p-nitrophenyl phosphate were 2.31mM and 1270s-1, respectively, under optimal conditions. The enzyme was strongly inhibited by vanadate, dithiothreitol, and SDS, but was highly resistant to urea and Triton X-100. Sb-phoK gene expression was regulated by the inorganic phosphate concentration in culture medium, and was induced at a low inorganic phosphate concentration. The deletion of Sb-phoB gene resulted in no induction of Sb-phoK gene even at a low inorganic phosphate concentration, confirming that Sb-PhoK is a member of Pho regulon.

Arsenic toxicity to Chlorella pyrenoidosa and its phycoremediation

Provision of clean drinking water remains a global requirement, so for arsenic-affected areas where various physico-chemical methods are used. These methods require regeneration of media requiring non-stop arsenic checking and it requires skilled operation. Otherwise arsenic contaminated water has to be discarded into the environment. This study examined the probability of using living microalgae Chlorella pyrenoidosa for phycoremediation of arsenic contaminated water (either As(III) or As(V). DO and pH cycles in presence of arsenic (either As(III) or As(V)) ions in the culture did not significantly differ from the control (pure media) indicating that the algae were still growing and photosynthesizing in presence of arsenic ions. As(V) was more noxious than As(III), particularly at pH7.0, but it was opposite at pH9.0. Phycoremediation efficiency of As(V) at pH9.0 by algal cells was greater than that As(III). Monod model has been employed for demonstrating growth kinetics of microalgae in pure media containing various concentrations of nitrate ions. Maximum specific growth rate and saturation constant have been found to be 0.146 d−1 and 8.29E–4g/L, respectively. With the increase in concentration of phosphate in growth medium the growth of microalgae increased. Media with 1.0g/L NaCl indicated the highest algal growth. Addition of NaHCO3 (1g/L) resulted in higher maximum biomass concentration.

Toxicity and bioremediation of As(III) and As(V) in the green microalgae Botryococcus braunii: A laboratory study

ABSTRACTWorldwide threats of fuel shortages in the near future and climate change because of greenhouse gas emissions are posing severe challenges and therefore it is vital to search for sustainable ways of preventing the consequences. The dual use of microalgae for phycoremediation and biomass production for sustainable biofuel production is a viable choice. Phycoremediation of As(III) and As(V) ions using microalgae was investigated in a two-staged batch reactor. Accumulation and toxicity of inorganic arsenic forms (As(III) and As(V)) to green microalgae Botryococcus braunii depend on environmental factors. Dissolved oxygen and pH cycles did not significantly differ due to the absence or presence of arsenic (either As(III) or As(V)) ions in the culture. Monod model was utilized for representing the growth kinetics of microalgae in pure media containing various concentrations of nitrate ions. Maximum specific growth rate and saturation constant were found to be 0.14788 d−1 and 0.00105 g/L, respectively. With the increase in concentration of phosphate in growth medium, the growth of microalgae increased. Media with NaCl (1.0 g/L) and NaHCO3 (1 g/L) resulted in higher maximum biomass concentration. Effect of coexisting ions on phycoremediation of As(III) and As(V) ions using microalgae was studied.

Phycoremediation Potential of Botryococcus braunii: Bioremediation and Toxicity of As(III) and As(V)

Worldwide threats of climate change because of greenhouse gas emissions and fuel shortages in near future are posing considerate challenges and therefore it is vital for exploring viable ways of preventing the consequences. The dual usage of microalgae for phycoremediation and biomass production for ecological biofuels production is a practicable choice. Accumulation and toxicity of inorganic arsenic forms (As(III) and As(V)) to the green microalgae Botryococcus braunii depends on various environmental factors. This study examined the possibility of using living algae B. braunii for phycoremediation of arsenic-enriched media (As(III) and As(V)). As(V) was more harmful than As(III), particularly at pH 7.0, but it was reverse at pH 9.0. The phycoremediation efficiency of As(V) at pH 9.0 by algal cells was higher than that As(III). An increase in concentration of phosphate in growth medium reduced the toxicity and phycoremediation of As(III) and As(V). Value of μ max remained almost constant after addition of arsenic (either As(III) or As(V)) in the media containing various concentration of phosphate ions, but the value of K s increased. The microalgae B. braunii can be employed for tertiary treatment of wastewater and the producing biomass can be utilized as source of renewable energy.

Regulation of osteoclast function by poly(ethylene sodium phosphate)

Event Abstract Back to Event Regulation of osteoclast function by poly(ethylene sodium phosphate) Yasuhiko Iwasaki1*, Sujit Kootala2* and Jöns Hilborn2* 1 Kansai University, Department of Chemistry and Materials Engineering, Japan 2 Uppsala University, Department of Chemistry, Sweden Introduction: An imbalance in bone resorption relative to bone formation is the origin of many metabolic bone diseases, including osteoporosis [1]. In the current field of clinical osteology, bisphosphonates (BPs), which reduce bone resorption, comprise a major class of drugs used in the treatment of osteoporosis. Although BPs are reliable molecules for bone treatment, medication-related osteonecrosis of the jaw has emerged as a serious complication in patients treated with high BP doses. Therefore, new therapeutic agents showing BP-like activity could be beneficial. Recently, we have clarified that polyphosphoester ionomers exhibited clearly superior physicochemical properties for mineralization and biocompatibility when compared with pamidronate [2]. Our hypothesis is that polyphosphoesters, in similarity to bisphosphonates, inhibit osteoclast growth without affecting osteoblasts. Therefore, we studied the effects of poly(ethylene sodium phosphate) (PEP·Na) treatment on bone slice-cultured osteoclasts. Materials and Method: Poly(ethylene sodium phosphate) (PEP·Na) was synthesized as previously reported [3]. Peripheral blood mononuclear cells (PBMCs) were seeded on one side of each bovine cortical bone slice placed in a 24-well cell culture insert; each culture contained a poly(ethylene terephthalate) (PET) trans well membrane and was maintained for 3 weeks with a change of medium every 3 days to induce monocyte differentiation into mature osteoclasts. Cultures of mature human osteoclasts on bone slices were exposed for 24 h to six different concentrations of PEP·Na to assess the toxicity. As a measure of cell viability the numbers of adherent cells were determined from microscopic images using Image J software. TRAcP activity was assessed with a leukocyte acid phosphatase kit. TRAcP-positive multinucleated cells were defined as cells with 3 or more nuclei. Results and Discussion: Optical microscopic data of osteoclasts incubated with different concentrations of PEP·Na are shown in Figure 1. The cells were stained for TRAcP and included giant cells. In the control, which was not treated with PEP·Na, many cells adhered to the bone slice and large, well-spread cells were observed. When a low concentration of PEP·Na was added to the cultivation medium, the number of mature osteoclasts that remained adhered increased, although many smaller adherent cells were also observed. By increasing the concentration of PEP·Na, the number of adherent cells decreased significantly, and their sizes were also reduced. Kanatani and colleagues reported that inorganic phosphate inhibited osteoclast differentiation both by upregulating osteoprotegrin (OPG) expression and through direct action on osteoclast precursor cells [4]. Polyphosphoester degradation is likely to increase the concentration of inorganic phosphate in the cultivation medium and upregulate both the intracellular osteoclast and extracellular inorganic phosphate concentrations. Conclusion: In the present study, the possibility of using polyphosphoesters for selective inhibition of human osteoclast functions was validated. Polyphosphoesters then have a potential to be used as a prodrug for treatment of osteoporosis. JSPS KAKENHI Grant Number 26107719

Secretion of non-cell-bound phytase by the yeast Pichia kudriavzevii TY13.

Mineral deficiencies cause several health problems in the world, especially for populations consuming cereal-based diets rich in the anti-nutrient phytate. Our aim was to characterize the phytate-degrading capacity of the yeast Pichia kudriavzevii TY13 and its secretion of phytase. The phytase activity in cell-free supernatants from cultures with 100% intact cells was 35-190 mU ml(-1) depending on the media. The Km was 0.28 mmol l(-1) and the specific phytase activity 0.32 U mg(-1) total protein. The phytase activity and secretion of extracellular non-cell-bound phytase was affected by the medium phosphate concentrations. Further, addition of yeast extract had a clearly inducing effect, resulting in over 60% of the cultures total phytase activity as non-cell-bound. Our study reveals that it is possible to achieve high extracellular phytase activity from the yeast P. kudriavzevii TY13 by proper composition of the growth medium. TY13 could be a promising future starter culture for fermented foods with improved mineral bioavailability. Using strains that secrete phytase to the food matrix may significantly improve the phytate degradation by facilitating the enzyme-to-substrate interaction. The secreted non-cell-bound phytase activities by TY13 could further be advantageous in industrial production of phytase.

Micropropagation of Endemic Scutellaria orientalis L. subsp. Bicolor Using Modified MS Medium and TDZ

Scutellaria orientalis L. subsp. Bicolor (HOCHST.) Edmondson is a perenniel endemic shrub bearing pale grey-white or pale grey-white violet flowers that grows on poor soils or marginal lands. They have high value as rock garden plants and in managed arid land scaping. They are also important medicinally for valuable secondary metabolites of pharmaceutical importance. The current study aimed to determine in vitro propagation of S.orientalis sub sp. Bicolor using cotyledon leaf, cotyledon petiole, cotyledon node and hypocotyl explants. Healthy green shoots were regenerated on above mentioned explants containing 1 and 2 × KH2PO4 concentrated MS medium containing variants of TD for shoot regeneration. Maximum shoot regeneration was noted on cotyledon node explants on 2 × KH2PO4 concentrated MS medium containing 0.04 µg/ml TDZ. Well developed shoots were rooted on 1 × KH2PO4 concentrated MS medium containing 0.50 µg/ml NAA and the developing plants were acclimatised in greenhouse. They flowered and set seeds. It was concluded that doubling potasium phosphate concentration in MS medium was helpful in reduction or non induction of hyperhydricity on TDZ regenerated shoots. The results of these studies may serve to provide basic information for scutellaria mass propagation, breeding and genetic transformation studies in future.

Screening and optimization of low-cost medium for Pseudomonas putida Rs-198 culture using RSM.

The plant growth-promoting rhizobacterial strain Pseudomonas putida Rs-198 was isolated from salinized soils from Xinjiang Province. We optimized the composition of the low-cost medium of P. putida Rs-198 based on its bacterial concentration, as well as its phosphate-dissolving and indole acetic acid (IAA)-producing capabilities using the response surface methodology (RSM), and a mathematical model was developed to show the effect of each medium component and its interactions on phosphate dissolution and IAA production. The model predicted a maximum phosphate concentration in medium containing 63.23 mg/L inorganic phosphate with 49.22 g/L corn flour, 14.63 g/L soybean meal, 2.03 g/L K₂HPO₄, 0.19 g/L MnSO₄ and 5.00 g/L NaCl. The maximum IAA concentration (18.73 mg/L) was predicted in medium containing 52.41 g/L corn flour, 15.82 g/L soybean meal, 2.40 g/L K₂HPO₄, 0.17 g/L MnSO₄ and 5.00 g/L NaCl. These predicted values were also verified through experiments, with a cell density of 10(13) cfu/mL, phosphate dissolution of 64.33 mg/L, and IAA concentration of 18.08 mg/L. The excellent correlation between predicted and measured values of each model justifies the validity of both the response models. The study aims to provide a basis for industrialized fermentation using P. putida Rs-198.

Copper tolerance mediated by polyphosphate degradation and low-affinity inorganic phosphate transport system in Escherichia coli

BackgroundMetal tolerance in bacteria has been related to polyP in a model in which heavy metals stimulate the polymer hydrolysis, forming metal-phosphate complexes that are exported. As previously described in our laboratory, Escherichia coli cells grown in media containing a phosphate concentration >37 mM maintained an unusually high polyphosphate (polyP) level in stationary phase. The aim of the present work was to evaluate the influence of polyP levels as the involvement of low-affinity inorganic phosphate transport (Pit) system in E. coli copper tolerance.ResultsPolyP levels were modulated by the media phosphate concentration and/or using mutants in polyP metabolism. Stationary phase wild-type cells grown in high phosphate medium were significantly more tolerant to copper than those grown in sufficient phosphate medium. Copper addition to tolerant cells induced polyP degradation by PPX (an exopolyphosphatase), phosphate efflux and membrane polarization. ppk−ppx− (unable to synthesize/degrade polyP), ppx− (unable to degrade polyP) and Pit system mutants were highly sensitive to metal even in high phosphate media. In exponential phase, CopA and polyP-Pit system would act simultaneously to detoxify the metal or one could be sufficient to safeguard the absence of the other.ConclusionsOur results support a mechanism for copper detoxification in exponential and stationary phases of E. coli, involving Pit system and degradation of polyP. Data reflect the importance of the environmental phosphate concentration in the regulation of the microbial physiological state.

Effect of nitrogen source and inorganic phosphate concentration on methanol utilization and PEX genes expression in Pichia pastoris.

Methylotrophic yeast Pichia pastoris has proved to be especially useful for production of various heterologous proteins. In biotechnology it is very important to maintain the balance between high levels of heterologous gene expression and cell viability. Decisive understanding of gene regulation mechanisms is essential for reaching this goal. In this study, we investigated the effect of different nitrogen sources and phosphate concentration in media on methanol utilization. It was shown that expression levels of main genes, which are involved in methanol utilization (MUT genes) and in functioning of peroxisomes (PEX genes), are maximal when ammonium sulphate is used as a nitrogen source. Expression of these genes is decreased in media with poor nitrogen sources, such as proline. Addition of rapamycin to the media completely removed repression of AOX1 promoter in media with proline, which allows proposing that Tor-kinase is involved in establishing of nitrogen regulation of this gene. It was also shown that MUT genes expression levels get higher, when the phosphate concentration in media is increased.

Solubilization of tricalcium phosphate by P(3HB) accumulating Azotobacter chroococcum MAL-201

Cells of Azotobacter chroococcum MAL-201 (MTCC 3853) are capable of accumulating the intracellular poly(3-hydroxybutyric acid) [P(3HB)], accounting for 65-71% of its cell dry weight and also capable of synthesizing the enzyme alkaline phosphatase (APase), when grown in glucose and tricalcium phosphate containing nitrogen-free modified Stockdale medium. The concentration of insoluble phosphate in broth medium was optimized as 0.25% (w/v) for growth and biosynthesis of APase. However, the suboptimal concentration of phosphate (0.1%, w/v) appeared as the best suited for accumulation of P(3HB) by the strain. The significant differences were observed in biosynthesis of polymer and APase enzyme under variable phosphate concentrations. Glucose, 3.0% (w/v) was recorded as the optimum concentration for all of the three parameters. The continuation of APase biosynthesis was observed during the period of significant decline in the cellular content of the polymer in the late phase of growth. In order to study the role of P(3HB), the rate of autodigestion of biopolymer and phosphate solubilization rate (k, mineralization constant) were determined in carbon-free medium under batch cultivation process and the parameters were found to be positively correlated. The maximum phosphate solubilization rate (k=0.0154) by the strain MAL-201 timed at the 10th hour of incubation when the rate of polymer degradation concomitantly attained its peak corresponding to 87mg/l/h and then declined gradually. Only a negligible amount of residual polymer remained undigested. These data strongly support the functional role of P(3HB) in response to multinutritional stress condition.

In vitro flowering red miniature rose

Using aseptic plantlets obtained from stem node explants of hybrid red miniature rose (Rosa hybrida cv. Fairy Dance), the effects of shoot physiological status, medium ingredients, and culture thermoperiod on in vitro flowering were evaluated. Shoot height, subculture media for shoot multiplication, sucrose concentration, plant growth regulators (PGRs), mineral substances in media, and thermoperiod had a significant effect on the percentage of in vitro flowering. Shoots 3 ± 0.2 or 2 ± 0.2 cm in height cultured on Murashige and Skoog (MS) medium containing 2.0 mg dm−3 6-benzyladenine (BA), 0.2 mg dm−3 α-naphthaleneacetic acid (NAA), and 20 g dm−3 sucrose were more suitable for in vitro flowering than shoots 4 ± 0.2, or 5 ± 0.2 cm in height. The most suitable sucrose concentration for in vitro flowering was 50 g dm−3 and the most suitable PGRs were a combination of 3.0 mg dm−3 BA and 0.1 mg dm−3 NAA. Increasing the potassium nitrate to ammonium nitrate ratio or increasing the phosphate concentration in MS medium had a positive effect on in vitro flowering. The percentage of in vitro flowering was significantly higher at day/night temperature of 28/20 °C than at other constant temperatures. The percentage of in vitro flowering shoots reached 68.33 % despite the occurrence of abnormal flowers and some unique developmental patterns. It makes miniature rose a potentially new in vitro experimental platform for research on the molecular mechanisms of flowering ornamental plants.

Polyphosphate degradation in stationary phase triggers biofilm formation via LuxS quorum sensing system in Escherichia coli.

In most natural environments, association with a surface in a structure known as biofilm is the prevailing microbial life-style of bacteria. Polyphosphate (polyP), an ubiquitous linear polymer of hundreds of orthophosphate residues, has a crucial role in stress responses, stationary-phase survival, and it was associated to bacterial biofilm formation and production of virulence factors. In previous work, we have shown that Escherichia coli cells grown in media containing a critical phosphate concentration >37 mM maintained an unusual high polyP level in stationary phase. The aim of the present work was to analyze if fluctuations in polyP levels in stationary phase affect biofilm formation capacity in E. coli. Polymer levels were modulated by the media phosphate concentration or using mutant strains in polyP metabolism. Cells grown in media containing phosphate concentrations higher than 25 mM were defective in biofilm formation. Besides, there was a disassembly of 24 h preformed biofilm by the addition of high phosphate concentration to the medium. These phenotypes were related to the maintenance or re-synthesis of polyP in stationary phase in static conditions. No biofilm formation was observed in ppk−ppx− or ppk−ppx−/ppk+ strains, deficient in polyP synthesis and hydrolysis, respectively. luxS and lsrK mutants, impaired in autoinducer-2 quorum sensing signal metabolism, were unable to form biofilm unless conditioned media from stationary phase wild type cells grown in low phosphate were used. We conclude that polyP degradation is required for biofilm formation in sufficient phosphate media, activating or triggering the production of autoinducer-2. According to our results, phosphate concentration of the culture media should be carefully considered in bacterial adhesion and virulence studies.

A protocol for the mass-micropropagation of carnation (Dianthus caryophyllus L.)

SummaryThe influence of increasing the phosphate concentration in Murashige and Skoog medium (MS), ventilation using 500 ml jars covered with rigid, vented polypropylene closures and 1, 2, or 3% (w/v) sucrose on the incidence of hyperhydricity and on shoot proliferation of carnation (Dianthus caryophyllus L.) were examined. Shoot proliferation from nodal explants was induced on MS medium containing 8.87 µM 6-benzylaminopurine (BAP), whereas shoot elongation and rooting were achieved on MS medium containing 9.8 µM indole-3-butyric acid (IBA). Hyperhydricity could be controlled, in part, by ventilation, and was completely eliminated by increasing the phosphate concentration of the MS growth medium to 6.45 mM. Moreover, explant growth and morphogenesis were also promoted by increasing the level of phosphate in the MS medium to 6.45 mM. In contrast to MS medium with the standard 1.25 mM phosphate, which produced 7.9 – 10.0 shoots explant–1, MS medium with an additional 5.20 mM phosphate produced 20.8 – 34.7 shoots explant–1, depending on providing ventilation and on the sucrose concentration. The water content of shoot clumps, which reflected a decrease in the proportion of hyperhydric shoots, decreased progressively by providing ventilation or by increasing the phosphate concentration. Plantlets were successfully acclimatised and transplanted in a greenhouse. The results suggested that container ventilation and MS medium enriched with 6.45 mM phosphate plus 3% (w/v) sucrose significantly improved the efficiency of mass micropropagation of D. caryophyllus cv. White Sim.

Phosphate assimilation by Chlorella and adjustment of phosphate concentration in basal medium for its cultivation

Assimilation of phosphate by Chlorella pyrenoidosa was 0.81-8.1 mg PO(4)-P/g dry weight for heterotrophic cultures and 0.81-16.1 mg/g for mixotrophic cultures. Optimal carbon:phosphorous (C/P) ratios were 206:1-2060:1 and 103:1-2060:1 for heterotrophic and mixotrophic cultivations, respectively. These requirements for phosphate for growth of C. pyrenoidosa under either heterotrophic or mixotrophic conditions are much less (6.25-62.5 or 3.12-62.5-fold at 10 g glucose/l) than its concentration in basal medium.