Effects Of Phosphate Limitation Research Articles

Engineering ergothioneine production in Yarrowia lipolytica.

Ergothioneine is a naturally occurring antioxidant that has shown potential in ameliorating neurodegenerative and cardiovascular diseases. In this study, we investigated the potential of the Crabtree‐negative, oleaginous yeast Yarrowia lipolytica as an alternative host for ergothioneine production. We expressed the biosynthetic enzymes EGT1 from Neurospora crassa and EGT2 from Claviceps purpurea to obtain 158 mg·L−1 of ergothioneine in small‐scale cultivation, with an additional copy of each gene improving the titer to 205 mg·L−1. The effect of phosphate limitation on ergothioneine production was studied, and finally, a phosphate‐limited fed‐batch fermentation in 1 L bioreactors yielded 1.63 ± 0.04 g·L−1 ergothioneine in 220 h, corresponding to an overall volumetric productivity of 7.41 mg·L−1·h−1, showing that Y. lipolytica is a promising host for ergothioneine production.

Effects of phosphate limitation on soluble microbial products and microbial community structure in semi‐continuous Synechocystis‐based photobioreactors

All bacteria release organic compounds called soluble microbial products (SMP) as a part of their normal metabolism. In photobioreactor (PBR) settings, SMP produced by cyanobacteria represent a major pool of carbon and electrons available to heterotrophic bacteria. Thus, SMP in PBRs are a major driver for the growth of heterotrophic bacteria, and understanding the distribution of SMP in PBRs is an important step toward proper management of PBR microbial communities. Here, we analyzed the SMP and microbial communities in two Synechocystis sp. PCC6803-based PBRs. The first PBR (PBRP0) became phosphate limited after several days of operation, while the second PBR (PBRP+) did not have phosphate limitation. Heterotrophic bacteria were detected in both PBRs, but PBRP0 had a much higher proportion of heterotrophic bacteria than PBRP+. Furthermore, PBRP+ had greater biomass production and lower SMP production per unit biomass than PBRP0. Carbohydrates that were most likely derived from hydrolysis of extracellular polymeric substances (EPS) dominated the SMP in PBRP0, while products resulting from cell lysis or decay dominated the SMP in PBRP+. Together, our data support that maintaining phosphate availability in Synechocystis-based PBRs is important for managing SMP and, thus, the heterotrophic community.

Effects of phosphate limitation in feed water on biofouling in forward osmosis (FO) process

Forward osmosis (FO) has been applied to various water treatment processes because of its low energy consumption and lower fouling propensity. Fouling, however, especially biofouling, is still problematic in FO. To resolve the biofouling issue, phosphate limitation was investigated to reduce biofouling instead of commonly used chemical cleaning. To establish the effects of phosphate limitation on biofouling formation, various phosphate concentrations and nutritional conditions were tested to seek a basic correlation between phosphate limitation and biofilm formation, and effects of phosphate limitation on performance of FO were checked. As a result, it was discovered that the phosphate limitation in feed water greatly reduced microbial growth and biofilm formation, thereby FO performance was maintained. This phenomenon was explained in accelerated biofilm-enhanced osmotic pressure (Accelerated BEOP) and Gibbs–Donnan equilibrium mechanism, and it shows that phosphate limitation can effectively control biofouling by alleviating the accelerated BEOP and balancing the Gibbs–Donnan equilibrium. Therefore, it may be feasible to use the phosphate limitation in FO pretreatment to control biofouling.

Exo-metabolome of Pseudovibrio sp. FO-BEG1 analyzed by ultra-high resolution mass spectrometry and the effect of phosphate limitation.

Oceanic dissolved organic matter (DOM) is an assemblage of reduced carbon compounds, which results from biotic and abiotic processes. The biotic processes consist in either release or uptake of specific molecules by marine organisms. Heterotrophic bacteria have been mostly considered to influence the DOM composition by preferential uptake of certain compounds. However, they also secrete a variety of molecules depending on physiological state, environmental and growth conditions, but so far the full set of compounds secreted by these bacteria has never been investigated. In this study, we analyzed the exo-metabolome, metabolites secreted into the environment, of the heterotrophic marine bacterium Pseudovibrio sp. FO-BEG1 via ultra-high resolution mass spectrometry, comparing phosphate limited with phosphate surplus growth conditions. Bacteria belonging to the Pseudovibrio genus have been isolated worldwide, mainly from marine invertebrates and were described as metabolically versatile Alphaproteobacteria. We show that the exo-metabolome is unexpectedly large and diverse, consisting of hundreds of compounds that differ by their molecular formulae. It is characterized by a dynamic recycling of molecules, and it is drastically affected by the physiological state of the strain. Moreover, we show that phosphate limitation greatly influences both the amount and the composition of the secreted molecules. By assigning the detected masses to general chemical categories, we observed that under phosphate surplus conditions the secreted molecules were mainly peptides and highly unsaturated compounds. In contrast, under phosphate limitation the composition of the exo-metabolome changed during bacterial growth, showing an increase in highly unsaturated, phenolic, and polyphenolic compounds. Finally, we annotated the detected masses using multiple metabolite databases. These analyses suggested the presence of several masses analogue to masses of known bioactive compounds. However, the annotation was successful only for a minor part of the detected molecules, underlining the current gap in knowledge concerning the biosynthetic ability of marine heterotrophic bacteria.

Effects of nitrate and phosphate on grazer‐induced toxin production in Alexandrium minutum

Two strains of Alexandrium minutum were exposed to waterborne cues from copepod grazers in factorial combinations of nitrate and phosphate concentrations to evaluate the importance of grazer‐induced paralytic shellfish toxin (PST) production under different nutrient regimes. In nitrate‐rich treatments, the presence of waterborne cues from grazers resulted in significantly increased cell‐specific PST content. In low nitrate treatments, however, waterborne cues from grazers did not result in any detectable increase in cell‐specific PST content. The grazer‐induced increase in cell‐specific PST content in nitrate‐rich treatments was comparable in size to the effects of nitrate in the absence of grazers. Effects of phosphate limitation were less consistent, resulting in increased PST content in nitrate‐rich treatments in one of the A. minutum strains, but had no significant effect in the other. The ability of dinoflagellates to sense and respond to the presence of grazers by increased PST production may be one of the most important factors affecting cell‐specific PST content under nitrogen replete conditions.

Effects of phosphate limitation on expression of genes involved in pyrimidine synthesis and salvaging in Arabidopsis

Arabidopsis seedlings grown for 14 d without phosphate (P) exhibited stunted growth and other visible symptoms associated with P deficiency. RNA contents in shoots decreased nearly 90%, relative to controls. In shoots, expression of Pht1;2, encoding an inducible high-affinity phosphate transporter, increased threefold, compared with controls, and served as a molecular marker for P limitation. Transcript levels for five enzymes (aspartate transcarbamoylase, ATCase, EC 2.1.3.2; carbamoyl phosphate synthetase, CPSase, EC 6.3.5.5); UMP synthase, EC 2.4.1.10, EC 4.1.1.23; uracil phosphoribosyltransferase, UPRTase, EC 2.4.2.9; UMP kinase, EC 2.7.1.14) increased 2–10-fold in response to P starvation in shoots. These enzymes, which utilize phosphorylated intermediates at putative regulated steps in de novo synthesis and salvaging pathways leading to UMP and pyrimidine nucleotide formation, appear to be coordinately regulated, at the level of gene expression. This response may facilitate pyrimidine nucleotide synthesis under P limitation in this plant. Expression of P-dependent and P-independent phosphoribosyl pyrophosphate (PRPP) synthases (PRS2 and PRS3, respectively) which provide PRPP, the phosphoribosyl donor in UMP synthesis via both de novo and salvaging pathways, was differentially regulated in response to P limitation. PRS2 mRNA levels increased twofold in roots and shoots of P-starved plants, while PRS3 was constitutively-expressed. PRS3 may play a novel role in providing PRPP to cellular metabolism under low P availability.

Population Dynamics of Phytoplankton and Viruses in a Phosphate-limited Mesocosm and their Effect on DMSP and DMS Production

The effect of phosphate limitation on viral abundance, phytoplankton bloom dynamics and production of dimethylsulphoniopropionate (DMSP) and dimethyl sulphide (DMS) was investigated in seawater mesocosm enclosures, in a Norwegian fjord, during June 1995. Daily estimates of viral concentrations, based on transmission electron microscope (TEM) counts, varied on an apparently random basis in each of the enclosures. A largeSynechococcusspp. bloom developed in an enclosure which was maintained at a high N:P ratio, simulating phosphate-deplete growth conditions. Following phosphate addition to this enclosure, there was a large increase in estimated virus numbers shortly before an apparent collapse of theSynechococcusbloom. It is tentatively suggested that lysogenic viruses were induced following phosphate addition to the phosphate-limited enclosures, and that these observations add to a growing body of evidence which supports the hypothesis that nutrient availability may be responsible for the switch between lysogeny and lytic production. High DMS concentrations and viral numbers were observed on the demise of the flagellate (predominantlyEmiliania huxleyi) and diatom blooms, but overall there was no significant correlation. Highest concentrations of DMSP were associated with blooms ofE. huxleyi, for which an intracellular concentration of 0·5pg cell−1(SD, 0·06) was calculated. Good correlation of DMSP withSynechococcusspp. cell numbers was observed, suggesting that these species of picoplankton may be significant producers of DMSP. No effects of phosphate limitation on DMS and/or DMSP production were evident from the data.

Effects of phosphate limitation on agarase production by Streptomyces lividans TK21

The dagA gene from Streptomyces coelicolor, encoding an extracellular agarase, has been expressed in Streptomyces lividans on a multicopy plasmid. Chemostat cultures were used to investigate the influence of phosphate limitation on the synthesis and secretion of this enzyme. The strain was cultivated with glucose as the carbon source and magnesium or phosphate as growth-limiting substrates and samples used to determine dagA transcription, agarase secretion and alkaline phosphatase activity under the various limitations. The data show that, compared to growth under magnesium limitation, phosphate-limited cells show increases in dagA transcription, the rate of agarase production and secretion, and alkaline phosphatase activity.

Bacterial-algal interactions in polysaccharide production

The effects of phosphate limitation and of the presence of marine bacteria during phosphate limitation on growth and polysaccharide production in the diatom Cylindrotheca fusiformis grown in batch cultures are described in this paper. Growth of C. fusiformis was inhibited under low Pi (inorganic phosphate) conditions, corresponding to an increasing N/P ratio, and higher amounts of polysaccharides were extruded in the medium, in particular during the stationary phase of growth. The presence of bacteria reduced phytoplankton cell density only when the phosphate added corresponded to 1/6 of the initial amount. Even when diatom cell growth was not affected, the presence of bacteria stimulated a higher polysaccharide production. These results are interpreted in the light of the fact that nutrient-stressed phytoplankton cells produced and released a higher amount of polysaccharides and, as bacteria exhibited a better utilization of phosphate than algal cells, their presence accentuated the Pi depletion, resulting in a higher polysaccharide production.

Properties ofAzotobacter vinelandii in phosphate-limited batch cultures

Batch cultures ofA. vinelandii in ammonium phosphate-limited and N-free phosphate-limited media were compared with control cultures (N-free phosphate-sufficient media). The effects of phosphate limitation on growth were determined by viable cells counts. Under phosphate-limitation conditions, growth inhibition and decreased viability were observed. Intracellular levels of RNA, poly-3-hydroxybutyrate, phosphate and oxygen uptake were significantly affected by phosphate limitation. When phosphate-limited cultures were examined microscopically, pleomorphism was more marked than in control cultures. Also phosphate-limited cells showed an increase in resistance to UV irradiation, mechanical disruption, desiceation and the combined action of ethylenediaminetetraacetie acid and lysozyme.

Growth ofBacillus megaterium in phosphate-limited medium

Batch cultures of Bacillus megaterium grown in phosphate-limited media were compared with control cultures grown in phosphate-sufficient media. The effects of phosphate limitation on growth were determined by viable cells counts. Intracellular levels of protein, RNA, poly-3-hydroxybutyrate, carbohydrate and oxygen uptake were significantly affected by phosphate limitation. Electron micrographs of sectioned cells revealed differences in the structure; in particular the thick, rigid cell wall was absent from cells grown in phosphate-limited media, and such cells were larger, pleomorphic, and after 2 d were insensitive to lysozyme.

Effect of Phosphate Limitation on the Morphology and Wall Composition of Bacillus licheniformis and Its Phosphoglucomutase-Deficient Mutants

Effect of Phosphate Limitation on the Morphology and Wall Composition of <i>Bacillus licheniformis</i> and Its Phosphoglucomutase-Deficient Mutants

Effect of phosphate limitation on the morphology and wall composition of Bacillus licheniformis and its phosphoglucomutase-deficient mutants.

Two very poorly lytic mutants of Bacillus licheniformis 6346 that had no teichuronic acid or glucose in their walls were phosphoglucomutase deficient. The walls of the mutants were less autolytic, and the lesion in the phosphoglucomutase gene and the formation of lytic amidase seemed to be interrelated. When phosphoglucomutase was regained or the effects of the deficiency were circumvented by the presence of galactose in the medium, the lytic enzyme was partially regained. When subjected to growth limitation by the supply of inorganic phosphate, the mutants ceased to make teichoic acid, and their walls contained a greatly increased proportion of mucopeptide. Under these conditions they formed irregular spheres which changed back to rods when inorganic phosphate was supplied. Both wall and protein synthesis were necessary for the changes in morphology. An intermediate crescent-shaped cell was formed in the change from sphere to a rod. The possible relationship of this morphological change to the distribution of biosynthetic sites is discussed.