Glucose-supplemented Cultures Research Articles

Nitrosative stress under microaerobic conditions triggers inositol metabolism in Pseudomonas extremaustralis.

Bacteria are exposed to reactive oxygen and nitrogen species that provoke oxidative and nitrosative stress which can lead to macromolecule damage. Coping with stress conditions involves the adjustment of cellular responses, which helps to address metabolic challenges. In this study, we performed a global transcriptomic analysis of the response of Pseudomonas extremaustralis to nitrosative stress, induced by S-nitrosoglutathione (GSNO), a nitric oxide donor, under microaerobic conditions. The analysis revealed the upregulation of genes associated with inositol catabolism; a compound widely distributed in nature whose metabolism in bacteria has aroused interest. The RNAseq data also showed heightened expression of genes involved in essential cellular processes like transcription, translation, amino acid transport and biosynthesis, as well as in stress resistance including iron-dependent superoxide dismutase, alkyl hydroperoxide reductase, thioredoxin, and glutathione S-transferase in response to GSNO. Furthermore, GSNO exposure differentially affected the expression of genes encoding nitrosylation target proteins, encompassing metalloproteins and proteins with free cysteine and /or tyrosine residues. Notably, genes associated with iron metabolism, such as pyoverdine synthesis and iron transporter genes, showed activation in the presence of GSNO, likely as response to enhanced protein turnover. Physiological assays demonstrated that P. extremaustralis can utilize inositol proficiently under both aerobic and microaerobic conditions, achieving growth comparable to glucose-supplemented cultures. Moreover, supplementing the culture medium with inositol enhances the stress tolerance of P. extremaustralis against combined oxidative-nitrosative stress. Concordant with the heightened expression of pyoverdine genes under nitrosative stress, elevated pyoverdine production was observed when myo-inositol was added to the culture medium. These findings highlight the influence of nitrosative stress on proteins susceptible to nitrosylation and iron metabolism. Furthermore, the activation of myo-inositol catabolism emerges as a protective mechanism against nitrosative stress, shedding light on this pathway in bacterial systems, and holding significance in the adaptation to unfavorable conditions.

Effect of type of anticoagulant, transportation time, and glucose in the culture media on neutrophil viability and function test results in dairy cattle.

In dairy cattle research, in vitro assessment of innate immune function is commonly evaluated by flow cytometry via the quantitative analysis of circulating polymorphonuclear leukocytes (PMN) functionalities specifically focusing on the capacities for phagocytosis (PC) and oxidative burst (OB). Variations in these PMN functions, however, may not only be influenced by the health status of the animals but also by technical, non-animal related factors. Our objectives were to assess the PMN viability, PC and OB capacities from blood samples collected in tubes coated with different anticoagulants (acid citrate dextrose (ACD) and ethylenediaminetetraacetic acid (EDTA)) and stored for 0, 3, 6, 9, and 12 h at 4°C (to mimic transportation timeframe). Furthermore, we evaluated the PMN functionalities (PC and OB) in samples incubated in culture medium with glucose (7.2 mM) versus no glucose. Over five replicates, coccygeal blood samples were collected from three nulliparous Holstein heifers (5 ACD and 5 EDTA per heifer) and allocated in a refrigerated container (4°C) for 0, 3, 6, 9, and 12 h. At each time point, PMN were isolated using gradient centrifugation. Immunolabeled PMN (CH138A) were subjected to a tricolor fluorescent staining to evaluate their viability (viable, apoptotic, and necrotic PMN). Phagocytosis and OB were assessed by incubating PMN with fluorescent beads and by phorbol 12-myristate 13-acetate stimulation, respectively. The effects of anticoagulant type, storage time, and presence of glucose in the culture medium on PMN viability and function parameters were fitted in mixed linear regression models. The proportion of viable PMN at 0 h was similar for ACD and EDTA (92 ± 4.6% and 93 ± 4.6%, respectively) but it decreased to 78 ± 4.6% for ACD and 79 ± 4.6% for EDTA after 6 h of storage. The proportion of viable PMN was not different between ACD and EDTA at any time point. The proportion of PMN that engulfed beads (PC percentage) and the PC median fluorescence intensity (MFI) reached their highest value after 3 h of storage compared with the other time points. However, the anticoagulant type (ACD versus EDTA) and the presence of glucose in the culture medium did not influence these PC parameters. Oxidative burst MFI was higher in PMN incubated in glucose-supplemented culture medium versus no glucose. We demonstrated that technical factors interfere with the evaluation of PMN viability and functionality, which can potentially lead to bias in the findings of a research hypothesis. To conclude, the present study showed that the optimal timeframe for performing PMN function analyses is within 3 hours after blood sampling. Furthermore, the presence of 7.2 mM glucose in the culture medium, a common concentration in formulation of cell culture medium, increases the in vitro OB capacity, potentially masking any impairments in in vivo PMN dysfunctionality.

Patterning of the Autotrophic, Mixotrophic, and Heterotrophic Proteomes of Oxygen-Evolving Cyanobacterium Synechocystis sp. PCC 6803.

Proteomes of an oxygenic photosynthetic cyanobacterium, Synechocystis sp. PCC 6803, were analyzed under photoautotrophic (low and high CO2, assigned as ATLC and ATHC), photomixotrophic (MT), and light-activated heterotrophic (LAH) conditions. Allocation of proteome mass fraction to seven sub-proteomes and differential expression of individual proteins were analyzed, paying particular attention to photosynthesis and carbon metabolism–centered sub-proteomes affected by the quality and quantity of the carbon source and light regime upon growth. A distinct common feature of the ATHC, MT, and LAH cultures was low abundance of inducible carbon-concentrating mechanisms and photorespiration-related enzymes, independent of the inorganic or organic carbon source. On the other hand, these cells accumulated a respiratory NAD(P)H dehydrogenase I (NDH-11) complex in the thylakoid membrane (TM). Additionally, in glucose-supplemented cultures, a distinct NDH-2 protein, NdbA, accumulated in the TM, while the plasma membrane-localized NdbC and terminal oxidase decreased in abundance in comparison to both AT conditions. Photosynthetic complexes were uniquely depleted under the LAH condition but accumulated under the ATHC condition. The MT proteome displayed several heterotrophic features typical of the LAH proteome, particularly including the high abundance of ribosome as well as amino acid and protein biosynthesis machinery-related components. It is also noteworthy that the two equally light-exposed ATHC and MT cultures allocated similar mass fractions of the total proteome to the seven distinct sub-proteomes. Unique trophic condition-specific expression patterns were likewise observed among individual proteins, including the accumulation of phosphate transporters and polyphosphate polymers storing energy surplus in highly energetic bonds under the MT condition and accumulation under the LAH condition of an enzyme catalyzing cyanophycin biosynthesis. It is concluded that the rigor of cell growth in the MT condition results, to a great extent, by combining photosynthetic activity with high intracellular inorganic carbon conditions created upon glucose breakdown and release of CO2, besides the direct utilization of glucose-derived carbon skeletons for growth. This combination provides the MT cultures with excellent conditions for growth that often exceeds that of mere ATHC.

Kinetics of petroleum oil biodegradation by a consortium of three protozoan isolates (Aspidisca sp., Trachelophyllum sp. and Peranema sp.)

Petroleum oil is a complex mixture of substances, the majority of which are hydrocarbons; the latter represent an extremely important and heterogeneous group of compounds that find their way into water resources by anthropogenic or natural ways. The majority of toxic hydrocarbon components of petroleum are biodegradable, where bioremediation using microbial species has become an integral process for the restoration of oil-polluted areas. In this study, three bioremediation processes, namely natural attenuation, nutrient supplementation by adding glucose and biostimulation by adding Tween® 80, were carried out in various petroleum hydrocarbon concentrations in polluted water media using a consortium of three protozoan isolates (Aspidisca sp., Trachelophyllum sp. and Peranema sp.). A first-order kinetics model was fitted to the biodegradation data to evaluate the biodegradation rate and to determine the corresponding half-life time. First-order kinetics satisfactorily described the biodegradation of the petroleum-based contaminants under abiotic conditions. The results showed an increase in the percentage removal of petroleum oil at the lower petroleum concentrations and a gradual percentage decrease in removing petroleum oil residues occurred when there was an increase in the initial concentrations of the petroleum oil: 39%, 27%, 22%, 12%, 10% for various petroleum oil concentrations of 50, 100, 150, 200, 250mg/L, respectively. A similar trend was also observed in the glucose-supplemented culture media where the reduction was 45% and 78% for petroleum concentrations of 250mg/L and 50mg/L, respectively. Biodegradation of between 33 and 90% was achieved at a Tween® 80 concentration of between 50mg/L and 250mg/L. The degradation rate constants for the natural attenuation process ranged between ≥0 to ≤0.50, ≥0 to ≤0.35, ≥0 to ≤0.25, ≥0 to ≤ 0.14 and ≥ 0 to ≤0.11 for petroleum oil concentrations varying from 50, 100, 150, 200 and 250mg/L, respectively, during the study after 30days. In the presence of glucose as a nutrient supplement, the degradation rate constants increased from 0day−1 to 0.8day−1 when exposed to the lowest oil concentration of 50mg/L, while the lowest rate constants (from 0day−1 to 0.25day−1) were observed at the highest petroleum oil concentration of 250mg/L for the same period. Overall enhancement of the degradation rates was achieved when adding the Tween® 80 surfactant compared to the first two biodegradation processes. The longest half-life was achieved after 217days during the natural attenuation process for samples with an oil concentration of 250mg/L and this was reduced to 85days using the glucose-supplemented process. There was a further decrease to 45days when Tween® 80 surfactant was added during the biostimulation process. The highest efficiency of ≥20% of Tween® 80 was observed between 6 and 18days and thereafter it decreased slightly to ≤20%.

Biomass and lipid production of a local isolate Chlorella sorokiniana under mixotrophic growth conditions

A local Chlorella sp. isolate with 97% rbcL sequence identity to Chlorella sorokiniana was evaluated in terms of its biomass and lipid production under mixotrophic growth conditions. Glucose-supplemented cultures exhibited increasing growth rate and biomass yield with increasing glucose concentration. Highest growth rate and biomass yield of 1.602day−1 and 687.5mgL−1, respectively, were achieved under 2gL−1 glucose. Nitrogen starvation up to 75% in the 1.0gL−1 glucose-supplemented culture was done to induce lipid accumulation and did not significantly affect the growth. Lipid content ranges from 20% to 27% dry weight. Nile Red staining showed more prominent neutral lipid bodies in starved mixotrophic cultures. C. sorokiniana exhibited enhanced biomass production under mixotrophy and more prominent neutral lipid accumulation under nitrogen starvation with no significant decrease in growth; hence, this isolate could be further studied to establish its potential for biodiesel production.

Effects of cell density, carbon dioxide and molybdenum concentration on biohydrogen production by Anabaena variabilis ATCC 29413

This paper aims to determine the effects of cell density, carbon dioxide, and molybdenum concentration towards hydrogen production rate. Batch cultures of Anabaenavariabilis sp. were incubated in anaerobic environment under continuous indoor illumination of 70μEm−2s−1 at 35°C. The optimal volumetric hydrogen production rate obtained was 44μmolH2mgchl a−1h−1 occurred at cells density of 110mgL−1, 5% carbon dioxide headspace concentration, and molybdenum concentration of 1.6mM. The effect of organic carbon source (glucose) was also evaluated in the present study and it was found that the additional carbon produced the highest hydrogen production rate in all conditions. An increased concentration of molybdenum significantly enhanced the hydrogen productivity rate almost to that of glucose-supplemented culture at 49μmolH2mgchl a−1h−1. However, further increase in molybdenum concentration beyond 1.6mM showed no further improvement in the amount of hydrogen produced.

Metabolomics-based component profiling of Halomonas sp. KM-1 during different growth phases in poly(3-hydroxybutyrate) production

To investigate the relationship between the production of poly(3-hydroxybutyrate) (PHB) and metabolic changes during different growth phases, a non-sterile batch fermentation process involving an alkalophilic and halophilic bacterium, Halomonas sp. KM-1, was used. Intracellular metabolites were analyzed using gas chromatography-mass spectrometry to characterize the metabolic profile. Significant changes relating to PHB production were observed in the TCA cycle, lipid-synthesis and amino acid biosynthetic pathways were found to shift dramatically between the exponential growth and stationary phases. During the stationary phase, 17 metabolites were upregulated and a cell dry mass of 17.8 g/L that included 44.8% PHB was observed at 24h in 5% glucose-supplemented cultures, whereas 11 metabolites were upregulated and a cell dry mass of 38.4 g/L that included 73.7% PHB was observed at 36 h in 10% glucose-supplemented cultures. This study provides pattern analysis of metabolite regulation during PHB accumulation, indicating that multicomponent and phase-specific mechanisms are involved.

Enhanced production of SCL-LCL-PHA co-polymer by sludge-isolated Pseudomonas aeruginosa MTCC 7925

To boost short-chain-length-long-chain-length polyhydroxyalkanoate (SCL-LCL-PHA) co-polymer yield in Pseudomonas aeruginosa MTCC 7925 by manipulating culture conditions. Stationary phase culture of P. aeruginosa MTCC 7925 was subjected to various doses of different carbons and, N and P deficiencies. Enhanced co-polymer yield was recorded under ethanol- and glucose-supplemented cultures. Interaction of ethanol with N-deficiency boosted co-polymer accumulation maximally. The sludge-isolated P. aeruginosa MTCC 7925 demonstrated good capability to synthesize SCL-LCL-PHA co-polymer from unrelated carbon sources. Supplementation of ethanol under N-deficiency boosted the co-polymer yield up to 69% of dry cell weight, which is significantly higher when compared with other SCL-MCL-PHA co-polymer accumulating bacterial species. This is the first report on P. aeruginosa MTCC 7925, which is capable of accumulating LCL 3-hydroxyhexadecanoate and 3-hydroxyoctadecanoate units with SCL 3-hydroxybutyrate and 3-hydroxyvalerate as constituents of PHAs. This opens up new possibilities for various industrial applications owing to the superior properties of this new co-polymer.

Interactions between organic and inorganic carbon sources during mixotrophic cultivation of Synechococcus sp.

Glucose at 3 g l(-1) markedly accelerated growth of Synechococcus sp. PCC 7002. The net photosynthesis rate was 263 micromol O2 (mg Chl a h)(-1) for mixotrophic culture and 146 micromol O2 (mg Chl a h)(-1) for photoautotrophic culture. Additing 1 g NaHCO3 l(-1) to the glucose-supplemented culture enhanced the photosynthetic rate by 18%, and the total carbon consumption rate was raised to 2.5 mg l(-1) (mg chl a h)(-1) from a previously negative value. An interaction between organic and inorganic carbon metabolism was established.

Ultrastructural observation of Helicobacter pylori in glucose-supplemented culture media.

Helicobacter pylori in the human gut can be divided morphologically into spiral and coccoid forms. The spiral form is known to change into the coccoid form in culture in vitro. The ultrastructural changes and culturability of H. pylori were studied in medium supplemented with different concentrations of glucose. H. pylori ATCC 43504(T) was cultured in liquid medium containing 10 % heat-inactivated horse serum supplemented with glucose (at 0, 10, 100, 300 and 500 mM) for 7 days. Bacterial ultrastructure and culturability were examined daily. With extended time in culture, the spiral forms had transformed into coccoid forms in all media. The coccoid forms could be further divided into two types, A and B, by electron microscopy. The type A coccoid form had an irregular surface with few flagella and indistinct cytoplasmic membrane. The type B coccoid form had a better-maintained integral membrane structure and was the dominant form in 300 mM glucose-supplemented medium. The highest culturability was obtained using 300 mM glucose-supplemented medium. Based on observations of ultrastructural changes in relation to the culturability data, the coccoid forms could be categorized into three stages: dying, viable but non-culturable and proliferating organisms. The optimal glucose concentration for H. pylori culture in this liquid medium culture experiment was approximately 300 mM.

Degradation of chlorinated guaiacols in anaerobic acetate enrichment conditions

Chlorinated guaiacols (4,5,6-trichloro-, 4,5-dichloro-, and 4-chloro-guaiacol) at 0.2 mM were completely degraded by anaerobic digester sludge within 4 d, but complete removal of TeCG could not be attained. The removal rates of chlorinated guaiacols by adsorption and biodegradation were in the following order: 4,5-dichloroguaiacol > 4-chloroguaiacol = 4,5,6-trichloroguaiacol > tetrachloroguaiacol. The most rapid initial TeCG degradation occurred in the glucose-supplemented cultures. However, the degradation rate of 4,5,6-TriCG was not increased significantly by supplementary glucose.

Proteinase Activity of Pseudomonas fluorescens Grown in Cold Milk Supplemented with Nitrogen and Carbon Sources

Extracellular proteinase activity varied when Pseudomonas fluorescens was grown at 7°C in UHT milk supplemented with different substrates. Addition of glutamine (5mM) and glutamic acid (10mM) reduced proteinase activity by 35 and 50%, respectively, and 6mM galactose and 10mM lactate supplements reduced activity to about one-half of that in the unsupplemented control. Glucose addition (9mM) delayed the onset of proteinase production by 24h compared with that for the control culture. These results suggest that the onset of proteinase production could depend on a repressor role of certain milk components or on a low energy status in Pseudomonas cells as a result of the exhaustion of the more easily used substrates. However, differences were not significant between intracellular concentrations of ATP for control and glucose-supplemented cultures.

Effects of Ketones, Acetate, and Glucose on In Vitro Immunoglobulin Secretion by Bovine Lymphocytes1,2

Individual and combined effects of ketones, acetate, and glucose on IgM secretion by bovine blood lymphocytes were evaluated in vitro. Supernatants from 14-d cultures of unstimulated and mitogen- or antigen-stimulated mono-nuclear leukocytes were harvested and analyzed for total and antigen-specific IgM. Ketones, acetate, and 1,3-butanediol individually added up to 6.25mM had no effect on total IgM secreted by cells grown in medium containing 11.1mM glucose. However, butyrate at 6.25mM inhibited IgM secretion. Addition of a mixture of ketones approximating plasma levels of severely ketotic cows inhibited mitogen-induced IgM Secretion in 11.1mM glucose-supplemented cultures. Results from experiments evaluating effects of glucose concentrations on IgM secretion indicated that plasma glucose concentration associated with the ketotic state (1.66mM), compared with normal plasma glucose concentration (3.33mM), did not affect total or antigen-specific IgM secretion. Supplementation of cultures containing up to 3.33mM glucose with ketones, acetate, or both either had no effect or a modest stimulatory effect. These data indicate that effects of ketones and acetate on IgM secretion are dependent on the concentration of glucose in culture and suggest that changes in plasma glucose, ketone, and acetate concentrations associated with bovine ketosis do not alter IgM secretion in vivo.

Biosynthesis of elsamicin A, a novel antitumor antibiotic.

The 1H noise-decoupled 13C-nmr spectrum of elsamicin A [1] prepared from the cultures of an unknown actinomycete species (ATCC 39417) supplemented with [1-13C]acetate and [2-13C]acetate showed enrichment of all 19-carbons in the aglycone. In addition, L-[methyl-13C]methionine- and D-[1-13C]glucose-supplemented cultures enriched the carbons of the two methyl groups on the disaccharide moiety and the C-1 carbons of the disaccharide, respectively. The results demonstrated that the aglycone of elsamicin A is derived entirely from acetate and the disaccharide portion is biosynthesized from two units of glucose and methionine.

Carbon catabolite regulation of neoviridogrisein production by glucose.

The production of neoviridogrisein II, a peptidolactone antibiotic, was suppressed by glucose in Streptomyces griseoviridus G-89. Pyruvic acid accumulated in the glucose-supplemented culture. The glucose effect was correlated with inhibition by pyruvic acid but not with low pH of the culture broth.