Effect Of Culture Age Research Articles

Effect of culture age and initial inoculum size on lipid accumulation and productivity in a hybrid cultivation system of Chlorella vulgaris

Chlorella vulgaris was cultivated in a hybrid (two-stage) system. The effect of the transferring time from nutrient-replete phase with a low light intensity (photobioreactor) to the nutrient deprivation phase (open pond) with a higher light intensity, as well as the effect of initial cell concentration in the deprivation phase, on the growth rate and lipid content of the microalgae was investigated. The microalgae were transferred to the nutrient deprived medium at different intervals with various initial cell concentrations. Transferring the cultivated medium of the 4th day with the initial cell concentration of 66mgL−1 into the deprivation phase resulted in a highest lipid productivity of 9.21±0.64mgL−1d−1 and lipid content of 53.97±4.30%. In a single-stage cultivation where a medium with nitrogen deficiency was used, despite a low lipid content (maximum 30%), a lipid productivity of 9.95±0.46mgL−1d−1 was observed that was slightly higher than that for the hybrid system.

The Effect of Culture Age and Initial Silver Concentration on Biosynthesis of Ag Nanoparticles

Many organisms or their extracts have the ability to reduce Ag + ions to Ag 0 and stabilize them what results in nanoparticle formation in solution. The aim of the article was to study the influence of two selected parameters - initial silver concentration and culture age, on Ag nanoparticles production by green algae Parachlorella kessleri. The presence of Ag nanoparticles in the solution was confirmed by the UV-vis spectroscopy and TEM analyses. Typical curve with the peak at app. 420 nm was found for nanoparticles produced by algae. While culture age did not have any significant effect, the initial silver concentration had significant influence on nanoparticle production which influenced the rate of nanoparticle production, their amount, their size and stability, as well.

Acute and sustained isoflurane neuroprotection: The effect of culture age and duration of oxygen and glucose deprivation

Objectives: Organotypic hippocampal slice (OHS) cultures provide the opportunity to dissect factors influencing volatile anaesthetic neuroprotective efficacy. It was hypothesized that three conditions—OHS culture age, oxygen glucose deprivation (OGD) duration and day of evaluation for cell death after OGD—influence isoflurane's ability to provide acute and sustained protection against OGD-induced cell death.Methods: OHS were prepared from PND 9–11 rat pups and maintained in vitro for 0.5, 1, 2 or 3 weeks. The slices were exposed to OGD for 0, 10, 30, 60 or 90 minutes with or without 1.5% isoflurane. Sytox staining was used to determine the amount of cell death on post-OGD days 1, 3 and 7 and was compared to the amount of cell death in culture–age matched controls (no OGD).Results: The duration of OGD necessary to produce cell death was inversely related to culture age. All culture ages showed evidence of both acute and sustained neuroprotection, but the magnitude of protection depended on OHS culture age, duration of OGD and post-OGD day of evaluation. In 1 and 2-week old slices early isoflurane neuroprotection was best observed with 90 minutes OGD and late isoflurane protection was best observed with 10 minutes OGD.Conclusions: In OHS, acute and sustained isoflurane neuroprotection in OGD-induced cell death is dependent on the conditions being studied.

In vitro callogenesis and detection of somaclonal variations in Plantago ovata L.

Planago ovata L. is an economically important species in the monotypic genus Plantago. It is a short-stemmed annual herb. The seed husk of this plant is commonly called psyllium or isabgol which is important in pharmaceutical formulation and food industry. In this study, callus induction was optimized using different explants of Plantago ovata. Callus DNA was utilized to access the somaclonal variations using the Random Amplification of Polymorphic DNA (RAPD) markers. The maximum callus growth was observed in Murashige and Skoog (MS) medium containing 4 mg L−1 2,4-D concentration for shoots, 0.5 mg L−1 for seeds and 2 mg L−1 for roots. Moreover, the effect of culture age was considered in assessing genetic variability. Maximum genetic variability was observed in the DNA samples of callus at the concentration of 2 mg L−1 2,4-D for all explants (roots, shoots, and seeds). Cluster analysis was performed based on 1) similarity coefficient between samples and 2) molecular data using the Numerical Taxonomy and Multivariate Analysis System (NTSYS) PC version 2.01; similarity index was generated by similarity for Quantitative Data (SIMQUAL). Our study indicated that Random Amplified Polymorphic DNAs can successfully be used to explore polymorphism among callus samples at different hormonal concentrations. This study can be useful for the production of callus from Plantago ovata and estimation of genetic variations due to tissue culture conditions. Evaluation of genetic variations can display novel features and manipulate genetic bottlenecks in Plantago ovata. New genetic variations in somaclones can bring vital insight for plant improvement.

Triacylglycerol accumulation and change in fatty acid content of four marine oleaginous microalgae under nutrient limitation and at different culture ages

Alteration of lipid biosynthesis is one of important biochemical changes when oleaginous microalgae grow under varied environmental conditions. The effects of culture age and nutrient limitation on triacylglycerol (TAG) accumulation and fatty acid content were investigated in four eicosapentaenoic acid (EPA)-rich marine microalgae. The amounts of TAGs in Chaetoceros sp., Phaeodactylum tricornutum and Nannochloropsis oculata increased sharply from day 4 to day 11, and then the former two remained nearly unchanged while the latter declined gradually during the batch culture. In contrast, no marked increase in TAG accumulation was observed in Pavlova viridis during the culture. Changes in total fatty acid (TFA) content mirrored those observed for TAG accumulation, while the EPA content reached a maximum generally at day 7 or 11 in the range of 11 - 32 mg g(-1) dry cell weight (DCW) and then declined. Nitrogen limitation led to a gradual increase in the amounts of TAGs from N. oculata pronouncedly but almost no change in other three species. The TFA content of the cultures after 5 days of nitrogen limitation was nearly twice that after 1 day in Chaetoceros sp., P. tricornutum and P. viridis, while the lowest increase (220 - 283 mg g(-1) DCW) was observed in N. oculata. TAGs increased gradually under phosphorus limitation in all four species but not sharply compared with that under nitrogen limitation in N. oculata. The TFA content increased gradually under phosphorus limitation and after 5 days of phosphorus limitation it was 1.5 - 2 times that after 1 day. The EPA content was generally not significantly affected by nitrogen or phosphorus limitation. Culture age and nutrient limitation could be useful variables for optimizing TAG accumulation and fatty acid content with potential for biodiesel production.

Fatty Acids Released by Chlorella vulgaris and Their Role in Interference with Pseudokirchneriella subcapitata: Experiments and Modelling

The role of extracellular fatty acids in the interference between two algae, Chlorella vulgaris Beijerink and Pseudokirchneriella subcapitata (Korshikov) Hindak, was assessed by the co-cultivation of the two selected strains, as well as by the chemical analysis of exudates from the culture media of single strain cultures. The effect of culture age and phosphate limitation was evaluated. The experiments showed that the composition and amount of fatty acids, released by C. vulgaris and by P. subcapitata, both in a batch and in a continuous monoculture, depend on the culture age and on the phosphate concentration in the culture medium. We also found that the amount of chlorellin generated in the two algae co-culture increased and was almost exclusively constituted by a mixture of C18 fatty acids. By using the evaluated concentrations of these fatty acids, an artificial chlorellin was prepared. The toxicity of this mixture to P. subcapitata appears to be similar to that of the natural chlorellin. For both algae, a stimulation of growth was observed at low concentrations of the natural chlorellin, whereas higher concentrations produced inhibitory effects on both species. However, P. subcapitata was much more sensitive than C. vulgaris. By using some of these new experimental results, two new mathematical models have been used to describe the toxicity of chlorellin to C. vulgaris and to the interference between C. vulgaris and P. subcapitata, respectively.

Culture age impacts Plectosporium alismatis propagule yields and subsequent desiccation and UV-radiation tolerance

The effect of culture age on yields, desiccation tolerance and resistance to ultraviolet radiation of Plectosporium alismatis, a potential mycoherbistat of aquatic weeds in Australian rice fields, was studied. P. alismatis was grown in a liquid basal medium supplemented with malt extract and sodium nitrate and harvested after 7, 14 or 21 days incubation. Although chlamydospore yields harvested from 14-day-old liquid cultures were significantly higher (29.2×105 chlamydospores mL−1) than chlamydospore yields harvested from 7-day-old liquid cultures (1.07×105 chlamydospores mL−1) or from 21-day-old liquid cultures, the germination of freshly-harvested chlamydospores from 7-day-old cultures (72.7%) was significantly reduced when propagules were grown for 14 days (55.3%). When exposed to UV-radiation, conidia and chlamydospores harvested from 14-day-old cultures germinated at a lower rate (<20%) than conidia and chlamydospores harvested from 7-day-old cultures (>40%). When conidia and chlamydospores were dried and subsequently exposed to UV, less than 30% of propagules harvested from 7-day-old cultures germinated, whereas less than 10% of propagules harvested from 14-day-old cultures germinated. A three-way analysis of variance including culture age, UV exposure and type of propagules confirmed that the culture age had more impact on the germination of fresh or dry propagules (P=0.00001 and P=0.0004, respectively) than the type of propagules considered (P=0.5). These results demonstrate that the culture age impacts significantly propagule yields and germination of P. alismatis conidia and chlamydospores, particularly after stress caused by dehydration and/or exposure to UV-B radiation.

Neutral Storage Lipids of Histoplasma capsulatum: Effect of Culture Age

Lipids contribute significantly to the pathogenesis of fungal infectious diseases and an understanding of lipid metabolism occurring in fungal pathogens can help the development of more efficient antifungal therapeutic strategies. In this study, the effect of culture age on the distribution of fatty acids among different neutral lipid (NL) classes in the dimorphic fungus Histoplasma capsulatum was investigated. Yeast cells of the G217B strain grown in two different media were collected after 4 and 7 days of growth, which roughly correspond to log and stationary culture growth phases, respectively. Neither culture age nor medium type had any influence on qualitative fatty acid (FA) profiles; however, the FA percentage composition varied with culture growth. A culture age-related decrease in the content of unsaturated FAs could be observed in all four of the NL classes examined, but the most intensive changes were detected in diacylglycerol and free FA fractions. Conversely, an increase in saturated FAs was observed. The transcriptional analysis of two major delta 9- and delta 12-FA desaturase genes, ode1 and sde1, showed no differences in their expression levels under experimental conditions. These results showing the dynamics of changes in FA composition in the NL fraction were concomitant with nutrient exhaustion in aging H. capsulatum cultures. Overall, the results presented in this work not only have implications for our knowledge of basic lipid biochemistry of H. capsulatum, but also will contribute to better understanding of biology and pathogenesis of this fungus and, consequently, can help in the discovery of more effective antifungal drugs.

Effect of culture age on mepanipyrim-mediated inhibition of pectinase secretion in Botrytis cinerea

The fungicide mepanipyrim tended to affect both mycelial growth and pectinase secretion of younger B. cinerea mycelia more strongly than older cultures, which is suggestive of its mode of action. Mepanipyrim was more effective in inhibiting pectinase secretion and in disease control activity against B. cinerea in the early stages of growth and infection. The uptake of mepanipyrim into the mycelia of B. cinerea was reduced as the culture aged and was inhibited by the mitochondrial uncoupling agent CCCP. This suggested that active energy metabolism results in greater mepanipyrim uptake, resulting in good inhibition of mycelial growth, pectinase secretion and disease control in younger B. cinerea.

Changes in the metabolome of associated with evolution in aerobic glucose-limited chemostats

The effect of culture age on intra- and extracellular metabolite levels as well as on in vitro determined specific activities of enzymes of central carbon metabolism was investigated during evolution for over 90 generations of Saccharomyces cerevisiae CEN.PK 113-7D in an aerobic glucose/ethanol-limited chemostat at a specific dilution rate of 0.052 h(-1). It was found that the fluxes of consumed (O2, glucose/ethanol) and secreted compounds (CO2) did not change significantly during the entire cultivation period. However, morphological changes were observed, leading to an increased cellular surface area. During 90 generations of chemostat growth not only the residual glucose concentration decreased, also the intracellular concentrations of trehalose, glycolytic intermediates, TCA cycle intermediates and amino acids were found to have decreased with a factor 5-10. The only exception was glyoxylate which showed a fivefold increase in concentration. In addition to this the specific activities of most glycolytic enzymes also decreased by a factor 5-10 during long-term cultivation. Exceptions to this were hexokinase, phosphofructokinase, pyruvate kinase and 6-phosphogluconate dehydrogenase of which the activities remained unchanged. Furthermore, the concentrations of the adenylate nucleotides as well as the energy charge of the cells did not change in a significant manner. Surprisingly, the specific activities of glucose-6-phosphate dehydrogenase (G6PDH), malate synthase (MS) and isocitrate lyase (ICL) increased significantly during 90 generations of chemostat cultivation. These changes seem to indicate a pattern where metabolic overcapacities (for reversible reactions) and storage pools (trehalose, high levels of amino acids and excess protein in enzymes) are lost during the evolution period. The driving force is proposed to be a growth advantage in the absence of these metabolic overcapacities.

Whole-organism Fingerprinting of the Genus Carnobacterium using Fourier Transform Infrared Spectroscopy (FT-IR)

Sixty seven strains of Carnobacterium, atypical Lactobacillus, Enterococcus durans, Lactobacillus maltaromicus and Vagacoccus salmoninarum were examined by Fourier transform infrared (FT-IR) spectroscopy. The effects of culture age and reproducibility over a six month period were also investigated. The results were analysed by multivariate statistics and compared with those from a previous numerical phenetic study, a pyrolysis mass spectrometry (PyMS) study and with investigations which used DNA-DNA and 16S rRNA sequencing homologies. Taxonomic correlations were observed between the FT-IR data and these studies. Culture age was observed to have little effect on the spectra obtained. The reproducibility study indicated that there was correlation between spectra produced on two occasions over the six month period. It was concluded that FTIR is a reliable method for investigating carnobacterial classification, and may have further potential as a rapid method for use in Carnobacterium identification.

Effects of Culture Age, Washing and Storage Conditions on Desiccation Tolerance of Colletotrichum truncatum Conidia

Colletotrichum truncatum conidia produced from a one week-old culture in a liquid semi-defined medium with a C:N ratio of 5:1 were more tolerant of desiccation than those harvested from two or three week-old cultures. Conidia washed with 20% (w/v) sucrose germinated better than unwashed conidia or those washed in 10% (w/v) sucrose, 10 and 20% (w/v) glucose or fructose, 0.1% (w/v) soluble starch, 0.9% (w/v) NaCl or deionized water. Washing with sucrose (20% w/v) also resulted in significantly longer germ tubes than those produced by unwashed conidia or conidia washed with deionized water or NaCl (0.9% w/v). Conidia washed twice in sucrose showed greater desiccation tolerance during storage at 15% relative humidity (RH) and 15°C than at 30% RH and 15 or 25°C or at 15% RH and 25, 5 or -10°C.

Effect of Culture Age on Protective Potency of a Vaccine against Bovine Ringworm Prepared of Trichophyton verrucosum

Rybnikafi A., J. Chumela, V. Vrzal: Effect of Culture Age on Protective Potency of a Vaccine against Bovine Ringworm Prepared of Trichophyton verrucosum. Acta Vet. Brno 2002, 71: 51-53. Calves vaccinated with live vaccine against bovine ringworm prepared of a two-week-old culture of Trichophyton verrucosum showed satisfactory protection against experimental dermatophytic infection. Vaccines prepared of cultures of the same strain aged 37, 65, or 92 days were less effective. Vaccination, Trichophyton verrucosum, challenge

The effect of thermal stress on Campylobacter coli.

Enteropathogenic Campylobacter jejuni, Camp. coli and Camp. lari are currently the most common cause of acute infectious diarrhoeal illness in the UK. Many domestic animals, including pigs, act as natural reservoirs for these organisms and infection may occur through the ingestion of contaminated foodstuffs. The safety of locally produced porcine liver was assessed in relation to the heat susceptibility of Campylobacter spp. present in eviscerated product. Heat susceptibility (D10) studies were performed on a wild-type strain of Camp. coli [NI39] isolated from porcine liver under standardized conditions. In addition, the effect of culture age and heating menstruum was determined. Thermal stress studies in phosphate-buffered saline showed Camp. coli NI39 to be heat sensitive (D10 = 8.-0, 30.8, 15.6, 10.3 s at 55.4, 57.4, 59.7, 61.2 degrees C, respectively; z = 6.10 degrees C). However, non-logarithmic biphasic survivor curves were observed at higher temperatures ( > 56 degrees C), indicating the presence of a heat-resistant subpopulation (10(4)-10(5) cfu) which was not demonstrated when examining either Salmonella typhimurium or Listeria monocytogenes. The use of D10 values may be limited. Therefore, porcine liver, under processing, must be treated as a potential source of Campylobacter spp., and clearly defined F-values should be quantified through the use of empirically 'spiked' samples to ensure the eradication of campylobacters from the product, for each individual process being evaluated. It is important to define safe processing parameters in the manufacture of products which receive mild thermal processes in order to eliminate the risk of disease to man.

Effect of culture age on the susceptibility of differentiating neuroblastoma cells to retinoid cytotoxicity

The cytotoxic effects of retinoids on neuroblastoma cells at various times during electrophysiological differentiation were evaluated. We used N1E-115, a clone of the murine neuroblastoma C1300 derived from the neural crest, and three retinoids: vitamin A (retinol), all-trans retinoic acid (tretinoin), and 13-cis-retinoic acid (isotretinoin). Differentiating N1E-115 cells exposed to retinoids at an isotretinoin EC(50) of 16 microM exhibited the greatest vulnerability in terms of cell death during a period (8 to 10 days) that was previously found to be the most sensitive for induction of gross malformations in rodents. This finding suggested possible similarities between the in vivo and in vitro retinoid mechanism(s) of action. The greatest period of vulnerability to retinoid cytotoxicity was also found to coincide with the rapid resting membrane potential (V(m)) development period, suggesting a linkage between neuronal V(m) and/or electrical excitability development and vulnerability to retinoid cytotoxicity.

Inhibitory metabolites production by the cyanobacterium Fischerella muscicola

Broad-spectrum inhibitory metabolites were produced by a benthic cyanobacterium Fischerella muscicola (UTEX 1829) in batch culture. These metabolites inhibited the growth of eukaryotic algae, cyanobacteria and eubacteria. The effect of culture age on the production and leakage of these inhibitory metabolites from the cyanobacterium was studied. Confirmation of the presence of an allelochemical, possibly fischerellin was achieved using high performance liquid chromatography (HPLC). The cyanobacterium produced inhibitory metabolites intracellularly at all stages of its growth cycle.

Effects of culture age on cryopreservation of marine microalgae

The effects of culture age (3, 7 and 14 days) on viability after cryopreservation of five marine microalgae were studied. Higher viability levels were generally found in older cultures, particularly when conditions of cryoprotectant concentration and salinity were not optimal. These differences decreased or disappeared when the algae were cryopreserved under optimal conditions, except for Rhodomonas baltica. Its cryopreservation using 15% dimethylsulphoxide in medium of 20%o salinity (optimal conditions) showed 451+1−3%, 424±133% and 350±13−7% viability respectively for cells from 3−, 7− and 14−day−old cultures. For the same culture ages, the viability of Chaetoceros gracilis was 257±7'7%, 2814−9% and 360±4−6%. This diatom could be recovered from −196°C without the use of any cryoprotectant only when cells were collected from 14−day−old cultures (mean viability of 31±3−7%). The cryopreservation of Tetraselmis chuii was relatively unaffected by culture age and was very close to 100% under optimum cryoprotedant concentration and salinity. Only in the absence of cryoprotectant were cells from the exponential phase (3 days) found to be more sensitive to cryopreservation (average viability 222±5−5%)than were cells from older cultures (372±149% and 42±126% for cells from 7− and 14−day−old cultures respectively). A lower resistance to cryopreservation was found in 3−day−old cultures of Nannochloris atomus (830±110%) in comparison with older cultures (100±78% and 949−±89% for 7− and 14−day−old cultures). The main increase in the viability of this alga with age was found in the absence of cryoprotectant and using a suboptimum salinity of 20%o. Three−day−old cultures of Nannochloropsis gaditana completely failed to recover after freezing under all conditions tested. Cells from 7− and 14−day−old cultures achieved mean viabilities of c. 67%.

Culture Age, Temperature, and pH Affect the Polyol and Trehalose Contents of Fungal Propagules

The growth and conidial physiology of the entomopathogenic fungi Beauveria bassiana, Metarhizium anisopliae, and Paecilomyces farinosus were studied under different conditions. The effects of culture age (up to 120 days), temperature (5 to 35(deg)C), and pH (2.9 to 11.1) were determined. Growth was optimal at pH 5 to 8 for each isolate and between 20 and 35(deg)C, depending on the isolate. The predominant polyol in conidia was mannitol, with up to 39, 134, and 61 mg g of conidia(sup-1) for B. bassiana, M. anisopliae, and P. farinosus, respectively. Conidia of M. anisopliae contained relatively small amounts of lower-molecular-weight polyols and trehalose (less than 25 mg g(sup-1) in total) in all treatments. Conidia of B. bassiana and P. farinosus contained up to 30, 32, and 25 mg of glycerol, erythritol, and trehalose, respectively, g(sup-1), depending on the treatment. Conidia of P. farinosus contained unusually high amounts of glycerol and erythritol at pH 2.9. The apparent effect of pH on gene expression is discussed in relation to the induction of a water stress response. To our knowledge, this is the first report of polyols and trehalose in fungal propagules produced over a range of temperature or pH. Some conditions and harvesting times were associated with an apparent inhibition of synthesis or accumulation of polyols and trehalose. This shows that culture age and environmental conditions affect the physiological quality of inoculum and can thereby determine its potential for biocontrol.

Characterization of Carnobacterium species by pyrolysis mass spectrometry

L.N. MANCHESTER, A. TOOLE AND R. GOODACRE. 1995. Forty‐eight strains of Carnobacterium were examined by pyrolysis mass spectrometry (PyMS). The effects of culture age and reproducibility over a 4 week period were also examined. The results were analysed by multivariate statistical techniques and compared with those from a previous numerical taxonomic study based on morphological, physiological and biochemical characteristics and with studies which used DNA‐DNA and 16S rRNA sequence homologies. Taxonomic correlations were observed between the PyMS data and the previous studies. Culture age was observed to have little effect on the mass spectra obtained and the reproducibility study indicated that there was very little variation over the 4 week period. It was concluded that PyMS provides a reliable method for studying carnobacterial classification and provides a rapid way for clarifying and refining subgeneric relationships within the genus Carnobacterium. Further work may also show that it offers a potentially very rapid and accurate method for the identification of Carnobacterium.

Improved transformation of Pseudomonas putida KT2440 by electroporation

An electric field-mediated transformation (i.e. electroporation) was performed to determine optimal conditions for P. putida transformation. The effects of culture age, electroporation buffer composition, electric field strength, pulse time constant and DNA concentration on transformation efficiency were examined. When plasmid DNA of 8 to 11 kb in size was used with an electroporation buffer containing 1 mM HEPES (pH 7.0), maximum transformation efficiency of 1.0 × 107 transformants/μg DNA was obtained at field strength of 12 kV/cm with pulse time of 2.5 millisecond. A linear increase in the number of transformants was observed as DNA concentration was increased over 4 orders of magnitude. A linear relationship was observed between growth phase and transformation efficiency up to OD600 = 2.0. This reliable and simple method should be useful for introduction of plasmid DNA into intact P. putida cells.