Seventh Day Of Cultivation Research Articles

Harnessing Diesel-Degrading Potential of an Antarctic Microalga from Greenwich Island and Its Physiological Adaptation.

Microalgae are well known for their metal sorption capacities, but their potential in the remediation of hydrophobic organic compounds has received little attention in polar regions. We evaluated in the laboratory the ability of an Antarctic microalga to remediate diesel hydrocarbons and also investigated physiological changes consequent upon diesel exposure. Using a polyphasic taxonomic approach, the microalgal isolate, WCY_AQ5_1, originally sampled from Greenwich Island (South Shetland Islands, maritime Antarctica) was identified as Tritostichococcus sp. (OQ225631), a recently erected lineage within the redefined Stichococcus clade. Over a nine-day experimental incubation, 57.6% of diesel (~3.47 g/L) was removed via biosorption and biodegradation, demonstrating the strain's potential for phytoremediation. Fourier transform infrared spectroscopy confirmed the adsorption of oil in accordance with its hydrophobic characteristics. Overall, degradation predominated over sorption of diesel. Chromatographic analysis confirmed that the strain efficiently metabolised medium-chain length n-alkanes (C-7 to C-21), particularly n-heneicosane. Mixotrophic cultivation using diesel as the organic carbon source under a constant light regime altered the car/chl-a ratio and triggered vacuolar activities. A small number of intracellular lipid droplets were observed on the seventh day of cultivation in transmission electron microscopic imaging. This is the first confirmation of diesel remediation ability in an Antarctic green microalga.

Исследования селекции микроорганизмов для разложения нефтепродуктов c повышенной нитрогеназной активностью в лабораторном биореакторе

The article presents the results of studies on decomposition of petroleum products, on the example of diesel fuel, the investigation having been carried out in a bioreactor with loading of immobilized microorganisms specially selected and adapted to the destruction of hydrocarbons. In the course of the investigation, 25 micro-organism culture strains were isolated from crude oil by selection, the strains had different destructive activity relative to hydrocarbons of the paraffin fraction of diesel fuel and nitrogen-fixing ability. Further, selection was carried out along with oxidation of hydrocarbons and according to the ability of microorganisms to nitrogen fixation. Such microorganisms during oxidation of hydrocarbons will allow reducing the content of biogens in purified water. Part of the microorganisms was identified and the nitrogenase activity of pseudomonades was investigated. When growing on diesel hydrocarbons in a bioreactor, nitrogen assimilation is most active in Rhodococcus erythopoltis R-2 culture and equals 10.52 nmol C2H4/mg protein per hour. The maximum nitrogenase activity has the association of bioreactor destructive cultures obtained by the authors, which, when growing on diesel oil products is 23.7 nmol C2H4/mg protein per hour, which is 2.3 times higher than the nitrogen-fixing capacity of Rhodococcus erythropoltis R-2. There was studied the rate of hydrocarbon degradation, оn the seventh day of cultivation, the degradation of paraffinic hydrocarbons in nitrogen-free media is 68%, whereas in the presence of ammonium nitrogen it reached - 87 %. These investigations have determined the ability of isolated cultures of oil-oxidizing bacteria to use hydrocarbons under atmospheric nitrogen assimilation conditions.

Evaluation of laccase production by Ganoderma lucidum in submerged and solid-state fermentation using different inducers.

Laccases are multicopper oxidases with high potential for industrial applications. Several basidiomycete fungi are natural producers of this enzyme; however, the optimization of production and selection of inducers for increased productivity coupled with low costs is necessary. Lignocellulosic residues are important lignin sources and potential inducers for laccase production. Pinus taeda, a dominant source of wood-based products, has not been investigated for this purpose yet. The aim of this study was to evaluate the production of laccase by the basidiomycete fungus Ganoderma lucidum in the presence of different inducers in submerged and solid-state fermentation. The results of submerged fermentation in presence of 5 μM CuSO 4 , 2 mM ferulic acid, 0.1 g/L P. taeda sawdust, or 0.05 g/L Kraft lignin indicated that although all the tested inducers promoted increase in laccase activity in specific periods of time, the presence of 2 mM ferulic acid resulted in the highest value of laccase activity (49 U/L). Considering the submerged fermentation, experimental design following the Plackett-Burman method showed that the concentrations of ferulic acid and P. taeda sawdust had a significant influence on the laccase activity. The highest value of 785 U/L of laccase activity on submerged fermentation was obtained on the seventh day of cultivation. Finally, solid-state fermentation cultures in P. taeda using ferulic acid or CuSO 4 as inducers resulted in enzymatic activities of 144.62 and 149.89 U/g, respectively, confirming the potential of this approach for laccase production by G. lucidum.

CU (II) ACCUMULATION BY MARINE NEUTROPHIL SULFUR-OXIDIZING BACTERIA

Aim: to determine the ability of neutrophilic sulfur-oxidizing bacteria to extract Cu (II) ions from aqueous solutions. Methods. The object of the study was the ability of marine sulfur-oxidizing bacteria isolated from the water of the Black Sea near the Odessa Bay to extract copper from aqueous solution. To study the metalaccumulating activity of sulfur-oxidizing bacteria, we have selected strains that were resistant to copper at the level of 0.01–0.02 mol/l. The initial concentration of Cu (II) in aqueous solution was 1 mM. The period of cultivation of microorganisms in aqueous solution containing copper was 10 days. The residual content of Cu (II) in aqueous solution was determined by the atomic absorption method on AAS-1 devices (Germany) and C-115PC Selmi (Ukraine) at the wavelength of 324.7 nm for Cu. The reliability of the results obtained was assessed on the Student's criterion with probability of p <0.05. Results. It was established that sulfur-oxidizing bacteria isolated from the water of the Black Sea capable of accumulating heavy metals from aqueous solutions, and Cu-accumulating activity depends on the duration of the interaction between microorganisms and solution. The maximum level of extraction of Cu (II) from aqueous solution was equal 89.24% was recorded on the 10th day of cultivation using a strain of Thiobacillus sp. DKZ_4. On the seventh day of cultivation, the extraction of Cu (II) from aqueous solution with using a strain of Thiobacillus sp. DKZ_2 reached 85.25%. It was shown that the level of resistance to copper, which had been previously defined, did not affect the ability of bacteria to accumulate copper. Conclusions. Neutral sulfur-oxidizing bacteria isolated from the water of the Black Sea are capable of extracting from aqueous solution from 22.83% to 89.24% of Cu (II) and therefore they can be used to develop biosorption biotechnology especially in the field of water purification from heavy metals. The ability to accumulate Cu depends on the strain, but does not depend on MIC.

CULTIVATION OF HUMAN LIVER CELLS AND ADIPOSE-DERIVED MESENCHYMAL STROMAL CELLS IN PERFUSION BIOREACTOR

Aim:to show the progress of the experiment of cultivation of human liver cells and adipose-derived mesenchymal stromal cells in perfusion bioreactor.Materials and methods.The cultivation of a cell-engineered construct, consisting of a biopolymer microstructured collagen-containing hydrogel, human liver cells, adipose-derived mesenchymal stromal cells, and William’s E Medium, was performed in a perfusion bioreactor.Results.On the 7th day large cells with hepatocyte morphology – of a polygonal shape and a centrally located round nucleus, – were present in the culture chambers of the bioreactor. The metabolic activity of hepatocytes in cell-engineered constructs was confi rmed by the presence of urea in the culture medium on the seventh day of cultivation in the bioreactor and by the resorption of a biopolymer microstructured collagen-containing hydrogel.

Effect of alcohol on insulin secretion and viability of human pancreatic islets

Introduction/Objective. There are controversial data in the literature on the topic of effects of alcohol on insulin secretion, apoptosis, and necrosis of the endocrine and exocrine pancreas. The goal of this research was to determine how alcohol affects the insulin secretion and viability of human adult pancreatic islets in vitro during a seven-day incubation. Methods. Human pancreatic tissue was digested with Collagenase XI, using a non-automated method. Cultures were incubated in Roswell Park Memorial Institute (RPMI) medium containing alcohol (10 ?l of alcohol in 100 ml of medium). Insulin stimulation index (SI) and viability of the islets were determined on the first, third, and seventh day of cultivation. Results. Analysis of the viability of the islets showed that there wasn?t significant difference between the control and the test group. In the test group, viability of the cultures declined with the time of incubation. SI of the test group was higher compared to the control group, by 50% and 25% on the first and third day of cultivation, respectively. On the seventh day, insulin secretion was reduced by 25%. The difference was not statistically significant (p > 0.05). In the test group, significant decline in insulin secretion was found on the third and seventh day of incubation (p ? 0.05). Conclusion. Alcohol can increase or decrease insulin secretion of islets cultures, which may result in an inadequate response of pancreatic ?-cells to blood glucose, leading to insulin resistance, and increased risk of developing type 2 diabetes.

Sulfur reducing bacteria from coal pits waste heaps of Chervonograd mining region

Sulfur reducing bacteria from coal pits waste heaps of Chervonograd minig region were isolated and their seasonal number changes were established. Sulfur reducing bacteria number increases during cold season both at the end of vegetation season, depending on gangue humidity as well as on substrate temperature. The forty sulfur reducing bacteria isolates were selected. According to the highest biomass acumulation and hydrogen sulfide production for the following identification two strains were chosen. The morpho-physiological characteristics of isolated strains SV 30 and SV 35 were investigated. In accordance with obtained data, we assumed isolated strain SV 30 to be identified as genus Desulfuromusa , meanwhile SV 35 – Geobacter . After the seventh day of cultivation, the highest sulfur reducing activity of both strains was observed. Due to the metabolization ability of wide range of pollutants isolated sulfur reducing bacteria are perspective for application in environmental remediation technologies with biological methods.

The effect of lead on the growth, content of primary metabolites, and antioxidant response of green alga Acutodesmus obliquus (Chlorophyceae)

Green unicellular alga Acutodesmus obliquus (Turpin) Hegewald et Hanagata (SAG strain no. 276-6) (Chlorophyceae) was used for determination of phytotoxicity of lead (Pb) at the range of concentrations 0.01-500 μM during 7 days of culture. The accumulation of Pb in algal cells was found to be increased in a concentration- and duration-dependent manner. The highest Pb uptake value was obtained in response to 500 μM Pb on the seventh day of cultivation. The decrease in the number and the size of cells and the contents of selected primary metabolites (photosynthetic pigments, monosaccharides, and proteins) in A. obliquus cells were observed under Pb stress. Heavy metal stimulated also formation of reactive oxygen species (hydrogen peroxide) and oxidative damage as evidenced by increased lipid peroxidation. On the other hand, the deleterious effects of Pb resulting from the cellular oxidative state can be alleviated by enzymatic (superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase) and non-enzymatic (ascorbate, glutathione) antioxidant systems. These results suggest that A. obliquus is a promising bioindicator of heavy metal toxicity in aquatic environment, and it has been identified as good scavenger of Pb from aqueous solution.

Effects of Candida on Insulin Secretion of Human Adult Pancreatic Islets and Possible Onset of Diabetes

This study aims to determine the origin of Candida contamination of pancreatic tissue cultures, as well as its influence on insulin secretory activity of the pancreatic islets. Pancreatic tissue was obtained after pancreatectomy in patients who had chronic pancreatitis or benign tumours. Islets were isolated under aseptic conditions by a manual method. Microbiological analysis was performed by standard procedures and secretory activity was determined on the first, third and seventh day of cultivation. Insulin stimulation index (SI) on the first day of incubation was 0.665±0.082 and 0.982±0.167 for sterile and infected cultures, respectively (expressed as means±SE). On the third day of cultivation, the SI for sterile cultures was 0.645±0.071 while these value were higher in contaminated cultures (1.252±0.413). On the seventh day, SI was 0.853±0.032 and 1.239±0.169 for sterile and infected cultures, respectively (P=0.05). Analysis of results for the first, third and seventh day of incubation and comparison of both groups showed that SI was 0.721±0.041 for sterile cultures, while for contaminated cultures it was higher by 37.68% (SI =1.157±0.154; P=0.01). The results show that cell culture contamination originates from an original pancreatic tissue infection, and that Candida can provoke an elevated level of insulin secretion in such patients, thus increasing chances for the onset of diabetes.

Production of Geotrichum candidum polygalacturonases via solid state fermentation on grape pomace

AbstractGeotrichum candidum CCY 16-1-29 (teleomorph Galactomyces geotrichum) is able to grow and produce polygalacturonase of remarkable activities on pectin or grape pomace as a sole carbon source. The highest activities of extracellular enzymes were found on the third and the seventh day of cultivation. After extraction and precipitation, polygalacturonases produced in these cultivation periods were characterized. Production of multiple forms of polygalacturonase was observed in both cultivation periods. Two major forms, polygalacturonase with random action pattern (endo-PGase, EC 3.2.1.15) and oligogalacturonate hydrolase (exoPGase, exopolygalacturonase preferring oligogalacturonides as substrates), as well as numerous minor forms were detected by IEF-PAGE using the print technique detection. EndoPGase was identified by mass spectrometry. The major forms have similar isoelectric points (below pH 6.0) and pH optima (4.6 and 4.8, respectively). pH optimum of 4.6 was associated with exoPGase and that of 4.8 with endoPGase. Both enzymes were stable after freeze-drying and storage at 4°C. EndoPGase had molecular mass of about 29 kDa (36 kDa by SDS-PAGE) as determined by gel filtration, temperature optimum of about 45°C and it was stable only below 35°C. Molecular mass of exoPGase was about 50 kDa, its temperature optimum was about 60°C, and it was stable to 60°C. Optimal substrate for exoPGase was a pentamer, for endoPGase it was a pectate. Values of K m for optimal substrate reached the values of 11.4 × 10−5 M for for exoPGase and 6.6 × 10−5 M for endoPGase. Pectin methylesterase as another pectolytic enzyme was also identified by mass spectrometry.

Effects of bacterial infection on insulin secretory capacity of human adult pancreatic islets

This study aims to determine the origin of bacterial contamination of pancreatic tissue cultures, as well as its influence on insulin secretory activity (expressed as stimulation index [SI]) of the pancreatic islets. Pancreatic tissue was obtained after pancreatectomy in patients who had chronic pancreatitis or benign tumours. Islets were isolated under aseptic conditions by a manual method. Microbiological analyses were performed by standard procedures and the SI was determined on the first and seventh day of cultivation. In cultures contamminated by Pseudomonas, SI was 1.58±1.16 on day 1 and 0.22±0.14 on day 7 (P<<0.01). Cultures contaminated by Enterobacter showed an SI of 0.21±0.1 on day 1, which increased to 1.19±0.66 on day 7 (P<0.01). In cases of Staphylococcus contamination, SI was 0.07±0.05 on day 1 and 0.33±0.21 on day 7 (P<<0.01). The study shows that cell culture contamination originates from an original pancreatic tissue infection. The presence of bacteria may reduce or increase insulin secretion in cell culture, depending on the type of microorganism, and this can provoke reduced or elevated levels of insulin secretion in recipients, thus increasing the chances for the onset of diabetes.

Interaction of Trace Elements and Ligninolytic Enzymes in Pleurotus eryngii

Considering the fact that some trace elements have influence on the growth and physiological processes in the fungi, the aim of this study was to resolve the question of whether Fe, Zn, and Se, added to the medium in the different forms, affect activity of selected Pleurotus eryngii ligninolytic enzymes participating in mandarine peels submerged fermentation. Compared with the control, Fe and Zn showed stimulatory effect on laccase activity. Reduction in the activity was emphasized with cultivation time, but it was slower in the presence of Zn than in the presence of Fe. Among studied Se forms, SeO2 was the most unfavorable Se source for laccase activity. All tested trace elements, independent on form, showed stimulatory effect on the activity of Mn-oxidizing peroxidases on the seventh day of cultivation. Based on the results, it could be concluded that the presence of the tested trace elements can advance various biotechnological processes among which is biodegradation of agricultural wastes, potential environmental pollutants.

Influence of the purification of human adult pancreatic islets on insulin secretion

The most effective method for human adult pancreatic islets purification is density-gradient centrifugation. The aim of this study was to analyze the effects of non-automated purification on preservation of functional capacity of human adult pancreatic islet cells. Human pancreata were obtained after pancreatectomy in the patients with chronic pancreatitis or benign tumors. Pancreatic islets were purified by non-automated method in discontinuous Ficoll density gradient. The samples were divided in 2 fractions: purified (P) and non-purified (NP) cultures. Islets were stained with diphenyl-thiocarbazone. The efficiency of separation was determined by comparing percentage of stained cells in P and NP cultures on day 1, 3 and 7 of shortterm cultivation. Glucose-stimulated insulin secretion was expressed as stimulation index (SI). The results obtained showed a statistically significant difference (p < 0.01) between P and NP cultures. P cultures had higher percentages of stained cells (70.43 +/- 3.97%, 73.77 +/- 4.22% and 71.34 +/- 4.69% on the first, third and seventh day of cultivation, respectively) than NP cultures (53.68 +/- 1.71%, 57.14 +/- 3.94% and 43.97 +/- 4.56%, respectively). P cultures had higher values of SI for the first, third and seventh day of cultivation than NP cultures (0.45 +/- 0.08, 0.80 +/- 0.21, 1.28 +/- 0.15 and 0.46 +/- 0.10, 0.752 +/- .0.16, 0.76 +/- 0.11 for P and NP cultures respectively). The difference was statistically significant on day seven (p = 0.01). Although during purification process islets were exposed to a number of insults that might result in cellular damage and functional impairment, our assessments showed that islets in P cultures preserved their functional capacity better than islets in NP cultures, since they had greater insulin secretion.

Glucocorticoid-induced growth inhibition of human neoplastic salivary gland duct cell line (HSG).

The purpose of this study was to examine the effect of glucocorticoid on human neoplastic salivary duct epithelial cell line (HSG). Dexamethasone was found to inhibit cell growth and to increase cell size and the ratio of protein content to DNA content in a cell. The inhibition of cell growth was dose-dependent; in comparison to the control (33.8 +/- 3.1 h), the population doubling time was 1.57-fold longer in 10(-5) M dexamethasone (P less than 0.01, N-K test). [3H] thymidine incorporation was inhibited in 45.5% of the control at 10(-5) M. Plating efficiency was 20.5 +/- 3.0% in 10(-5) M and 47.0 +/- 4.4% in the absence of dexamethasone. Cell diameters increased 1.29 fold in 10(-5) M dexamethasone in comparison to the control size (16.0 +/- 2.1 micron). The ratio of total protein content of DNA content increased 1.46 fold in 10(-5) M dexamethasone-treated cells on the seventh day of cultivation. Scatchard plot analysis using [6, 7-3H]-triamcinolone revealed that the HSG cells had apparent cytosolic glucocorticoid receptors with an equilibrium dissociation constant (Kd value) of 6.48 nM, whose number of binding sites (NBS) was 57.8 fmol/mg protein.

Citrate regulation of NADP+-specific isocitrate dehydrogenase of Aspergillus niger [proceedings

Many theories have been proposed to account for the accumulation of citrate in selected strains of Aspergillus niger (Berry et al., 1977). Citrate accumulation was attributed by Ramakrishnan et al. (1955) to loss of aconitase and isocitrate dehydrogenase activity, but the presence of these enzymes was clearly demonstrated by La Nauze (1966). The presence of tricarboxylic acid-cycle enzymes throughout the fermentation was further demonstrated by Ahmed et al. (1972), using intact mitochondria. Mattey (1977) reported the presence of an NADP+-specific isocitrate dehydrogenase in mitochondria of an A . niger strain during the phase of growth in which citrate was produced, which was inhibited by citrate, but was not affected by other tricarboxylic acid-cycle intermediates. He postulated that the regulation of isocitrate dehydrogenase activity by citrate could form the basis for the control of citrate production. If this is correct, then citrate inhibition of isocitrate dehydrogenase should be found in other strains of A. niger. In the present communication we report on the effect of citrate on NADP+-specific isocitrate dehydrogenase enzymes from two strains, one of which (72-4) can yield commercially useful quantities of citrate under suitable conditions, the other normally cultured for cellulolytic enzymes (50-565 ii). Stock cultures of A. niger strain 72-4 of Shu & Johnson (1947) and A . niger Van Tieghem strain C.M.I.50-565 ii (Commonwealth Mycological Institute, Kew, Surrey, U.K.) were maintained on Czapek-Dox agar. The organisms were grown in a highsucrose medium (Ahmed et al., 1972) in shake culture from an inoculum of 5 x lo7 conidia. Changes in mycelial weight, medium pH and citrate concentrations in the medium and mycelium were determined over an incubation period of 14 days. Citrate was determined by the method of Saffran & Denstedt (1948), modified by Spencer & Lowenstein (1967). Both strains showed similar batch culture growth kinetics. A . niger strain 72-4 showed a marked increase in citrate concentration in the mycelium from an initial value of 0.5mM to a maximum of 4 . 6 6 m ~ by the fifth day of cultivation, when citrate accumulation in the medium became significant. A. niger strain C.M.I. 50-565 ii showed no significant variation in citrate concentration in the mycelium, reaching 0.55 mM by the seventh day of cultivation. There is thus an 8-fold difference in internal citrate concentrations between the two strains. The pH of the medium fell from 3.1 to 1.9 during the first 4 says in both strains. The internal pH of the mycelium did not seem to be affected by the external pH under these conditions. Strain 72-4 produced 2.5g of citrate per g of dry mycelium by day 10 of cultivation, and strain C.M.I. 50-565 ii produced 0.609g of citrate per g of dry mycelium at this time. It would therefore appear that citrate concentrations in the medium and the mycelium are related. Mitochondria were isolated and disrupted as previously described (Mattey, 1977). Isocitrate dehydrogenase activity was assayed by the method of Ochoa (1948), with NADP+ and D,-threo-isocitrate as substrates. The NADP+-specific isocitrate dehydrogenase activity from mitochondria, isolated from day-6 mycelia, was inhibited by citrate, but was not affected by malate, fumarate, succinate, a-oxoglutarate or pyruvate. Fig. 1 shows a double-reciprocal (LineweaverBurk) plot of NADP+-specific isocitrate dehydrogenase activity versus substrate concentration, in the presence and absence of citrate, from both strains. It was previously reported that citrate inhibition of the NADP+-specific isocitrate dehydrogenase appeared to be competitive, but it is apparent that at higher isocitrate concentrations the enzyme does not obey conventional Michaelis-Menten kinetics. The kinetic data suggested that two similar enzymes were acting on the same substrate, but

An interferon-like substance released from tickborne encephalitis virus-infected chick embryo fibroblast cells.

AN interference phenomenon between tick-borne encephalitis and Western equine encephalomyelitis viruses in chick embryo fibroblast monolayers has been described1. When chick embryo tissue cultures are inoculated with tick-borne encephalitis virus, the virus multiplies readily, but no cytopathic effect appears until the sixth or seventh day of cultivation. If cultures thus infected had been superinfected with a convenient dose of Western equine encephalomyelitis virus, which normally completely destroys the cells within 48–72 hr., the following events could be observed : (1) complete degeneration of cells caused by this virus and, hence, no interference if tick-borne encephalitis virus-infected cultures were challenged with Western equine encephalomyelitis immediately or 24 hr. after primary inoculation; (2) partial interference if cultures were challenged 48 hr. after inoculating them with tick-borne encephalitis virus ; (3) complete suppression of the cytopathic effect caused by Western equine encephalomyelitis virus if challenged 72 hr. after primary inoculation. No interference was established if interfering virus previously neutralized by immune serum was added to cultures.