Ammonia In Cultures Research Articles

Oleaginous yeast Cryptococcus curvatus for biofuel production: Ammonia’s effect

Abstract Culturing oleaginous yeast with organic residues can provide cheap feedstock for biodiesel production, but the high content of nitrogen in some feedstocks (such as food residues) can lead to inhibition to cell growth and lipid synthesis, and this may be caused by ammonia. Thus, the inhibitory effect of ammonia on the growth of yeast Cryptococcus curvatus and its possible mechanism were evaluated in this study. The biomass production, lipid contents, affected metabolic pathways, and activities of the related pathway enzymes were investigated. The results showed that the yeast biomass production on acetate decreased from 5.9 kg m −3 to 2.0 kg m −3 when the initial ammonia concentration (in nitrogen equivalent terms) increased from 131 g m −3 to 3140 g m −3 . Ammonia significantly inhibited the yeast growth with volatile fatty acids (VFA) as carbon sources, but not in the culture with pyruvate generating substances (PGS) as carbon sources. However, the fatty acid synthesis was suppressed by ammonia in cultures with either VFA or PGS. The study on mechanism of ammonia inhibition suggested it might include an inhibition on the acyl-CoA synthetase activity, since a 61% decrease in the enzyme activity was found when 3140 g m −3 of ammonia (in nitrogen equivalent terms) was added. This study firstly reported the effect of ammonia with high concentrations to oleaginous yeast growth and lipid accumulation, and provided preliminary evidences on its possible mechanism of acyl-CoA synthetase inhibition.

Production of ammonia by Tritrichomonas foetus and Trichomonas vaginalis.

Production of ammonia is difficult to find among the various studies of amino acid metabolism in protozoa. Several studies suggest that catabolism of arginine to ammonium is important for the growth of trichomonads. Trichomonads are amitochondriate zooflagellates that thrive under microaerophilic and anaerobic conditions. The authors were able to detect accumulation of ammonium ions and ammonia in cultures of Tritrichomonas foetus and Trichomonas vaginalis, including those resistant to metronidazole. Ammonium ions and ammonia were detected using the indophenol colorimetric method. Cells incubated overnight under an ambient oxygen gas phase had 0.9 mM soluble ammonium (NH(4)(+) and NH(3)) or a 20 % greater concentration of ammonium relative to sterile growth medium that had been incubated similarly. Production of ammonia itself was confirmed by analysis of a wick that was moistened with sulfuric acid (20 mM) and placed above the liquid in sealed cultures of a strain of Trichomonas vaginalis. The wicks from these cultures captured the equivalent of 0.048 mM volatile ammonia (NH(3)) from the liquid as compared to 0.021 mM volatile ammonia from sterile medium after overnight incubation. Intact trichomonads, 0.7 x 10(6) cells ml(-1) equivalent to 0.7 mg protein ml(-1), incubated in Doran's buffer with or without (1 mM) L-arginine produced significant amounts of soluble ammonium (0.07 mM and 0.04 mM, respectively) during 60 min. The results indicate that ammonium ions and the more irritating ammonia are significant metabolites of trichomonads. In addition, based upon end-product amounts, it appears that the rate of arginine metabolism is of the same order of magnitude as that for carbohydrate metabolism by trichomonads.

Effects of ammonia and glucosamine on the heterogeneity of erythropoietin glycoforms.

Recombinant human erythropoietin (EPO) is a glycoprotein produced as a therapeutic agent from mammalian cell cultures for the treatment of anemia associated with severe kidney damage. The EPO structure has a high glycan content which is essential for bioactivity but shows considerable molecular heterogeneity. The cell culture conditions that affect the heterogeneity of the glycoforms of EPO are not well understood. However, the accumulation of ammonia in culture is one factor that has been associated with an enhanced heterogeneity of glycoforms. In this report we investigate the metabolic perturbations associated with ammonia and glucosamine that may give rise to an altered pattern of EPO glycosylation. Recombinant human erythropoietin was synthesized in serum-free cultures of transfected Chinese hamster ovary (CHO) cells. The molecular heterogeneity of erythropoietin was increased by supplementation of cultures with either ammonia or glucosamine. The enhanced molecular heterogeneity was shown to be due to variable glycosylation that resulted in EPO with an enhanced molecular weight and isoelectric point range. Enzymatic removal of the glycan moiety of EPO in all cases resulted in a single molecular form with a molecular weight of 18 000, which corresponded to non-glycosylated EPO. The variable glycosylation was consistent with reduced sialylation and antennarity of the carbohydrate structures present on the three N-linked sites of EPO. In the presence of ammonia (>30 mM) the proportion of tetrasialylated and tetraantennary glycan structures were reduced by 73% and 57%, respectively, as determined by HPLC analysis. Such changes were also observed, although to a lesser extent (41% and 37%), by an increase in the glucosamine concentration (>10 mM) in the medium. The enhanced heterogeneity of the glycan structures coincided with a significant increase in the intracellular UDP-N-acetylhexosamine (UDP-GNAc) pool. The measured UDP-GNAc level was up to 2 orders of magnitude higher in the presence of either glucosamine or ammonia. However, the changes in the glycosylation profiles induced by either glucosamine or ammonia were significantly different even at the same intracellular UDP-GNAc concentration. This suggests that the enhanced EPO heterogeneity could not be mediated solely by the increased UDP-GNAc level. Glucosamine (but not ammonia) was shown to cause significant inhibition of glucose transport into the cells, which could induce a different pattern of primary metabolism.

The effect of steer sera generating low or high concentrations of ammonia in culture on bovine embryo development and cell number in vitro

Ovine embryos produced in synthetic oviduct fluid (SOF) medium or in coculture with granulosa cell monolayers supplemented with low (A; 120 μmol/l) and high (B; 190 μmol/l) ammonia-producing steer sera caused different degrees of fetal oversize (Carolan et al., 1998). The objective of the present study was to determine whether the effects on fetal growth induced by these sera were associated with alterations in early embryo development.A total of 911 bovine oocytes, used in 8 replicates to test the effect of three culture treatments on embryo development, were matured and fertilized in vitro (IVF= Day 0). Presumptive zygotes were allocated on Day 1 to culture in SOF supplemented with 10% v/v steer serum (SOF+A, n=308; SOF+B, n=302) or with amino acids plus 0.4% w/v crystalline BSA (SOFaaBSA, n=301). All cultures were in 20 μl droplets under oil (38.5°C; 5% CO2, 5% O2; 4 zygotes per drop) and droplets were renewed every 48 h. Cleavage rate was recorded on Day 3. On Days 7 and 8, blastocyst yields, grade 1 and 2 blastocysts, their cell numbers (by staining with Hoechst 33342) and their stage and diameter were determined.

Growth inhibition in animal cell culture

Eight independent cell lines accumulated ammonia in culture to concentrations between 1.3 and 2.9 mM. The growth inhibition of such concentrations of ammonium chloride when added to culture medium was variable. The cell lines tested could be divided into 3 groups depending on their growth response to 2 mM added NH4Cl. In the first group (293, HDF, Vero, and PQXB1/2) little (less than 14%) or no growth inhibition occurred. In the second group (McCoy and MDCK) a reduction in final cell yield of 50-60% was observed. The third group (HeLa and BHK) was most sensitive to the effects of NH4Cl with growth inhibition (greater than 75%) compared to controls. The growth inhibitory effect of added lactate up to 20 mM was negligible (less than 10%) for 3 cell lines, although one cell line (PQXB1/2) showed greater sensitivity. The interactive effects of ammonia and lactate were determined in a matrix experiment. At lactate (greater than 12 mM) and ammonia (1-4 mM), the growth inhibitory effects of the two components were synergistic. However, at low concentrations of lactate (less than 12 mM) the toxic effect of ammonia was reduced. A proposed mechanism for the sparing effect of lactate on ammonia toxicity is discussed. This may have importance in developing strategies for the optimal growth of ammonia-sensitive cell lines.

Ecotypic differentiation in response to nutritional factors in the algal genusStigeocloniumKütz. (Chlorophyceae)

Growth responses of several Stigeoclonium strains to increasing levels of phosphate, ammonia and chloride in the culture medium were studied in an attempt to explain the distribution of the strains according to the nutrient levels of their habitats. The strains show relatively high saturation values and tolerances towards phosphate and ammonia, which is in accordance with the often severe eutrophication at their locations. No close correlation between demand or tolerance to phosphate and ammonia in culture and the field situation was found, apart from some incidental cases. With regard to chloride a positive correlation between salinity tolerances of the strains and mean chloride levels in the field could be established. Apparently chloride is an important factor in the distribution of at least several of the tested strains. One strain, originating from a brackish pond (5·5 g 1-1 chloride), was found to be clearly adapted. It grew well in medium with 10 g 1-1 chloride, a concentration at which the other strains did not survive.

A Salicylate–Hypochlorite Method for Determining Ammonia in Seawater

Total ammonia-nitrogen (NH4-N) in artificial seawater was determined spectrophotometrically by a method in which indophenol blue is produced by the reaction of ammonia with salicylate and hypochlorite, in the presence of sodium nitroprusside. Recoveries of 10 to 1000 μg NH4-N/L by the salicylate– and phenol–hypochlorite methods compared well (r = 0.99); the coefficient of variation for 25 μg NH4-N/L and above was less than 3% for the salicylate–hypochlorite method. The new method is safer than the phenol–hypochlorite method because phenol is not used, but it may not be suitable for field determinations because of photosensitivity. The characteristic colors produced by increasing concentrations of ammonia make the assay useful for the direct, visual estimation of ammonia in culture systems.Key words: ammonia, analytical methods, seawater analysis