Gluconic Acid Fermentation by Pullularia pullulans

During the study on the sugar metabolism of molds, several strains of Pullularia pullulans were found to produce large amounts of gluconic acid from glucose. Thirty seven strains of P. pullulans were then tested for their acid-producing abilities. Seven strains did not produce any amount of gluconic acid. However, all of the other strains were shown to be capable of producing this acid. The superior strains produced yiclds of gluconic acid as high as about 90%, based on glucose available, in shaking cultures at 30°C after 2 days. The yields were increased up to approximately 100% during later stages. In addition to high yields, gluconic acid was produced exclusively by these strains. Glutamic acid and inorganic ammonium salts, such as (NH₄)₂SO₄, NH₄Cl and (NH₄)₂HPO₄, were favorable nitrogen sources for acid production. In the case of (NH₄)₂SO₄, the optimum concentration was 0.05%. The addition of CaCO₃ was essential for gluconic acid production by P. pullulans and a 3% concentration of CaC0₃ appeared to be desirable for the maximum conversion to gluconic acid in a medium containing 10% glucose.     

Nε-Methylated Lysines from Formaldehyde-treated Casein

Phospholipase A activity of Pseudomonas aeruginosa was found only in the membrane fraction under normal conditions, but a part of that activity was converted into soluble form after polymyxin B treatment. This soluble phospholipase A was further activated by addition of 80% saturated (NH₄)₂SO₄ or incubation at 70 ± 5°C for 5 minutes. The phenomenon was confirmed in terms of kinetic parameters. Phospholipase A activity in the sonicates of P. aeruginosa was also markedly enhanced by (NH₄)₂SO₂ at 80% saturated concentration. The mechanism of the activation is discussed.     

Salt‐enhanced cultivation as a morphology engineering tool for filamentous actinomycetes: Increased production of labyrinthopeptin A1 in Actinomadura namibiensis

Salt‐enhanced cultivation as a morphology engineering tool for the filamentous actinomycete Actinomadura namibiensis was evaluated in 500‐mL shaking flasks (working volume 100 mL) with the aim of increasing the concentration of the pharmaceutically interesting peptide labyrinthopeptin A1. Among the inorganic salts added to a complex production medium, the addition of (NH₄)₂SO₄ led to the highest amount of labyrinthopeptin A1 production. By using 50 mM (NH₄)₂SO₄, the labyrinthopeptin A1 concentration increased up to sevenfold compared to the non‐supplemented control, resulting in 325 mg L^?1 labyrinthopeptin A1 after 10 days of cultivation. The performance of other ammonium‐ and sulfate‐containing salts (e.g., NH₄Cl, K₂SO₄) was much lower than the performance of (NH₄)₂SO₄. A positive correlation between the uptake of glycerol as one of the main carbon sources and nongrowth‐associated labyrinthopeptin productivity was found. The change in the cell morphology of A. namibiensis in conjunction with increased osmolality by the addition of 50 mM (NH₄)₂SO₄, was quantified by image analysis. A. namibiensis always developed a heterogeneous morphology with pellets and loose mycelia present simultaneously. In contrast to the non‐supplemented control, the morphology of (NH₄)₂SO₄‐supplemented cultures was characterized by smaller and circular pellets that were more stable against disintegration in the stationary production phase.     

Nitrogen regulation of gibberellin biosynthesis in Gibberella fujikuroi

Summary In Gibberella fujikuroi, ammonium (NH₄ ⁺) interfered with the production of gibberellic acid (GA₃). Optimal production occurred at 19 mm (NH₄)₂SO₄ and the synthesis of GA₃ was reduced threefold in a medium with 38 mm (NH₄)₂SO₄. Using a resting cell system with mycelia previously grown on two concentrations (19 mm and 38 mm) of (NH₄)₂SO₄, it was found that NH₄ ⁺ depressed synthesis of the gibberellin-synthesizing enzymes. Furthermore, addition of NH₄ ⁺ to a producing system shut off gibberellin formation, indicating that the negative effect of NH₄ ⁺ ions is also due to inhibition of one or more enzymes in the gibberellin biosynthesis pathway. The onset of gibberellin biosynthesis in media with high (38 mm) and low (19 mm) concentrations of (NH₄)₂SO₄ was studied by addition of cycloheximide to batch cultures of various ages. Offprint requests to: B. Brückner     

A new approach to ammonium sulphate feeding for fed‐batch Arthrospira (Spirulina) platensis cultivation in tubular photobioreactor

Arthrospira platensis was cultivated in tubular photobioreactor using different photosynthetic photon flux densities (PPFD) and protocols of (NH₄)₂SO₄ fed‐batch supply. Results were evaluated by variance analysis selecting maximum cell concentration (X_m), cell productivity (P_x), nitrogen‐to‐cell conversion factor (Y_X/N) and biomass, protein and lipid contents as responses. At PPFD of 120 and 240 μmol‐photons/m² s, a parabolic profile of (NH₄)₂SO₄ addition aiming at producing biomass with 7% nitrogen content ensured X_m values (14.1 and 12.2 g/L, respectively) comparable to those obtained with NaNO₃. At PPFD of 240 μmol‐photons/m² s, P_x (1.69 g/Ld) was 36% higher, although the photosynthetic efficiency (3.0%) was less than one‐half that at PPFD of 120 μmol‐photons/m² s. Biomass was shown to be constituted by about 35% proteins and 10% lipids, without any dependence on PPFD or kind of nitrogen source. These results highlight the possible use of (NH₄)₂SO₄ as alternative, cheap nitrogen source for A. platensis cultivation in tubular photobioreactors. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Organic and inorganic nitrogen source ratio effects onBacillus thuringiensis var.israelensis delta-endotoxin production

The influence of different organic and inorganic nitrogen source combinations and CN ratios was studied in connection with growth and protein production ofBacillus thuringiensis var.israelensis. Protein production was assumed to be proportional to delta-endotoxin production. Delta-endotoxin concentration increased when media were supplemented with (NH₄)₂SO₄, but the delta-endotoxin: biomass dry weight ratio was unaffected by different CN ratios. Organic nitrogen source, yeast extract, could be partially replaced by (NH₄)₂SO₄ with a significant increase in delta-endotoxin production.

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Résumé On a étudié l'influence de diverses sources d'azote organique et inorganique et des rapports CN en relation avec la croissance et la production de protéine parBacillus thuringiensis var.israelensis. On fait l'hypothèse que la production de protéines est proportionnelle à la production de delta-endotoxine. La concentration de delta-endotoxine croît quand on ajoute au milieu du (NH₄)₂SO₄, mais le rapport delta-endotoxine: poids sec de biomasse n'est pas affecté par différents rapports CN. On peut remplacer partiellement la source organique d'azote, l'extrait de levure, par du (NH₄)₂SO₄ avec une augmentation significative de production de delta-endotoxine.

     

Yeasts isolated from olive mill wastewaters from southern Italy: technological characterization and potential use for phenol removal

Olive mill wastewater (OMW) samples from two traditional varieties (Peranzana and Ogliarola Garganica) of Apulian region (southern Italy) and produced through continuous and traditional methods were microbiologically and chemically examined; thus, 104 yeasts were isolated and selected for further analyses. The strains were identified as Candida boidinii, Pichia holstii, Pichia membranifaciens, and Saccharomyces cerevisiae and analyzed to assess their suitability to metabolize phenols. Based on phenol metabolism, 27 strains were selected and inoculated into OMW aliquots to determine their ability to reduce phenols in vivo; then, five strains (identified with the codes 682—C. boidinii and 625, 642, 647, and 941—P. holstii) were used as a cocktail in wastewaters for a final validation step. In this last experiment, the effects of the temperature (10–30°C) and (NH₄)₂SO₄ (0.0–6.0 g l⁻¹) were studied through a central composite design approach, and the results highlighted that the cocktail was able to reduce phenols by 40% at 10°C with 6.0 g l⁻¹ of (NH₄)₂SO₄ added.     

Determination of the specific growth of molds on semi-solid cultures

The determination of growth constant of Aspergillus niger were obtained for semisolid cultures on cassava flour, Manihot esculenta, as a sole carbon source. As a consequence, a technique was developed that consisted of the use of a packed-bed microfermentor with a working volume of 16 cm³. The bed consisted of gelatinized and granulated cassava flour containing mineral nutrients and mold spores. The carbon dioxide produced during the respiration was drawn off with a current of air and then absorbed in a solution of sodium hydroxide. The absorption of CO₂, P, was correlated with the specific growth rate μ, by means of the equation P=Ke^μt, where t is time and K is a constant. Ammonium nitrogen was used as a limiting substrate and its concentration was varied from 0.039 to 2.5% in dry base. The maximum growth rate, μ_max, and the saturation constant, K_s, were 0.31 hr^?1 and 0.065 mmol (NH₄)₂SO₄/g total dry solids. Substrate inhibition was presented and the constant k_i gave a value of 1.5 mmol (NH₄)₂SO₄/g total dry solids. The proposed method is highly recommended for the evaluation of the semisolid fermentation of molds and for strictly aerobic bacteria and yeasts. It can be used especially in the evaluation of the growth of microorganisms on peanut shells, coffee residues, sugar cane bagasse, and other agricultural wastes.     

Optimal concentration of ammonium ion in a minimal synthetic medium for the growth of Candida albicans

Candida albicans strain B 311-10 with and without starvation was cultivated in the minimal synthetic medium of Shepherd et al. [18], modified without biotin, aminoacids, low glucose concentration [20] and with decreasing amounts of (NH₄)₂SO₄, to determine the optimal growth requirement for this strain. All the experiments were carried out under sterile conditions at 25 °C in a thermostat with initial O.D.s (675 nm) of 0.500 and 0.100. Cell growth was generally monitored everyday for six days with a spectrophotometer by determining the absorbance of the cultures at 675 nm. All the experiments were repeated three times and a statistical analysis of the data with a probability of 99% and 1% of error was performed to confirm the validity of the results. Best growth was obtained with starved cells at an initial O.D. of 0.100 and with a 0.1 g/L concentration of (NH₄)₂SO₄. At this concentration, the growth of C. albicans B 311-10 was best between the first and the fourth day with the maximum at the third day. With (NH₄)₂SO₄ concentrations of 0.05 and 0.5 g/L, cell growth was the same.     

Effect of pretreatment chemicals on xylose fermentation by Pichia stipitis

Pretreatment of biomass with dilute H₂SO₄ results in residual acid which is neutralized with alkalis such as Ca(OH)₂, NaOH and NH₄OH. The salt produced after neutralization has an effect on the fermentation of Pichia stipitis. Synthetic media of xylose (60 g total sugar/l) was fermented to ethanol in the presence and absence of the salts using P. stipitis CBS 6054. CaSO₄ enhanced growth and xylitol production, but produced the lowest ethanol concentration and yield after 140 h. Na₂SO₄ inhibited xylitol production, slightly enhanced growth towards the end of fermentation but had no significant effect on xylose consumption and ethanol concentration. (NH₄)₂SO₄ inhibited growth, had no effect on xylitol production, and enhanced xylose consumption and ethanol production.     

Light and ethylenediamine tetraacetic acid mediated differential effects of ammonium on nitrate reductase activity in maize seedlings

Abstract Effect of ammonium on in vivo activity of nitrate reductase in roots, shoots and leaves of maize (Zea mays L.) seedlings was studied in relation to light/dark conditions and EDTA supply. Supply of 5 mM (NH₄)₂SO₄ increased the steady state level of enzyme only in leaves and in light, while it had no effect in roots and shoots and in the dark. The substrate induction of enzyme was also little affected by 1 to 10 mM (NH₄)₂SO₄ in roots and shoots. In the leaves the activity in the dark was either inhibited (minus EDTA) or stimulated (plus EDTA) by 5 to 10 mM (NH₄)₂SO₄. The activity was stimulated in the light also in the presence of EDTA at higher concentrations of ammonium. When different concentrations of ammonium were supplied without any exogenous nitrate in the light, the enzyme activity increased at low concentration and was either inhibited or unaffected at higher concentrations depending upon the tissue used. Supply of EDTA with ammonium modified its effect to some extent. It is suggested that the effect of ammonium on nitrate reductase activity depends upon the tissue used and the effective concentration of the ammonium.     

Application of statistically-based experimental designs in medium optimization for spore production of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> from distillery effluent

Spore production of Bacillus subtilis from distillery effluent was optimized using statistically-based experimental designs. The two-level Plackett–Burman design was applied to choose the nutrient supplements significantly influencing spore production. Among the seven variables we tested, the most significant variables influencing spore production were statistically elucidated for optimization, and included (NH₄)₂SO₄, corn flour and MgSO₄. The optimum concentration of each significant variable was then predicted using Box–Behnken design. A second-order polynomial was determined by the multiple regression analysis of this experimental data. The optimum values for the critical nutrient supplements for the maximum were obtained as followed: (NH₄)₂SO₄, 4.54%; corn flour, 1.2%; MgSO₄, 0.56% with the corresponding value of maximum spore production of 7.24 × 10⁸ spores/ml. A verification experiment performed under the optimum conditions resulted in 6.95 × 10⁸ spores/ml. The determination coefficient (R ²) was 0.98, which ensure an adequate credibility of the model.     

The growth conditions of cephalosporium eichhorniae for the conversion of starch substrates to protein

The protein synthesis by Cephalosporium eichhorniae on substrates containing starch was evaluated at different pH, moisture content, ammonium sulphate and potassium dihydrogen phosphate supplementation. The optimum pH level was about 4.2 and the moisture content 65%. The optimum level of supplementation of medium containing sweet potato substrate with (NH₄)₂SO₄ was smaller than that for cassava substrate (5.2 g/l < 6.7 g/l in submerged culture, and 4.0% < 5.2% in solid state fermentation). The total crude protein yields were about 7.6 g/l for submerged cultures and 12% DM for solid state fermentations.     

Comparison of fractionation of groundnut proteins by two different methods

The salt-soluble proteins of groundnut meal were fractionated by precipitation with (NH₄)₂SO₄ by increasing the (NH₄)₂SO₄ saturation in steps of 10%. The sharp separation into arachin and conarachin claimed by earlier workers was not achieved, as protein was precipitated at each stage from 20 to 100% saturation with (NH₄)₂SO₄. The fractions so obtained were examined by disc electrophoresis on polyacrylamide gel and the amino acid compositions were determined by ion-exchange chromatography. Differences in both electrophoretic pattern and amino acid composition were found. The protein precipitated by CaCl₂ solution was similar in yield, nitrogen content, electrophoretic pattern, and amino acid composition to the fraction precipitating at 10–20% (NH₄)₂SO₄ saturation. The main differences in amino acid composition of the various fractions precipitated by (NH₄)₂SO₄ were found in the amino acids cystine, methionine, and lysine, which increased with increase in (NH₄)₂SO₄ saturation. The electrophoretic pattern and amino acid composition of “conarachin” varied according to the method of preparation.     

Extracellular protease from Bacillus coagulans,a psychrotrophic,Antarctic bacterium

Bacillus coagulans, when grown on casein at 20°C, produced an inducible, metalloprotease of 28 kDa at 1.6 U/mg cell protein. (NH₄)₂SO₄ at 2 g/l decreased enzyme production irrespective of carbon source.The authors are with the Defence R & D Establishment, Tansen Road, Gwalior-474 002, India.     

Partial purification and some properties of lipoxygenase from Saccharomyces cerevisiae

A partially purified lipoxygenase extract was obtained from the yeast Saccharomyces cerevisiae by precipitation with solid (NH₄)SO₄ at 20% to 80% saturation. The enzyme had two pH optima, at pH 8.0 and 10.0, with respective apparent K _m values of 13 and 9.5 m. At both pH optima, the lipoxygenase demonstrated highest substrate specificity towards linoleic acid, followed by linolenic acid; although the enzyme had less specificity towards mono-linolein than di-linolein at pH 8.0, the reverse was true at pH 10.0.     

Pollution control of swine manure and straw by conversion to Chaetomium cellulolyticum SCP feed

Swine manure has a very high pollution potential and obnoxious odor. Large farms particularly are confronted with a manure disposal problem since environmentally acceptable solutions are now required by government regulations. Swine manure was found to be a good source of supplementary nutrients to ferment wheat straw into single-cell protein (SCP) with Chaetomium cellulolyticum when 0.13g (NH₄)₂SO₄/g solid was used as an additional source of N. In batch fermentations, inhibitory effects, possibly due to soluble released from the straw during alkali or acid pretreatment, were overcome by starting the fermentation at about pH 7.0 and then reducing it to 5.0 during growth. An overall protein productivity of up to 66 mg/L h was obtained from a slurry mixture of 1% w/v solids of manure and straw. This compares favorably with 99 mg/L h when manure was fermented with glucose instead of straw as the main carbon source. A high protein productivity of 200 mg/L h was obtained from a slurry mixture containing anaerobically prefermented swine manure liquor and 1.5% w/v solids from straw. The final products of the manure and straw fermentations contained 25–30% DW crude protein and 6–20% DW cellulose and the materials were free of the original obnoxious odor and undesirable microbial contamination.     

Effect of some nitrogenous salts on nitrogen transfer and protease activity in germinatingZea mays L. seeds

Maize seeds were allowed to germinate in the presence of different nitrogenous salts for 72 h. Changes in the ethanol soluble and insoluble nitrogen were studied in the embryo and in the endosperm. Supply of Ca(NC₃)₂ enhanced germination and protease activity in the endosperm resulting in greater solubilisation of protein to soluble nitrogen in the seeds. NH₄NO₃ and (NH₄)₂SO₄ were less effective as compared to Ca(NO₃)₂. Cycloheximide inhibited germination and protease activity. Pretreatment also resulted in increase in growth, soluble and insoluble nitrogen, and nitrate reductase activity in the primary leaves. Ca(NO₃)₂ was more effective than NH₄NO₃ and (NH₄)₂SO₄.     

STUDIES ON THE NATURAL SUBSTRATE FOR PROTEIN METHYLASE II IN MAMMALIAN BRAIN AND BLOOD

—The activity of protein methylase II (S-adenosylmethionine:protein-carboxyl methyltransferase, EC 2.1.1.24), which methylates (esterifies) free carboxyl groups in the substrate protein, was measured in several mammalian organs in an effort to elucidate the nature of the natural substrate for the enzyme. The highest endogenous substrate activity was found in posterior and anterior pituitary glands, possibly in association with neurosecretory granules. In other parts of the brain endogenous substrates are lacking, although the cytosol fractions contain high activity of the enzyme which methylates exogenously added substrates. Rat whole blood also contains endogenous substrate protein. The protein precipitated by 50% (NH₄)₂SO₄ contained active substrate protein whereas blood protein methylase II is localized exclusively in the erythrocytes. Cohn fractions I, II and III are more active as substrate for protein methylase II than fraction V.     

Glutamic acid production byArthrobacter globiformis

Two hundred and fiftyArthrobacter strains were tested in a basal salts-glucose medium for their ability to produce glutamic acid; 50 strains produced small amounts of glutamic acid and alanine, as well as traces of other amino acids. Five biotin-dependent strains produced extraordinarily large amounts of glutamic acid. One of these, which was identified asA. globiformis, was selected for further study. Glutamic acid was only produced by this organism at biotin levels suboptimal for growth; maximal production (0.45 moles of glutamic acid per mole of glucose consumed) occurred at a biotin level of 10^–5 µg/ml. Other factors which markedly influenced glutamic acid production were temperature, (NH₄)₂SO₄ concentration, and pH of the growth medium.The taxonomy of glutamic acid-producing bacteria and the correlation between biotin deficiency and glutamic acid production are discussed.