Use of an organic buffer for the selection of acid tolerantRhizobium meliloti strains

Summary Nine media used to grow rhizobia were examined for their ability to maintain a stable low pH during the growth ofR. meliloti Large fluctuations in the pH of all media were recorded within 72 h, indicating their unsuitability for use in the selection of acid tolerant rhizobia. Morpholino-ethanesulphonic acid (MES) was assessed for its ability to buffer the pH of the media whilst still permitting rapid growth ofR. meliloti, R. trifolii, andBr. lupini. With 30.7 mM MES, the pH of a defined medium containing galactose, arabinose, and glutamate did not change from the initial value of 5.5 even though rhizobial numbers increased from 10⁴ to 10⁹ cells.ml^–1. Even at a buffer concentration of 15.3 mM, pH only increased from 5.5 to 5.6. There was no effect of the buffer on rhizobial growth.     

The effect of pH value on the toxicity of culture filtrates ofHelminthosporium Sativum and on its possible correlation to extracellular nitrogen accumulation

Summary Helminthosporium sativum P.K. & B. was grown on modified Fries No. 3 medium buffered at pH 4.7, 5.5, 6.7, and 7.8. The nitrogen content of the culture filtrate, mycelial growth and the toxicity of the filtrate (as measured by its wilt inducing capacity to lupin seedlings) were traced over 24 days. The correlation between nitrogen accumulation and the toxicity of the filtrates was clear in case of media originally buffered at 4.7, 5.5 and 7.8. On the other hand, there was no correlation between growth of the mycelial mats and toxin production. The toxicity of the filtrate was attributed to two substances at least, one of unknown composition produced during the accelerated growth phase, while the other is nitrogenous and produced during the decline growth phase i.e. autolytic in nature. Acidity of the medium favoured toxin production during the first two weeks of incubation. The toxicity of the filtrate remained stable within a wide range of pH between pH 3–9.4.     

The Effects of Bacterial Leaching on Metal Partitioning in Sewage Sludge

The partitioning of Mn, Al, Zn, Cu and Ti ions in municipal sewage sludge was investigated before and after bioleaching processes effectuated by Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans. Oxidation–reduction potential increase and pH decrease were obtained as a result of bacterial activity. A less pronounced and constant decrease was obtained with A. ferrooxidans, whereas A. thiooxidans presented a lag phase before a steep pH decrease. Metal solubilization was accomplished in experimental systems supplemented with energy source, Fe²⁺ for A. ferrooxidans and S⁰ for A. thiooxidans. Solubilization efficiency differed for each metal except for Al, and was relatively similar for either organism. Metal partitioning was conducted using a five-step sequential extraction procedure before and after the bioleaching. The results indicated that Zn and Mn ions were mostly associated with the organic fraction, whereas Cu, Al and Ti ions with the sulphide/residue fraction. The bioleaching process caused prompt solubilization of metals mostly associated with the more labile fractions (exchangeable, adsorbed and organically bound metals), whereas those associated to the less labile ones (EDTA and sulphide/residue fractions) were exchanged towards more labile fractions.     

Biomass relationship to growth and phosphate uptake ofPseudomonas fluorescens,Escherichia coli andAcinetobacter radioresistens in mixed liquor medium

The ability ofPseudomonas fluorescens, Escherichia coli andAcinetobacter radioresistenns to remove phosphate during growth was related to the initial biomass as well as to growth stages and bacterial species. Phosphate was removed by these bacteria under favourable conditions as well as under unfavourable conditions of growth. Experiments showed a relationship between a high initial cell density and phosphate uptake. More phosphate was released than removed when low initial cell densities (10²–10⁵ cells ml^–1) were used. At a high initial biomass concentration (10⁸ cells ml^–1), phosphate was removed during the lag phase and during logarthmic growth byP. fluorescens. Escherichia coli. at high initial biomass concentrations (10⁷ cells ml^–1), accumulated most of the phosphate during the first hour of the lag phase and/or during logarithmic growth and in some cases removed a small quantily of phosphate during the stationary growth phase.Acinetobacter radioresistens, at high initial cell densities (10⁶, 10⁷ cells ml^–1) removed most of phosphate during the first hour of the lag phase and some phosphate during the stationary growth phase.Pseudomonas fluorescens removed phosphate more thanA. radioresistens andE. coli with specific average ranges from 3.00–28.50 mg L^–1 compared to average ranges of 4.92–17.14 mg L^–1 forA. radioresistens and to average ranges of 0.50–8.50 mg L^–1 forE. coli.     

Analysis of growth of Gluconobacter oxydans in glucose containing media

Gluconobacter oxydans oxidizes glucose via alternative pathways: one involves the non-phosphorylative, direct oxidation route to gluconic acid and ketogluconic acids, and the second requires an initial phosphorylation and then oxidation via the pentose phosphate pathway enzymes. During growth of G. oxydans in glucose-containing media, the activity of this pathway is strongly influenced by (1) the pH value of the environment and (2) the actual concentration of glucose present in the culture. At pH values below 3.5 the activity of the pentose phosphate pathway was completely inhibited resulting in an increased requirement of the organism for nutrient substances, and a poor cell yield. At pH 5.5 a triphasic growth response was observed when G. oxydans was grown in a defined medium. Above a threshold value of 5–15 mM glucose, oxidation of both glucose and gluconate by the pentose phosphate pathway enzymes was repressed, causing a rapid accumulation of gluconic acid in the culture medium. When growing under these conditions, a low affinity for the oxidation of glucose was found (K _s=13 mM). Below this threshold glucose concentration, pentose phosphate pathway enzymes were synthesized and glucose was actively assimilated via this pathway. It was shown that de novo enzyme synthesis was necessary for increased pentose phosphate pathway activity and that assimilation of gluconate by washed cell suspensions was inhibited by glucose.     

Abilities of organic acids to support growth and anthocyanin accumulation by suspension cultures of wild carrot cells using ammonium as the sole nitrogen source

Summary Fifteen organic acids were examined for their abilities to support the growth and anthocyanin accumulation by suspension cultures of wild carrot (Daucus carota L.) using ammonium as the sole nitrogen source. Glutarate, adipate, pimelate, azelate, cinnamate, and phthalate were toxic to the culture. They prevented growth and anthocyanin accumulation at 5 mM or less in media that were otherwise adequate for growth. Succinate, fumarate, malate, α-ketoglutarate, glutamate, maleate, malonate, tartarate, and citrate all supported growth and anthocyanin accumulation but in varying amounts. The growth achieved in medium containing 20 mM acid was higher at an initial pH of 5.5 than at an initial pH of 4.5. The growth achieved was dependent on the organic acid used, its concentration, and the initial pH of the medium. When growth occurred the final pH was higher than the initial pH with most of the acids. Anthocyanin accumulation was greatest with succinate at 14 to 20 mM at an initial pH of 4.2 or 4.3 and declined when the initial pH was above 4.3. These studies were supported by grants from General Foods Corporation and the W. Alton Jones Foundation.     

Selection and Optimization of Culture Medium for Exopolysaccharide Production by Coriolus (Trametes) Versicolor

Summary Coriolus versicolor is a medicinal fungus producing exopolysaccharides (EPS). Five well-defined culture media were studied to select the medium that maximizes production of EPS by C. versicolor. Biomass, reducing sugars and EPS concentrations along with the rheological behaviour of the broth were followed during fermentations lasting 9 days. The yeast malt extract medium (YM) was shown to yield the highest production of EPS. Fermentation conditions with YM medium were further investigated to optimize EPS production by C. versicolor. An experimental design to do this was adopted, in which the effects of pH and initial substrate concentration were considered. The effects of initial glucose concentration (5, 15 and 25 g l⁻¹) and pH (4.0, 5.5 and 7.0) were evaluated. The initial glucose concentration was found to be the most important factor in EPS production and also cell growth.     

Energy-dispersive X-ray analysis of the extracellular cadmium sulfide crystallites of Klebsiella aerogenes

Klebsiella aerogenes forms electron-dense partieles on the cell surface in response to the presence of cadmium ions in the growth medium. These particles ranged from 20 to 200 nm in size, and quantitative energy dispersive X-ray analysis established that they comprise cadmium and sulfur in a 1:1 ratio. This observation leads to the conclusion that the particles are cadmium sulfide crystallites. A combination of atomic absorption spectroscopy, inductively coupled plasma mass spectrometry, and acid-labile sulfide analysis revealed that the total intracellular and bound extracellular cadmium:sulfur ratio is also 1:1, which suggests that the bulk of the cadmium is fixed as extracellular cadmium sulfide. The tolerance of K. acrogenes to cadmium ions and the formation of the cadmium sulfide crystallites were dependent on the buffer composition of the growth medium. The addition of cadmium ions to phosphate-buffered media resulted in cadmium phosphate precipitates that remove the potentially toxic cadmium ions from the growth medium. Electrondense particles formed on the surfaces of bacteria grown under these conditions were a combination of cadmium sulfide and cadmium phosphates. The specific bacterial growth rate in the exponential phase of batch cultures was not affected by up to 2mM cadmium in Tricine-buffered medium, but formation of cadmium sulfide crystallites was maximal during the stationary phase of batch culture. Cadmium tolerance was much lower (10 to 150 M) in growth media buffered with Tris, Bistris propane, Bes, Tes, or Hepes. These results illustrate the importance of considering medium composition when comparing levels of bacterial cadmium tolerance.Abbreviations EDXA Energy dispersive X-ray analysis - AAS Atomic absorption spectroscopy - TEM Transmission electron microscopy - SEM Scanning electron microscopy - ICP-MS Inductively coupled plasma mass spectrometry - ALSA Acid-labile sulfide analysis     

Isolation and characterization of a thermophilic acetotrophic strain of Methanothrix

An enrichment culture which converted acetate to methane at 60°C was obtained from a thermophilic anaerobic bioreactor. The predominant morphotype in the enrichment was a sheathed gas-vacuolated rod with marked resemblence to the mesophile Methanothrix soehngenii. This organism was isolated using vancomycin treatments and serial dilutions and was named Methanothrix sp. strain CALS-1. Strain CALS-1 grew as filaments typically 2–5 cells long, and cultures showed opalescent turbidity rather than macroscopic clumps. The cells were enclosed in a striated subunit-type sheath and there were distinct cross-walls between the cells, similar to M. soehngenii. The gas vesicles in cells were typically 70 nm in diameter and up to 0.5 m long, and were collapsed by pressures over 3 atm (ca. 300 kPa). Stationary-phase cells tended to have a higher vesicle content than did growing cells, and occasionally bands of cells were seen floating at the top of the liquid in stationary-phase cultures. Acetate was the only substrate of those tested which was used for methanogenesis by strain CALS-1, and acetate was decarboxylated by the aceticlastic reaction. The optimum temperature for growth of strain CALS-1 was near 60°C (doubling time=24–26 h), with no growth occurring at 70°C and 37°C. The optimum pH value for growth was near 6.5 in bicarbonate/CO₂ buffered medium and no growth occurred at pH 5.5 or pH 8.4. No growth was obtained below pH 7 when the medium was buffered with 20 mM phosphate. Strain CALS-1 grew in a chemically defined medium and required biotin. Sulfide concentrations over 1 mM were inhibitory to the culture, and growth was more rapid with 1 mM 2-mercaptoethane sulfonate (coenzyme M) or 1 mM titanium citrate as an accessory reductant than with 1 mM cysteine. It is likely that strain CALS-1 represents a new species in the genus Methanothrix.     

Glycerophosphate as a phosphorus source in a defined medium for Pichia pastoris fermentation

Pichia pastoris has emerged as a commercially important yeast for the production of a vast majority of recombinant therapeutic proteins and vaccines. The organism can be grown to very high cell densities using a defined basal salts media (BSM). However, BSM contains bi-cation or tri-cation phosphate, which precipitates out of the medium at pH above 5.5, although the optimal fermentation pH of most recombinant protein fermentation varies between 5.5 and 7.0. In this article, the application of glycerophosphates was investigated as a substitute phosphate source in an effort to eliminate precipitation. The solubility of BSM containing sodium or potassium glycerophosphates was examined before and after autoclaving at various pHs. Sodium glycerophosphate was found stable at autoclave temperature but formed complexes with coexisting magnesium and calcium ions that were insoluble above pH 7.0. Medium where sodium glycerophosphate was autoclaved separately and then added to the growth medium did not produce any precipitate up to pH 10.5. The performance of P. pastoris fermentations expressing α-galactosidase and ovine interferon-τ using a glycerolphosphate-based medium was found to be comparable to a conventional BSM. The results from this work demonstrate that sodium glycerophosphate can be assimilated by the P. pastoris strains and can be employed as a reliable phosphorus source for both cell growth and recombinant protein production.     

Temperature dependency of induction of sexual agglutinability by α pheromone in the yeast Saccharomyces cerevisiae

When pheromone-pretreated cells of an inducible a strain of Saccharomyces cerevisiae carrying the inducible gene saa1 were incubated in a growth medium at 28°C, induction of sexual agglutinability began after a 10 min lag period. If the cells were incubated at 38°C during the lag period, no induction occurred even after incubation at 28°C. Contrary to this, if the cells were incubated at 28°C during the lag period, almost complete induction occurred, even after transfer to 38°C. Temperature shift experiments revealed that 5 min incubation at 28°C was necessary for the initiation of the temperature-sensitive period and further 5 min incubation for the completion of the period. The temperature-sensitive period was sensitive to phenylmethylsulfonyl fluoride.Non-common abbreviations PBS 10^-2 M phosphate buffer solution, pH 5.5 - PMSF phenylmethylsulfonyl fluoride     

Axenic mass cultivation of the free-living soil amoeba, Acanthamoeba castellanii in a laboratory fermentor

Axenic mass cultivation of Acanthamoeba castellanii in laboratory fermentors (14 l) yielded after 20 days approximately 3 g cells (wet weight). After a short lag phase amoebal cell numbers increased exponentially to a maximum of 3.5×10⁵ cells per ml until cell death occurred after 20 days. Optical density and protein concentrations revealed identical patterns. During amoebal growth only 12–19% of the initially added glucose (100 mM) as sole carbon source was used. Large amounts of ammonia (1 g in 10.5 l culture volume) were excreted into the medium which subsequently raised the pH from 6.6 to 7.7, and from 6.6 to 6.8 in 2 and 20 mM buffered media, respectively. Growth inhibition and cell death could not be explained by a depletion of glucose or oxygen limitations during growth. The production of ammonia had a growth inhibitory effect, however, the sudden termination of the exponential growth phase and cell death could not be explained by the toxic influence of ammonia only.     

Influence of Phosphate on the Growth and Nodulation Characteristics of Rhizobium trifolii

The growth and nodulating characteristics of Rhizobium trifolii 6 and 36 differed under different external phosphate conditions. Under growth conditions designed to deplete the internal phosphate content of the rhizobia, strain 6 maintained a generation time of 5 h during the exponential phase over two cycles of growth in phosphate-depleted medium. In contrast, the generation time of strain 36 was extended from 3.5 to 9.8 h over two cycles of phosphate-depleted growth, although the organism eventually achieved the same cell density and cellular phosphate content as that of strain 6 at stationary phase. Phosphate-depleted strain 6 required 0.51 ± 0.08 μM phosphate to commence proliferation, whereas phosphate-depleted strain 36 required 0.89 ± 0.04 μM phosphate under the same conditions. Phosphate-depleted strain 6 maintained viability when exposed to external phosphate concentrations subcritical for growth to occur, whereas phosphate-depleted strain 36 lost viability within 48 h when exposed to medium containing phosphate at concentrations subcritical for growth. Phosphate-depleted strain 36 was inferior to phosphate-depleted strain 6 at nodulating subterranean clover (Trifolium subterraneum L. cv. Mt. Barker) by taking 2 to 4 days longer to develop nodules in phosphatedepleted plant grown medium at pH 5.5. Nodulation by phosphate-depleted strain 36 was accelerated either by including phosphate in the plant growth medium at pH 5.5 or by raising the solution pH of phosphate-depleted plant growth medium to pH 6.5. External phosphate and pH effects were not observed on the nodulating capabilities of phosphate-depleted strain 6 or on luxury phosphate-grown cells of either strain. Phosphatedepleted strains 6 and 36 proliferated to a similar extent on the rhizoplanes even under stringently low external P_i concentrations. The phosphatase activities of both phosphate-depleted strains were significantly (P = 0.05) higher at pH 6.5 than at pH 5.5, and the activity of strain 6 was significantly higher (P = 0.05) than that of strain 36 at pH 5.5 and 5.0.     

Characterization of arsenopyrite oxidizing Thiobacillus. Tolerance to arsenite,arsenate, ferrous and ferric iron

Two strains of Thiobacillus, T. ferrooxidans and T. thiooxidans, have been isolated from a bacterial inoculum cultivated during a one-year period in a 1001 continuous laboratory pilot for treatment of an arsenopyrite/pyrite concentrate. The optimum pH for the growth of both strains has been found to be between 1.7 and 2.5. Because of the high metal toxicity in bioleach pulps, the tolerance of T. ferrooxidans and T. thiooxidans with respect to iron and arsenic has been studied. The growth of both strains is inhibited with 10 g/l of ferric ion, 5 g/l of arsenite and 40 g/l of arsenate. 20 g/l of ferrous iron is toxic to T. ferrooxidans but 30 g/l is necessary to impede the growth of T. thiooxidans.     

Aluminium toxicity and binding to Escherichia coli

The toxicity and binding of aluminium to Escherichia coli has been studied. Inhibition of growth by aluminium nitrate was markedly dependent on pH; growth in medium buffered to pH 5.4 was more sensitive to 0.9 mM or 2.25 mM aluminium than was growth at pH 6.6–6.8. In medium buffered with 2-(N-morpholino)ethanesulphonic acid (MES), aluminium toxicity was enhanced by omission of iron from the medium or by use of exponential phase starter cultures. Analysis of bound aluminium by atomic absorption spectroscopy showed that aluminium was bound intracellularly at one type of site with a K _m of 0.4 mM and a capacity of 0.13 mol (g dry wt)^-1. In contrast, binding of aluminium at the cell surface occurred at two or more sites with evidence of cooperativity. Addition of aluminium nitrate to a weakly buffered cell suspension caused acidification of the medium attributable to displacement of protons from cell surfaces by metal cations. It is concluded that aluminium toxicity is related to pH-dependent speciation [with Al(H₂O) ₆ ³⁺ probably being the active species] and chelation of aluminium in the medium. Aluminium transport to intracellular binding sites may involve Fe(III) transport pathways.     

Factors influencing the activity of cellular alkaline phosphatase during growth and sporulation ofBacillus cereus

Alkaline phosphatase (EC 3.1.3.1) is synthesized in media with a low phosphate concentration (0.37 mmm of total and 19 μm of inorganic phosphate, respectively) already during the exponential phase of growth ofBacillus cereus. The enzyme is repressed by higher phosphate concentrations (3.7 mm) during the whole growth period; during sporogenesis the enzyme activity in cells slightly increases even under these conditions. During growth the enzyme is not secreted into the medium, a minor amount being released after cessation of growth. The enzyme activity can be increased by adding Zn²⁺ ions (10 μm). When during growth without phosphate the pH of the medium decreases below 5.0, the enzyme activity temporarily decreases and growth is slowed down, followed by a subsequent increase of the enzyme activity. In this case the onset of sporulation is also delayed.     

Effect of culture conditions on batch growth ofPseudomonas fluorescens on olive oil

Summary Batch cultures ofPseudomonas fluores-cens were grown in minimal medium with olive oil as the sole carbon source. When olive oil me-dium was inoculated with cells from nutrient broth there was an initial lag phase followed by logarithmic growth. The duration of the lag phase was influenced by the incubation temperature and the growth phase of the inoculum. Both factors are known to affect lipase induction during growth in fat-free media. Maintenance of condi-tions reported to be conducive to lipase produc-tion in cultures used for inoculation ensured a minimal lag before logarithmic growth com-menced on olive oil. Growth on oil occurred when the culture was maintained at pH 6 or 7, but did not occur at pH 5 or 8.     

Phosphatase production byAspergillus ficuum

Summary Effects of nutritional and cultural conditions on cell growth and phosphatase production byAspergillus ficuum were studied.A. ficuum produced high levels of phosphatases when grown on a basal medium that contained a minimal amount (2 mg/100 ml) of phosphorus in an acidic growth medium. The organism produced a nonspecific acid phosphomonoesterase rather than phytin-specific phosphatase. The enzyme hydrolyzed a variety of phosphates and produced orthophosphate. The rate of phosphate hydrolysis was dependent on the pH of the reaction, where the pH optimum for acid phosphatase was 2.5 and that for phytase was 5.0. The organism slowly released the phosphatase, and the enzyme activity in the growth medium increased continually during a one-month growth period. For a high level of phosphatase production, low levels (1–5 mg%) of initial phosphorus were necessary and polyphosphates were the desired form rather than the monophosphate. The addition of surfactants, such as polyoxyethylene ethers and sodium oleate, to fungal culture medium markedly increased the level of phosphatase production.     

Assays of invertase activity in acidic soils: Influence of buffers

Summary The influence of buffered and unbuffered systems for assays of invertase activity in a range of acidic soils (pH4.9–6.8), and a neutral soil (pH 7.1), from under pasture was determined. The buffers were those recently recommended in other studies,viz. a modified universal buffer (MUB) and a potassium phosphate buffer. The optimum pH for the invertase activity of a moderately acid soil (pH 5.5) wasc 4.0 and for the neutral soil was 5.0 With the acidic soils, invertase activity was lower in the assay system with MUB (initial pH 5.0) than in the unbuffered system, and decreased with increasing MUB molarity. The phosphate buffer was more satisfactory, even though the pH (5.0) was below its most effective range. Generally, either phosphate buffer or unbuffered systems appear suitable for measuring invertase activity in these acidic soils.     

Transcellular ion currents and extension ofNeurospora crassa hyphae

Summary Hyphae ofNeurospora crassa, like many other tipgrowing organisms, drive endogenous electric currents through themselves such that positive charges flow into the apical region and exit from the trunk. In order to identify the ions that carry the current, the complete growth medium was replaced by media lacking various constituents. Omission of K⁺ or of phosphate diminished the zone of inward current, effectively shifting the current pattern towards the apex. Omission of glucose markedly reduced both inward and outward currents; addition of sodium azide virtually abolished the flow of electric current. Growing hyphae also generate a longitudinal pH gradient: the medium surrounding the apex is slightly more alkaline than the bulk phase, while medium adjacent to the trunk turns acid. The results suggest thatNeurospora hyphae generate a proton current; protons are expelled distally by the H⁺-ATPase and return into the apical region by a number of pathways, including the symport of protons with phosphate and potassium ions. Calcium influx may also contribute to the electric current that enters the apical region. There seems to be no simple obligatory linkage between the intensity of the transcellular electric current and the rate of hyphal extension. Calcium ions, however, are required in micromolar concentrations for extensions and morphogenesis of hyphal tips.