Morphology of Ureaplasma urealyticum (T-mycoplasma) organisms and colonies.

The morphology of Ureaplasm urealyticum in broth cultures was studied by phase-contrast microscopy. Most organisms appeared singly or in pairs. Long filaments and long chains of cocci, common in classical mycoplasma cultures, were not observed. On solid medium, U. urealyticum produced "fried-egg" colonies which developed according to the scheme suggested by Razin and Oliver (J. Gen. Microbiol., 1961) for the morphogenesis of the classical mycoplasma colonies. The formation of the peripheral zone of the colonies followed that of the central zone only when growth conditions were adequate, Hence, the appearance of peripheral zones, and consequently the larger colony size, can be taken as an indicator of improved growth conditions. Incubation in an atmosphere of 100% CO2 resulted in significantly larger colonies than in an atmosphere of N2, O2, or air. CO2 acts as a buffer, keeping the pH at the optimal range for Ureaplasma growth (pH 6.0 to 6.5) in the presence of the ammonia produced from the urea hydrolyzed by the organisms. The addition to the medium of 0.01 M urea together with 0.01 M putrescine enabled better growth than with urea alone. Small amounts of phosphate improved growth in an atmosphere of CO2, apparently fulfilling a nutritional role. Under nitrogen, higher phosphate concentrations were required for good growth, apparently serving as a buffer as well as a nutrient. Sodium chloride and sucrose which had been added to increase the tonicity of the medium inhibited growth above 0.1 M. An increase in the agar concentration above 2% resulted in decreased colony size. Likewise, prolonged drying of the agar plates caused a marked decrease in colony size, mostly affecting the peripheral zone. The addition of both urea and putrescine to the growth medium and incubation in a humidified CO2 atmosphere are recommended for improved growth and formation of fried-egg colonies of U. ureaplyticum on agar. It must be emphasized that these experiments were carried out with a laboratory-adapted strain.     

Effect of ammonium ion concentration and osmotic pressure on growth of Ureaplasma urealyticum (T-strain mycoplasma).

The effect of ammonium ion concentration and osmotic pressure on growth of Ureaplasma urealyticum type VIII was determined by using a well-buffered broth medium containing 10 mM urea. The addition of NH4Cl to the medium at concentrations up to 10 mM did not affect growth; however, addition of larger quantities progressively decreased both the specific growth rate (mu) and the maximum yield of the culture, with concentrations of 80 mM completely inhibiting growth. Addition of either 150 mM KCl or NaCl to the medium did not inhibit growth, indicating that the growth-inhibitory effect was specific to NH4+ and was neither a result of increased Cl- concentration nor increased osmotic pressure. Concentrations of NH4Cl as high as 100 mM did not affect growth of either Acholeplasma laidlawii or Mycoplasma hominis. U. urealyticum was more sensitive to osmotic pressure: osmotic pressures of 710 to 780 mosmol/kg (with KCl, NaCl, or sucrose) resulted in both a substantially lower growth rate and a 5- to 10-fold lower peak yield of organisms. Both A laidlawii and M. hominis were less sensitive to increased osmotic pressure.     

Multiplication and Fermentation of Saccharomyces cerevisiae under Carbon Dioxide Pressure in Wine

Conditions for rapid fermentation of sugar in wine under pressure were sought for use in continuous production of naturally fermented sparkling wine. Wine yeast growth and fermentation were measured under CO(2) pressure. The medium was white wine with added glucose. Pressure was very inhibitory to growth, especially at low pH or high alcohol concentration. Use of various strains of wine yeast, cultures of various ages, or cells adapted to wine did not give more rapid growth. Addition of nutrients increased growth, but under no conditions was growth rapid enough to bring about sufficiently rapid fermentation rates. Conditions for rapid fermentation were sought by use of high levels of cells as inocula. Fermentation rates in wine also were inhibited by pressure, and were dependent on pH and alcohol levels. Addition of nutrients did not increase the fermentation rate, but rapid fermentation rates were obtained, under pressure, by inoculation with high levels of cells adapted several weeks to the base wine. Thus, continuous sparkling-wine production might be practical with proper amounts of adapted cells used as inocula, or perhaps with reuse of the fermentation culture.     

Effect of nitrogen-mediated changes in alkalinity on pH control and CO(2) supply in intensive microalgal cultures

The freshwater alga Scenedesmus obliquus was grown in continuous culture at a fixed dilution rate of 0.5/day, but at varying pH in the range 4.17-10.67. The pH was regulated in the range 4.17-7.67 by continuously bubbling 1% CO(2)-enriched air into the cultures and by varying the source of nitrogen (NO(3) (-), NH(4) (+), or urea) in the growth medium, which, in turn, led to changes in culture alkalinity. Culture alkalinity and P(CO(2) ) were the sole determinants of pH. A pH-stat system, together with NO(3) (-) in the medium, was used to regulate the pH in the range 7.92-10.67. Maximum productivity, which occurred at pH 6.6, was dependent on N source only to the extent that culture alkalinity was a function of nitrogen uptake. The results demonstrate that the choice of N is a critical factor in controlling the pH of large-scale algal cultures. NH(4) (+) is a poor source of N because it leads to destruction of culture alkalinity and concomitant growth-inhibiting reductions in pH, whereas NO(3) (-) has an opposite effect, although pH is not so severely affected in this case. Urea is, by far, the most suitable N source for maximizing algal yield when it is supplied in combination with the proper amounts of HCO(3) (-) alkalinity in the growth medium and percent CO(2) in the bubbled gas that will lead to an equilibrium pH near the optimum pH.     

Effect of Urea Concentration on Growth of Ureaplasma urealyticum (T-Strain Mycoplasma)

The effect of urea on growth of Ureaplasma urealyticum type VIII was studied by cultivating the organisms in a dialysate broth, prepared from soy peptone and autoclaved yeast, supplemented with 5% dialyzed horse serum, 100 mM 2-(N-morpholino)ethane sulfonic acid buffer (pH 5.75), and defined amounts of urea. Without urea, growth did not occur. Total growth was directly related to urea concentration. The least amount of urea that supported growth was 0.032 mM, which resulted in 3 × 10⁴ colony-forming units per ml. The maximum yield of organisms, 8.0 × 10⁷ colony-forming units per ml, was observed at 32 mM urea. Growth was limited not only by urea concentration, but also by the buffer capacity of the medium. The maximum amount of 2-(N-morpholino)ethane sulfonic acid buffer that could be employed was 100 mM; at higher concentrations, growth was inhibited. The yield of U. urealyticum was small even in medium with 32 mM urea and 100 mM 2-(N-morpholino)ethane sulfonic acid buffer: 0.63 mg of protein per liter of culture containing 5 × 10¹⁰ total colony-forming units. The molar growth yield was 20 mg of protein per mol of urea. The growth rate was also a function of urea concentration. Generation times ranged from 8 h at 0.032 mM urea to 1.6 h at 3.2 mM urea, where the substrate level was saturating. The K_s value for growth was 2.0 × 10⁻⁴ M urea. Thus, urea is a growth-limiting factor for U. urealyticum, but remarkably large amounts of this substrate are required.     

Responsiveness of mouse calvaria to parathyroid hormone after explant cryopreservation: 45Ca release in vitro

Newborn mouse calvaria prelabeled with 45Ca and cryopreserved at -196 degrees C in serum-free medium containing dimethylsulfoxide were compared to unpreserved explants for response to parathyroid hormone during subsequent culture. After short-term cryopreservation followed by rapid thawing, the viable explants continued to release 45Ca to the culture medium but additions of parathyroid hormone to the medium did not cause increased bone resorption. The data suggest that cryopreservation and thawing impairs mechanisms responsible for parathyroid hormone action on bone cells.     

Fermentation of cellulose and cellobiose by Clostridium thermocellum in the absence of Methanobacterium thermoautotrophicum.

The fermentation of cellulose and cellobiose by Clostridium thermocellum monocultures and C. thermocellum/Methanobacterium thermoautotrophicum cocultures was studied. All cultures were grown under anaerobic conditions in batch culture at 60 degrees C. When grown on cellulose, the coculture exhibited a shorter lag before initiation and growth and celluloysis than did the monoculture. Cellulase activity appeared earlier in the coculture than in the monoculture; however, after growth had ceased, cellulase activity was greater in the monoculture. Monocultures produced primarily ethanol, acetic acid, H2 and CO2. Cocultures produced more H2 and acetic acid and less ethanol than did the monoculture. In the coculture, conversion of H2 to methane was usually complete, and most of the methane produced was derived from CO2 reduction rather than from acetate conversion. Agents of fermentation stoppage were found to be low pH and high concentrations of ethanol in the monoculture and low pH in the coculture. Fermentation of cellobiose was more rapid than that of cellulose. In cellobiose medium, the methanogen caused only slight changes in the fermentation balance of the Clostridium, and free H2 was produced.     

Bioenergetic properties of alkalophilic Bacillus sp. strain C-59 on an alkaline medium containing K2CO3.

Alkalophilic Bacillus sp. strain C-59 could grow well on an alkaline medium containing K2CO3, as well as Na2CO3, but did not grow on K+-depleted medium. Right-side-out membrane vesicles, energized in the absence of Na+, however, could not take up [14C]methylamine actively, while vesicles equilibrated with 10 mM NaCl actively took up [14C]methylamine. The uptake of [14C]serine was also stimulated by the addition of Na+, and the imposition of a sodium gradient caused transient uptake. These results indicated that an Na+/H+ antiporter was involved in pH homeostasis and generation of an electrochemical sodium gradient in strain C-59 even though a growth requirement for Na+ was not evident. The efflux of 22Na+ from 22Na+-loaded vesicles was more rapid at pH 9.5 than at pH 7 in the presence of an electron donor. On the other hand, vesicles at pH 7 showed more rapid efflux than at pH 9.5 when the antiporter was energized by a valinomycin-mediated K+ diffusion potential (inside negative).     

Growth of and omega-3 fatty acid production by Phaeodactylum tricornutum under different culture conditions

Detailed studies were carried out on the effects of nitrogen source, phosphate, sodium chloride, growth factors, precursors, CO2, temperature, initial pH, and inoculum size on biomass and eicosapentaenoic acid (EPA) production by Phaeodactylum tricornutum. The EPA content of total fatty acids increased with increasing concentrations of nitrate and urea. Sodium chloride was not required for growth or EPA production. While vitamins B1 and B12 did not affect growth significantly, EPA yield was increased by 65% by B12 supplementation. Maximum EPA production occurred when the air gassing supply was supplemented with 1% CO2. Optimum culture temperature and initial pH for EPA production were 21.5 to 23 degrees C and 7.6, respectively. EPA yields of up to 133 mg/liter of culture were observed. EPA constituted up to 30 to 40% of total fatty acids.     

Cytotoxic effect of a T-strain mycoplasma (Ureaplasma urealyticum) on cultured normal human cells (WI-38)

Normal human embryonic lung fibroblasts (WI-38) were infected with Ureaplasma urealyticum, a urea hydrolysing mycoplasma. It was possible to observe reduced rates of multiplication of infected cells and reduced plating efficiency as well as the morphological changes usually associated with mycoplasma infection of animal cells in vitro. The cytotoxic effect on multiplication was sensitive to aureomycin but not penicillin. It was not related to depletion of amino acids or nucleic acid precursors from the cell culture medium but appeared to require that the host cells be growing. Ureaplasmas could not be recovered from cell culture medium after 4 days post infection and their characteristic urease activity could not be demonstrated either in cell culture medium or associated with the cells after the first cell subcultivation. [³H]TdR was incorporated into the nuclei of infected cells and the percent labelled nuclei was reduced compared with uninfected cells. Nuclear labelling indices of infected cells increased as the cells were subcultivated by trypsinization suggesting that the ureaplasmas were removed from the host cell surface by this treatment. In general, the effects of ureaplasmas on WI-38 cells do not appear to be as pronounced as effects of other mycoplasmas on animal cells in culture. It is clear, nonetheless, that the urea hydrolysing mycoplasmas can infect cells in culture and cause discernible effects on the growth and metabolism of these cells.     

Growth of and omega-3 fatty acid production by Phaeodactylum tricornutum under different culture conditions.

Detailed studies were carried out on the effects of nitrogen source, phosphate, sodium chloride, growth factors, precursors, CO2, temperature, initial pH, and inoculum size on biomass and eicosapentaenoic acid (EPA) production by Phaeodactylum tricornutum. The EPA content of total fatty acids increased with increasing concentrations of nitrate and urea. Sodium chloride was not required for growth or EPA production. While vitamins B1 and B12 did not affect growth significantly, EPA yield was increased by 65% by B12 supplementation. Maximum EPA production occurred when the air gassing supply was supplemented with 1% CO2. Optimum culture temperature and initial pH for EPA production were 21.5 to 23 degrees C and 7.6, respectively. EPA yields of up to 133 mg/liter of culture were observed. EPA constituted up to 30 to 40% of total fatty acids.     

Determination of carbon dioxide production rates for mammalian cells in 24-well plates

In this report, we describe a method for the quantitative determination of carbon dioxide production rates of mammalian cells. Custom-made, reusable, optically clear plugs are used to seal the wells of a 24-well plate. These plugs prevent the loss of CO2 produced by the mammalian cells cultured in bicarbonate-free medium. Measurements of pH, total liquid phase CO2, and viable cell density are used to estimate the average CO2 production rate during a 6-h incubation period. Using this method, four chemicals well-characterized in regards to toxicity, 2,4-dinitrophenol, antimycin A, rotenone, and cyanide, were found to elicit significant changes in CO2 production for given concentrations within 6 h, without inducing a decline in culture viability. Over longer exposure times, similar concentrations caused growth inhibition but not cell death. An assay based on metabolic change corresponding to growth inhibition that is more sensitive than traditional measures of cell death is a feasible complement to existing methods in drug discovery and toxicity testing.     

Utilization of seawater-urea as a culture medium for Spirulina maxima.

The possibilities of utilization of seawater enriched with ureas as the culture medium for a blue-green alga, Spirulina maxima, were investigated. Pretreatment by precipitation with NaHCO3 and (or) Na2CO3 was found essential to remove the excess amounts of Ca2+ and Mg2+ present in seawater prior to cultivation. A culture medium as good as the synthetic medium reported in the literature for the growth of S. maxima was obtained after treating seawater with NaHCO3 (19.2 g/L) at pH 9.2 and 35 degrees C for 2 h, filtering to remove precipitates, and enriching with K2HPO4 (0.5 g/L), NaNO3 (3.0 g/L), and FeSO4 (0.01 g/L). The same results were obtained by substituting a small amount (0.2 g/L or less) of either crystalline or polymerized urea for the NaNO3 in the above medium. Growth of S. maxima was inhibited at higher concentration of urea in the culture medium. The inhibition effect was due to the partial decomposition of urea into ammonia in alkali medium. Tests conducted on the 130-L cultivation open pond also confirmed that the seawater-urea medium supports growth of S. maxima as well as the best known synthetic medium.     

Urea as sole source of nitrogen for plant growth

Summary Spirodela oligorrhiza grown in sterile culture was able to use urea as sole source of nitrogen but only when the pH of the culture medium was below 4.3. Plants inoculated into urea media at pH 6.4 initially made little growth and became nitrogen-deficient in appearance and composition although they contained about 100 grams of urea per gram fresh weight of tissue. After a period the pH of the medium usually fell below 4.3 and growth commenced. Growth with other compounds, e.g. ammonium, nitrate or allantoin, as sources of nitrogen was not similarly affected by the pH of the culture medium.Urease activity could always be detected in the tissues of Spirodela oligorrhiza growing on urea. Plants with little or no urease activity soon developed significant activity when inoculated into urea media at pH 4.0. When the pH of the medium was higher there was no increase in urease activity and no growth ensued. Plants growing on urea possessed an activity of about 50 milliunits per gram fresh weight of tissue, but if the pH of the medium fell to 3.5 or lower, the activity present rose to 10 times this level.Urease activity also appeared, in the absence of supplied urea, as plants became increasingly nitrogen-deficient.     

比较研究白蚁巢上生长的担子菌——鸡(土从)菌及其分生孢子阶段小白球菌的生长与其基质的生态关系

鸡(土从)菌Termitomyces albuminosus(Berk.)Heim..及其分生孢子阶段小白球菌在基础培养基上生长缓慢,而在基础培养基上分别添加乌头酸、延胡索酸、柠檬酸或者添加生物素、VB_1、VB_2等都能促进鸡(土从)菌和小白球菌菌丝体及其分生孢子的生长。实验还表明维生素混合使用效果比单一添加为好。鸡(土从)菌和小白球菌生长的合适的碳源是麦芽糖、葡萄糖、果糖、蔗糖,而淀粉、甘露醇效果不良;合适的氮源是蛋白胨、花生粉、黄豆粉、甘氨酸和铵盐,但是小白球菌生长的培养基中含氮量不宜过高,一般为0.2％左右,而鸡(土从)菌要求含氮量较高,最佳的氮源浓度范围为0.4—0.6％。鸡(土从)菌和小白球菌菌丝体生长的pH值范围为4.0—5.5之间,鸡(土从)菌最适的pH值在4.0—4.5之间,而小白球菌最适pH值随着培养基组成和CO_2浓度的不同会有所变化。小白球菌具有忍耐高浓度CO_2(15％)的特性,静置培养比摇瓶培养生长更好。鸡(土从)菌菌丝体却需要较低的CO_2浓度方能生长良好,它更适宜于摇瓶培养。目前鸡(土从)菌和小白球菌菌丝体在实验室条件下生长都非常良好,液体培养已获得成功,为人工栽培和利用鸡(土从)菌提供了一条可能的途径。     

Occurrence of urease in T strains of Mycoplasma

A previously unknown metabolite necessary for growth of T strains of Mycoplasma in artificial culture media has been identified as urea. The source of this metabolite was the mammalian plasma or serum enrichment of the culture medium. Normal horse serum was the most satisfactory native protein enrichment for cultivation of T strains of mycoplasma, and it is believed that its superior performance in agar and fluid culture media is associated with its relatively high urea content (approximately 40 mg/100 ml). T-strain urease activity was maximal at pH 6.0 +/- 0.5. This is also the optimal pH for growth of T strains. Substrate concentrations greater than 1.0% urea were inhibitory to growth and urease activity of T-strain organisms, and optimal urea concentrations in fluid media appeared to lie within the range of 0.008 to 0.01 m. This range of urea concentration permitted maximal growth of T-strain organisms without rapid loss of viability due to excessive ammonia accumulation and rise in pH to lethal levels. T strains of Mycoplasma were cultivated in a serum-free fluid medium containing urea as the only added metabolite and nitrogen source. T strains are the only known human mycoplasmas which exhibit urease activity, and this biochemical marker can be employed as an aid in the detection and identification of T strains of Mycoplasma (urease color test) and in distinguishing T strains from other members of the human Mycoplasma group.     

Determination of dissolved CO(2) concentration and CO(2) production rate of mammalian cell suspension culture based on off-gas measurement

The determination of dissolved CO(2) and HCO(3)(-) concentrations as well as the carbon dioxide production rate in mammalian cell suspension culture is attracting more and more attention since the effects on major cell properties, such as cell growth rate, product quality/production rate, intracellular pH and apoptosis, have been revealed. But the determination of these parameters by gas analysis is complicated by the solution/dissolution of carbon dioxide in the culture medium. This means that the carbon dioxide transfer rate (CTR; which can easily be calculated from off-gas measurement) is not necessarily equal to carbon dioxide production rate (CPR). In this paper, a mathematical method to utilize off-gas measurement and culture pH for cell suspension culture is presented. The method takes pH changes, buffer and medium characteristics that effect CO(2) mass transfer into account. These calculations, based on a profound set of equations, allow the determination of the respiratory activity of the cells, as well as the determination of dissolved CO(2), HCO(3)(-) and total dissolved carbonate. The method is illustrated by application to experimental data. The calculated dissolved CO(2) concentrations are compared with measurements from an electrochemical CO(2) probe.     

Regulation of CO on heterocyst differentiation and nitrate uptake in the cyanobacterium Anabaena sp. PCC 7120

AIMS: The aim of the present investigation was to study the effects of different inorganic carbon and nitrogen sources on nitrate uptake and heterocyst differentiation in the culture of cyanobacterium Anabaena sp. PCC 7120. METHODS AND RESULTS: Anabaena was cultivated in media BG11 containing combined nitrogen and supplementary NaHCO3 or CO2. Cell growth, heterocyst differentiation, nitrate reductase (NR, EC 1.7.7.2), glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) and NO uptake were analysed. The cells cultivated in BG11(0) medium with aeration were taken as reference. Experimental results showed that the differentiation frequency of heterocysts when the cells were cultivated with elevated CO2 was higher than that of the cells grown with air or bicarbonate. Heterocysts appeared unexpectedly when CO2 was introduced into the medium containing nitrate. However, no heterocysts emerged when CO2 was added to medium containing NH or urea, or when NaHCO3 was supplied to the medium with nitrate. Both nitrate uptake rate and nitrate reduction enzyme activity were depressed by the supplement of CO2 to the culture. The activity of G6PDH was enhanced with the increase in heterocyst differentiation frequency. CONCLUSION: CO2 might compete with NO for energy and electrons in the uptake process and CO2 appears favoured. This led to a high intracellular C/N ratio and a relative N limitation. So the process of heterocyst differentiation was activated to supplement nitrogen uptake. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provided an attractive possibility to form more heterocysts by rapid growth of Anabaena cells cultivated in the medium containing nitrate in order to increase nitrogen fixation and hydrogen production.     

Growth response of axenic Entamoeba histolytica to hydrogen, carbon dioxide, and oxygen.

Entamoeba histolytica required CO2 for growth in axenic culture while growth was inhibited by H2. The organism was tolerant to 5% O2 in the gas phase and it was able to detoxify products of O2 reduction in the medium. The ameba did not require a negative oxidation-reduction potential for axenic growth. However, little or no free O2 was present in media exposed to 5% O2 in the gas phase. Growth was improved by adding yeast extract to the medium.