NUTRITION OF CHYTRIOMYCES AND ITS INFLUENCE ON MORPHOLOGY

The nutrition and its influence on some morphological characteristics of one isolate of Chytriomyces aureus Karling and two isolates of C. hyalinus Karling, one with and one without resting bodies, was studied. All three isolates required exogenous thiamine for optimum growth. Glucose, fructose, mannose, maltose, cellobiose, and starch supported good or moderate amounts of growth of all the isolates. Galactose, arabinose, xylose, ribose, sucrose, lactose, and raffinose supported little or only trace amounts of growth of C. hyalinus, with resting bodies, but failed to produce any growth of C. aureus and C. hyalinus, without resting bodies. All isolates utilized ammonium [NH₄NO₃, (NH₄)₂SO₄, NH₄Cl] as nitrogen sources but were unable to utilize nitrate (KNO₃) and nitrite (KNO₂). All isolates were able to grow at pH ranges of 5–8; the optimum pH range was 6.5. Both isolates of C. hyalinus were able to grow at a temperature of 10–30 C, whereas C. aureus did not grow below 15 C or above 25 C. Sporangial size was influenced by varying temperature levels, pH ranges, vitamins, carbohydrates, and nitrogen sources, while the morphology and sizes of zoospores, lipoid globules, apophyses, and rhizoids were little affected. The similarity of response exhibited by both species of Chytriomyces to various nutrients may suggest that these are closely related taxa.     

Utilization of nitrogen compounds and ammonia assimilation by chromatiaceae

Chromatium vinosum strain D, Thiocapsa roseopersicina strain 6311 and Ectothiorhodospira mobilis strain 8112 were grown anaerobically in the light with various single nitrogen sources. When substituted for NH₄Cl only glutamine and casamino acids supported good growth of all strains tested. Peptone and urea were utilized by C. vinosum and T. roseopersicina, glutamate, asparagine and nitrate only by C. vinosum. The strains were able to grow with molecular nitrogen; complete inhibition of this growth was observed in the presence of alanine with E. mobilis, and of alanine or asparagine with T. roseopersicina.Glutamate dehydrogenase, requiring either NADH or NADPH, NADH-linked glutamate synthase, and glutamine synthetase were demonstrate in the above organisms grown on NH₄Cl.     

Physiological studies on Phymatotrichum omnivorum X. Influence of nitrogen sources on free and bound amino acid pools

The effect of nitrogen source on the free and bound amino acids of mycelium of Phymatotrichum omnivorum (Shear) Dugg was investigated. The largest free amino acid pool was present in the natural medium and the smallest in the synthetic medium. Phymatotrichum omnivorum was able to utilize different nitrogen sources with the best growth occurring with NH₄NO₃. The ratio of glycine to alanine and aspartic to glutamic was around 0.25 in the free amino acid pool and around 1 in the bound amino acid pool. The free pool of glutamic acid ranged from 5.6 % to 27.2 % depending upon the nitrogen source in the media. The free pool of alanine ranged from 35.7 % to 17.2 % in relation to the nitrogen source. Most other amino acid ratios did not vary significantly between the free amino acids and the bound amino acids.     

The capability of the algaeChlorella vulgaris andScenedesmus obliquus of utilizing amino acids as the sole nitrogen source

The capability of utilizing 20 amino acids and 2 amides as the sole nitrogen source for growth was studied in two green algae (Chlorophyceae). A comparison was made of the growth rate of algae in a mineral nutrient solution containing nitrate as the nitrogen source, with that in the same solution in which nitrogen in the form of nitrate was substituted by an equivalent nitrogen amount in the form of various amino acids. In addition to this, another series of experiments was carried out in whioh both culture media were supplied with glucose. The results show that both algae utilize a series of amino acids in dependence of their structure (mostly 3-carbon amino acids). The growth rate ofChlorella in the presence of these sources is the same as in nitrate, that ofScenedesmus even much higher. In the cultures containing glucose both algal species exhibit a higher growth rate in the media with the nitrate nitrogen source than in those with amino acids (with the exception of glycine inScenedesmus).     

氨氮对3种吸附态亚硝化单胞菌的抑制动力学

[背景] 氨氮浓度会明显影响亚硝化单胞菌的活性,但氨氮浓度对吸附态亚硝化单胞菌菌种的抑制动力学尚缺乏研究。[目的] 研究氨氮浓度对3种吸附态亚硝化单胞菌（Nitrosomonas eutropha CZ-4、Nitrosomonas halophila C-19和Nitrosomonas europaea SH-3）的影响。[方法] 以碳酸钙作为吸附基质,设定氨氮浓度为25-1 000 mg/L,测定3种亚硝化单胞菌（N.eutropha CZ-4、N. halophila C-19和N. europaea SH-3）的亚硝氮积累速率与最大比生长速率,并通过Edwares2模型建立氨氧化的抑制动力学方程。[结果] N. halophila C-19在初始氨氮浓度为50-100 mg/L时的亚硝氮积累最快,N. europaea SH-3的亚硝氮积累则在初始氨氮浓度为50-200 mg/L时最快,而N. eutropha CZ-4则适于在初始氨氮浓度为50-400 mg/L时积累亚硝氮;N. eutropha CZ-4的最大比生长速率出现在初始氨氮浓度为50-400 mg/L时,明显高于N. halophila C-19（25-100 mg/L）,而N. europaea SH-3的生长速度在初始氨氮浓度为50-800 mg/L区间内无显著差异;N. europaea SH-3的K_I（922.76 mg/L）显著高于N. eutropha CZ-4（597.88 mg/L）,而CZ-4的K_I又显著高于N. halophila C-19（186.24 mg/L）,N. europaea SH-3的K_m（72.06 mg/L）显著高于N. halophila C-19（23.23 mg/L）。[结论] 3种吸附态亚硝化单胞菌的生长和氨氧化对氨氮浓度变化的响应存在明显差异,对于认识不同亚硝化单胞菌在不同氨氮浓度污水中的功能并开发相应的工程技术具有重要意义。     

Field assessment of efficacy of nitrogen salts to control the root-knot nematode (Meloidogyne javanica) on tomato

Influence of different nitrogen salts at electrical conductivity levels (EC2, 4 and 8?mmhos/cm) on tomato and root-knot nematode (Meloidogyne javanica) and their interactions was evaluated under field conditions. It was found that both diammonium phosphate ((NH₄)₂HPO₄) and ammonium sulphate ((NH₄)₂SO₄) were more effective than ammonium chloride (NH₄Cl) in causing an obvious suppression of M. javanica infection on tomato through reducing root galling and nematode reproduction and improving tomato growth and yield and their suppressive effect was similar to that of oxamyl or ethoprophos. At higher ECs, the tested nitrogen salts did not greatly affect pH, EC and salinity of rhizospheric soil except NH₄Cl at EC8 that caused higher EC and salinity over the untreated control which makes NH₄Cl less suitable candidate. Therefore, the use of (NH₄)₂HPO₄ and (NH₄)₂SO₄ alone or in combination with other control measures could control M. javanica and improve the growth and yield of tomato under field conditions.     

New Bistriazole Derivatives and the Biological Activity of the Thermophilic Cyanobacterium Mastigocladus laminosus Cohn.

The conditions of cultivation and the composition of medium for Desulfovibrio vulgaris Miyazaki F (DvMF) were examined to obtain cytochrome c₃ labeled with a stable isotope. The growth of DvMF was steady and reproducible under purging with N₂ and under pH control. DvMF was able to grow on a defined medium without natural products. The composition of medium containing a small amount of NH₄Cl as sole nitrogen source was established. Then, [U-¹⁵N]cytochrome c₃ was obtained during the culture of DvMF in a defined medium with ¹⁵NH₄Cl; it was confirmed by ¹H?¹⁵N HMQC. This is the first report of [U-¹⁵N]cytochrome c₃.     

Microbial production of ethanol from acetate by engineered <Emphasis Type="Italic">Ralstonia eutropha</Emphasis>

This study was performed to produce ethanol from acetate using a genetically engineered Ralstonia eutropha. In order to genetically modify R. eutropha H16, phaCAB operon encoding metabolic pathway genes from acetyl-CoA to polyhydroxybutyrate (PHB) was deleted and adhE encoding an alcohol dehydrogenase from Escherichia coli was overexpressed for conversion of acetyl-CoA to ethanol. The resulting strain produced ethanol up to 170 mg/L when cultivated in minimal media supplemented with 5 g/L of acetate as a sole carbon source. Growth and ethanol production were optimized by adjusting nitrogen source (NH₄Cl) content and repetitive feeding of acetate into the bacterial culture, by which the ethanol production was reached to approximately 350 mg/L for 84 h.     

Optimization of cell growth and poly(3-hydroxybutyrate) accumulation on date syrup by a Bacillus megaterium strain

Optimal growth and PHB accumulation in Bacillus megaterium occurred with 5% (w/v) date syrup or beet molasses supplemented with NH₄Cl. When date syrup and beet molasses were used alone without an additional nitrogen source, a cell density of about 3gl^–1 with a PHB content of the cells of 50% (w/w) was achieved. NH₄NO₃ followed by ammonium acetate and then NH₄Cl supported cell growth up to 4.8gl^–1, whereas PHB accumulation was increased with NH₄Cl followed by ammonium acetate, NH₄NO₃ and then (NH₄)₂SO₄ to a PHB content of nearly 42% (w/w). Cultivation of B.megaterium at 30l scale gave a PHB content of 25% (w/w) of the cells and a cell density of 3.4gl^–1 after 14h growth.     

Lack of effect of NH4Cl on protein synthesis in cultured fibroblasts

In experiments with isolated hepatocytes, Seglen [1] has shown that in the combined presence of NH₄Cl and high concentrations of valine, incorporation of this amino acid into cell protein is inhibited. He has proposed that NH₄Cl, in addition to inhibiting protein degradation in lysosomes, inhibits protein synthesis in these cells as part of a general toxic effect. To determine if NH₄Cl inhibits protein synthesis in cultured cells we incubated rat embryo fibroblasts, prelabeled with [¹⁴C]leucine, in the presence of 10 mM NH₄Cl and 15 mM leucine in both growth and serum-free media. We did not detect any effect of NH₄⁺ on protein synthesis or cell growth over a 3-day period. A partial inhibition of protein degradation was observed, particularly during the first 24 h of the experiment. In pulse-labeling experiments, NH₄Cl had no effect on the incorporation of [³H]leucine in the media. High concentrations of leucine, however, reduced re-utilization of endogenously derived leucine and inhibited the transport of valine into the cellular acid-soluble pool.These experiments show that at least in cultured fibroblasts 10 mM NH₄Cl shows no significant toxicity beyond an inhibition of lysosomal function. In addition these data suggest the possibility that high chase concentrations of one amino acid in the medium may be saturating a common transport mechanism, in effect reducing the transport of other amino acids utilizing this mechanism. A combined blockade by both NH₄Cl and a high concentration of a single amino acid may in certain sensitive cells result in a significant reduction in protein synthesis.     

Denitrifikation bei Hydrogenomonas eutropha Stamm H16

Zusammenfassung Hydrogenomonas eutropha (syn. Alcaligenes eutrophus) Stamm H 16 wächst anaerob mit Fructose und Nitrat bzw. Nitrit. Autotrophanaerobes Wachstum unter einer H₂-CO₂-Atmosphäre (90+10 Vol.-%) mit Nitrat als einzigem Wasserstoff-Acceptor ist minimal.Während des anaeroben Wachstums mit Nitrat sind zwei Phasen zu unterscheiden. In der ersten Phase erfolgt die Zellvermehrung auf Kosten der Reduktion von Nitrat zu Nitrit; dieses wird angehäuft. In der zweiten Phase wird Nitrit unter Bildung von Stickstoff reduziert.Gewaschene, anaerob gewachsene Zellen reduzieren Nitrat und Nitrit unter Bildung von N₂. Stöchiometrische Experimente mit H₂ oder Fructose als H-Donatoren lassen darauf schließen, daß Stickstoff das einzige Produkt der Denitrifikation durch die Zellen ist. Diese Schlußfolgerung wurde durch eine massenspektrometrische Analyse des gebildeten Gases bestätigt. Aerob gewachsene Zellen reduzieren Nitrat nur zu Nitrit. In Gegenwart von Ammonium-Salz gewachsene Zellen reduzieren Nitrat mit sehr geringer Rate.Die Ergebnisse deuten darauf hin, daß Stamm H 16 über nur eine Nitratreductase verfügt. Die Bildung des Enzyms ist durch Ammonium reprimierbar; O₂ ist ohne Einfluß. Die Nitritreductase fand sich sowohl in der löslichen Fraktion als auch in den gereinigten Partikeln lokalisiert. Das Nitritreductase-System wird nur unter anaeroben Bedingungen gebildet.

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Denitrification in Hydrogenomonas eutropha strain H16

Summary The hydrogen bacterium Hydrogenomonas eutropha (syn. Alcaligenes eutrophus) strain H 16 is able to grow anaerobically with fructose and nitrate or nitrite, respectively. Autotrophic anaerobic growth under a gas atmosphere of hydrogen and carbon dioxide (90+10 vol-%) with nitrate as the sole hydrogen acceptor is minimal.During anaerobic growth with nitrate as H-acceptor, two growth phases are distinguishable: During the first phase cell growth occurs with the reduction of nitrate to nitrite, which is accumulated; on the second phase nitrite is reduced with the formation of gaseous nitrogen.Washed, anaerobically grown cells reduce nitrate and nitrite with the formation of N₂. Stoichiometric experiments employing hydrogen or fructose as the hydrogen donors are consistent with the conclusion that nitrogen is the sole product of denitrification by these cells. This was confirmed by mass spectrometric analysis of the gas formed. Aerobically grown cells are able to reduce nitrate only to nitrite; when grown in the presence of ammonia, the reduction rate is very low.The results indicate that strain H 16 contains only one nitrate reductase. The formation of this enzyme system is not influenced by oxygen, however, is repressed by ammonia.When employing a purified soluble fraction and particles, nitrite reductases were found in both fractions. The nitrite reductase system is formed only under anaerobic conditions.

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Abkürzungen MB Methylenblau - PMS Phenazinmethosulfat     

Sources of Nitrogen Supporting Growth and Embryogenesis in Cultured Wild Carrot Tissue

This paper seeks to calarify conflicting reports on the nitrogen requirements for in vitro embryogenesis in Daucus carota. Tissue derived from petiole explants of the wild strain of this species were tested with a variety of sources of cellular nitrogen under conditions otherwise favorable for in vitro embryogenesis. The use of very small, sieved and well-washed inocula reduced the carry-over of soluble materials with the inoculum. Embryo yield was quantified by direct counting of samples. Nitrate at concentrations ranging from 5 to 95 mM KNO₃ supportes only weak growth and very low embryogenesis under the exacting conditions of these experiments. As little as 0.1 mM NH₄Cl added to a nitrate medium allows some embryogenesis and 10 mM NH₄Cl is near optimal when KNO₃ is in the range of 12 to 40 mM concentration. Glutamine, glutamic acid, urea and alanine can individually partially replace NH₄Cl as a supplement to KNO₃. Glutamine, alanine, and possibly glutamic acid can serve as sole sources of nitrogen supporting both good growth and embryogenesis. It was concluded that a reduced nitrogen source is required, at least as a supplement to nitrate, for rapid growth and for in vitro embryogenesis of cultured wild carrot tissue. The relationship of pH of the culture medium to growth and embryogenesis was explored and optima observed at approximately pH 5.4 for both processes.     

Effects of nitrogen fertilisers on the Javanese root-knot nematode Meloidogyne javanica and its interaction with cucumber

The influence of different nitrogen salts at five electrical conductivity levels (EC 2, 4, 6 and 8 mmhos/cm) on the Javanese root-knot nematode (RKN) (Meloidogyne javanica) and its interaction with cucumber was evaluated under in vitro, growth chamber and greenhouse conditions. Percentages of egg-hatching and second-stage juvenile viability of M. javanica were greatly reduced when NH₄Cl, (NH4)₂SO₄ and NH₄NO₃ were used especially at the higher levels of EC and accompanied with reduction in cucumber root galling. The lower root galling (less than 2.5) was accompanied with NH₄Cl, (NH₄)₂SO₄ and (NH₄)₂HPO₄, while KNO₃ and NH₄NO₃ resulted in moderate root galling. In contrast to the nitrogen salts, NaCl caused a reduction in both nematode infection and root growth especially at higher EC levels and this could be due to salinity effect. Diammonium phosphate was superior over the tested salts in increasing plant and root fresh and dry weights and cucumber phosphorus content, while KNO₃ was superior in increasing in plant content of potassium.     

Ammonium-nitrogen metabolism and nitrogen fixation in azotobacter vinelandii

The probable effect of increasing levels of ammonium nitrogen on the growth, efficiency of nitrogen fixation, and main cellular constituents of Azotobacter vinelandii was studied under shaking and static culture conditions. The presence of NH₄⁺-N up to 50 mgl^-1 level has no harmful effect on the multiplication as well as the yield efficiency ratio of the tested organism. A. vinelandii was able to fix dinitrogen in the presence of NH₄⁺-N when both nitrogen sources were available in the culturing medium. The efficiency of nitrogen fixation was affected by the initial presence of NH₄⁺-N in the medium, it was quite low at the highest level. The crude protein efficiency ratio was correlated inversely with the initial NH₄⁺-N concentration, whereas the total carbohydrate efficiency ratio as well as the total lipid efficiency ratio were positively correlated with the NH₄⁺-N concentration. The presence of NH₄⁺-N in the culturing medium has no essential influence on the qualitative composition of the amino acids in the Azotobacter cells.     

Chemoorganoheterotrophic Growth of <Emphasis Type="Italic">Nitrosomonas europaea</Emphasis> and <Emphasis Type="Italic">Nitrosomonas eutropha</Emphasis>

The ammonia oxidizers Nitrosomonas europaea and Nitrosomonas eutropha are able to grow chemoorganotrophically under anoxic conditions with pyruvate, lactate, acetate, serine, succinate, α-ketoglutarate, or fructose as substrate and nitrite as terminal electron acceptor. The growth yield of both bacteria is about 3.5 mg protein (mmol pyruvate)⁻¹ and the maximum growth rates of N. europaea and N. eutropha are 0.094 d⁻¹ and 0.175 d⁻¹, respectively. In the presence of pyruvate and CO₂ about 80% of the incorporated carbon derives from pyruvate and about 20% from CO₂. Pyruvate is used as energy and only carbon source in the absence of CO₂ (chemoorganoheterotrophic growth). CO₂ stimulates the chemoorganotrophic growth of both ammonia oxidizers and the expression of ribulose bisphosphate carboxylase/oxygenase is down-regulated at increasing CO₂ concentration. Ammonium, although required as nitrogen source, is inhibitory for the chemoorganotrophic metabolism of N. europaea and N. eutropha. In the presence of ammonium pyruvate consumption and the expression of the genes aceE, ppc, gltA, odhA, and ppsA (energy conservation) as well as nirK, norB, and nsc (denitrification) are reduced.     

Conversion of sodium cyanide to carbon dioxide and ammonia by immobilized cells ofPseudomonas putida

Summary Pseudomonas putida, isolated from contaminated industrial wastewaters and soil sites, was found to utilize sodium cyanide (NaCN) as a sole source of carbon and nitrogen. Cells, immobilized in calcium alginate beads (1–2 mm diameter) were aerated in air-uplift-type fluidized batch bioreactor containing 100–400 ppm of NaCN. Degradation of NaCN was monitored for 168 h by analyzing gaseous and dissolved ammonia (NH₃), CO₂, pH and optical density. The results indicated that the alginate-immobilized cells ofP. putida were able to degrade NaCN into NH₃ and CO₂ in a time-dependent manner.     

The Effect of Several Protein Amino Acids and Some Inorganic Nitrogen Sources on the Growth of Sphagnum nemoreum

The nitrogen metabolism of a bog moss, Sphagnum nemoreum Scop., has been studied in aseptic cultures. The effect of several protein amino acids, especially those found in peat, has been investigated. NH₄NO₃ (1.25 mM) was the best nitrogen source but NH₄⁺ ions were more effectively utilized than NO₃⁻ ions when given as the only nitrogen source. Some of the amino acids (2.5 mM) allowed fairly satisfactory growth (arginine and alanine) when given as the only nitrogen source, but some of them were not utilized at all (leucine, lysine, isoleucine and methionine). Given at low concentrations (0.001 and 0.25 mM) together with NH₄NO₃ (2.5 mM), most of the protein amino acids failed to reveal any growth-promoting or -inhibiting effect. Only lysine (0.25 mM) clearly inhibited growth under these conditions. The nitrogen metabolism of Sphagnum nemoreum seems to be rather flexible and this species is more tolerant of organic nitrogen, especially hydroxyproline, than the higher plants.     

Physiological conditions conducive to high cell density and high cyanophycin content in <Emphasis Type="Italic">Ralstonia eutropha</Emphasis> strain H16 possessing a KDPG aldolase gene-dependent addiction system

The recombinant strain of Ralstonia eutropha H16-PHB⁻4-∆eda (pBBR1MCS-2::cphA ₆₃₀₈/eda _H16) presenting a 2-keto-3-desoxy-phosphogluconate (KDPG) aldolase (eda) gene-dependent catabolic addiction system for plasmid maintenance when using gluconate or fructose as sole carbon source was used in this study. The effects of the initial pH, the nitrogen-to-carbon ratio, the inorganic components of medium, the oxygen supply, and the different carbon and nitrogen sources on the cell dry matter (CDM) and the cyanophycin granule polypeptide (CGP) content of the cells were studied in a mineral salts medium (MSM) without any additional amino acids or CGP precursor substrates. The experiments were designed to systematically find out the optimal conditions for growth of cells to high densities and for high CGP contents of the cells. Maximum contents of water-insoluble CGP and water-soluble CGP, contributing to 47.5% and 5.8% (w/w) of CDM, respectively, were obtained at the 30-L scale cultivation when cells were cultivated in MSM medium containing sufficient supplements of fructose, NH₃, K₂SO₄, MgSO₄⋅7H₂O, Fe(Ш)NH₄-citrate, CaCl₂⋅2H₂O, and trace elements (SL6). The molecular masses of water-insoluble and water-soluble CGP ranged from 25 to 31 kDa and from 15 to 21 kDa, respectively. High cell densities of up to 82.8 g CDM/L containing up to 37.8% (w/w) water-insoluble CGP at the 30-L scale cultivation were also obtained. This is by far the best combination of high cell density and high cellular CGP contents ever reported, and it showed that efficient production of CGP at the industrial scale in white biotechnology could be achieved.     

l‐cysteine desulfhydrase‐related H2S production is involved in OsSE5‐promoted ammonium tolerance in roots of Oryza sativa

Previous studies revealed that rice heme oxygenase PHOTOPERIOD SENSITIVITY 5 (OsSE5) is involved in the regulation of tolerance to excess ammonium by enhancing antioxidant defence. In this study, the relationship between OsSE5 and hydrogen sulfide (H₂S), a well‐known signalling molecule, was investigated. Results showed that NH₄Cl triggered the induction of l ‐cysteine desulfhydrase (l ‐DES)‐related H₂S production in rice seedling roots. A H₂S donor not only alleviated the excess ammonium‐triggered inhibition of root growth but also reduced endogenous ammonium, both of which were aggravated by hypotaurine (HT, a H₂S scavenger) or dl ‐propargylglycine (PAG, a l ‐DES inhibitor). Nitrogen metabolism‐related enzymes were activated by H₂S, thus resulting in the induction of amino acid synthesis and total nitrogen content. Interestingly, the activity of l ‐DES, as well as the enzymes involved in nitrogen metabolism, was significantly increased in the OsSE5‐overexpression line (35S:OsSE5), whereas it impaired in the OsSE5‐knockdown mutant (OsSE5‐RNAi). The application of the HT/PAG or H₂S donor could differentially block or rescue NH₄Cl‐hyposensitivity or hypersensitivity phenotypes in 35S:OsSE5‐1 or OsSE5‐RNAi‐1 plants, with a concomitant modulation of nitrogen assimilation. Taken together, these results illustrated that H₂S function as an indispensable positive regulator participated in OsSE5‐promoted ammonium tolerance, in which nitrogen metabolism was facilitated.     

Growth inhibition of hybridoma cells by ammonium ion: correlation with effects on intracellular pH

The effect of NH₄Cl addition on intracellular pH (pH _i ) was determined by flow cytometric measurements of the fluorescence of a pH-sensitive dye. The effects of NH₄Cl on growth were determined for batch growth of cells in flasks in an incubator. The addition of NH₄Cl caused a cytoplasmic acidification. A new lower steady-state value of pH _i was attained within 20–40 min of NH₄Cl addition. A correlation was found between the effects of NH₄Cl on growth and on pH _i : whereas 3 mM NH₄Cl had little effect on growth and on pH _i , 10 mM NH₄Cl caused a substantial growth inhibition and a pH _i decrease of 0.2–0.3 units. The effects of NH₄Cl on growth and on pH _i were found to be independent of the external pH value (pH _e over the range 6.8 to 7.6, except that 10 mM NH₄Cl was more toxic at pH _e 7.6. The addition of NH₄Cl caused an increase in the average cell volume at pH _e 7.6, but had no effect on the average cell volume at pH _e 's 6.8 and 7.2. For comparison, the effects of pH _e alone on growth and on pH _i were determined. There was little difference in cell growth at pH _e 's 6.8, 7.2 and 7.6. At pH _e 6.6, there was a substantial growth inhibition. Some measurements of the effects of pH _e on pH _i were made, although the steady-state value of pH _i as a function of pH _e was not determined due to limitations in the pH _i -measuring technique. These measurements showed that pH _i remained constant from pH _e 7.6 to 6.8, but fell by 0.2 units at pH _e 6.6, in agreement with the growth results.