Inhibitory effects of titanium (III) citrate on enumeration of bacteria from rumen contents.

Titanium citrate (TC) or L-cysteine-sodium sulfide was added as a reducing agent to buffers and agar media used for enumeration of bacteria from rumen contents of high-forage-fed steers. Approximately equal colony counts were found on TC and L-cysteine-sodium sulfide-reduced media with rumen contents taken 8 h postfeeding, when active bacterial growth was occurring. The colony counts on TC medium were only 56% of those with L-cysteine-sodium sulfide medium with rumen contents taken 1 h prefeeding when bacterial growth was minimal. When colonies from L-cysteine-sodium sulfide medium were transferred to TC medium and vice versa, almost all colonies grew. The data indicate that TC can be inhibitory to bacteria upon their initial isolation from natural habitats, particularly when growth rates are low in these habitats.     

Relationship between the cytoplasm and the vacuole phosphate pool in Acer pseudoplatanus cells

The Pi concentration of Acer pseudoplatanus cells in the two major intracellular compartments, the cytoplasm and the vacuole, has been studied using 31P NMR. For sycamore cells containing approximately 2 mM of total Pi, the cytoplasmic Pi and the vacuolar Pi concentrations were approximately 6 and 1.5 mM, respectively. When the cells were transferred to a phosphate-deficient medium, the vacuolar Pi decreased rapidly while the cytoplasmic Pi decreased slowly during the first 48 h, indicating that Pi in the cytoplasm was maintained at the expense of the vacuolar Pi. When the Pi-starved cells (i.e., those containing less than 0.5 mumol of total Pi/g wet wt) were transferred to a medium containing 300 microM Pi, Pi entered the cells rapidly and accumulated in the cytoplasm. Once the cytoplasmic Pi pool was filled, Pi was taken up in the vacuole until the vacuole Pi pool was filled. On the contrary when the non-Pi-starved cells were transferred to a phosphate-rich medium (i.e., containing 45 mM Pi), Pi entered the cells slowly by diffusion and accumulated in the vacuole but not in the cytoplasm. These results demonstrate that the Pi content of the cytoplasm is maintained at the expense of the vacuolar Pi pool when sycamore cells are transferred to either a phosphate-deficient or a phosphate-rich medium.     

On the effects of phosphorus on zinc uptake by barley

Summary The response of barley to phosphate application and the effect of applied phosphorus on the uptake of soil zinc by the crop were tested in pot, Neubauer and incubaticn experiments on four soils differing in native phosphate status.There was a response of the yield to phosphorus application in alluvial soils from Clementina (Ecuador) and Bangla Desh. Barley grown on red soil from Bangla Desh and glacial clay from Uppsala did not show any response to P application.Applied P³² was fixed to a great extent in all the soils studied. But in phosphate-deficient soils, a much higher degree of sorption of P³² was recorded than in phosphate-rich soils. Compared to phosphate-rich soils, the utilization of fertilizer phosphorus is higher than of native soil P in the case of phosphate-deficient soils. It was observed that uptake of Zn⁶⁵ by the crop is counteracted by phosphorus application at the three stages of crop growth studied.This work was financially supported by the International Atomic Energy Agency, Vienna, Austria, and by the National Council for Forestry and Agricultural Research, Sweden.     

Changes in the concentration of phenolic compounds and exudation induced by phosphate deficiency in bean plants (Phaseolus vulgaris L.)

The effect of prolonged phosphate starvation of bean plants (Phaseolus vulgaris L.) on the concentration of phenolics and their exudation by roots was studied. Plants cultured on phosphate-deficient media maintained a steady concentration of total phenolics in the leaves, whereas in the leaves of plants grown on complete nutrient media the phenolic concentration decreased. After 18 days of culture, higher total phenolics and anthocyanin concentrations in phosphate-deficient leaves compared with control leaves were observed. The divergent trends in total phenolic concentrations between phosphate-deficient and control leaves corresponded to the changes in the activity of L-phenylalanine ammonia-lyase. In the roots, the concentration of total phenolics was lower in phosphate-deficient plants compared with control plants. However, after 18 days of culture of bean plants, the amount of exuded phenolics from phosphate-deficient roots was 5-times higher than that from the roots of control plants. The activity of L-phenylalanine ammonia-lyase was twice as high in the roots of phosphate-starved plants. Comparable rates in the exudation of phenolics by bean roots observed after 18 days of culture on nitrogen-deficient or phosphate-deficient medium may suggest a similar system of signal transduction for phenolics release. The results are discussed in relation to the possible functions of phenolics in nutrient uptake and as chemical signals in root-soil microbe interactions to enhance the plant adaptation to particular environmental conditions.     

Effects of cadmium, copper, magnesium, and zinc on the decomposition of citrate by a Klebsiella sp

The effects of Cd2+, Cu2+, Mg2+, and Zn2+ on the decomposition of citric acid by a Klebsiella sp. were studied by monitoring the degradation of [14C]citrate. The carbon concentration used was 10 micrograms of C liter-1, and the media were designed to provide at least 95% of the citrate complexed to the metal studied. After 72 h of incubation, 80% of the uncomplexed citric acid and 76% of the magnesium citrate had been decomposed. A marked inhibition was observed when Cd2+, Cu2+, or Zn2+ was bound to the organic anion; only 23% of the cadmium citrate, 14% of the zinc citrate, and 5% of the cuprous citrate had been decomposed. The effects were not the result of toxicity, since experiments run with [14C]glucose (nonchelating compound) instead of citrate resulted in similar decomposition rates regardless of the presence of the metal. To examine whether the binding of a metal to citrate enhanced its uptake by the Klebsiella sp., we studied the relative uptake of 65Zn in citrate- and in glucose-containing media. No such effect could be observed, with the uptake of Zn2+ being higher in the glucose-containing media. The study shows that metals may render low-molecular-weight organic acids, such as citric acid, resistant to bacterial degradation. This stresses the importance of metals in influencing microbial decomposition of organic compounds, not only as a result of toxicity.     

Effects of cadmium, copper, magnesium, and zinc on the decomposition of citrate by a Klebsiella sp.

The effects of Cd2+, Cu2+, Mg2+, and Zn2+ on the decomposition of citric acid by a Klebsiella sp. were studied by monitoring the degradation of [14C]citrate. The carbon concentration used was 10 micrograms of C liter-1, and the media were designed to provide at least 95% of the citrate complexed to the metal studied. After 72 h of incubation, 80% of the uncomplexed citric acid and 76% of the magnesium citrate had been decomposed. A marked inhibition was observed when Cd2+, Cu2+, or Zn2+ was bound to the organic anion; only 23% of the cadmium citrate, 14% of the zinc citrate, and 5% of the cuprous citrate had been decomposed. The effects were not the result of toxicity, since experiments run with [14C]glucose (nonchelating compound) instead of citrate resulted in similar decomposition rates regardless of the presence of the metal. To examine whether the binding of a metal to citrate enhanced its uptake by the Klebsiella sp., we studied the relative uptake of 65Zn in citrate- and in glucose-containing media. No such effect could be observed, with the uptake of Zn2+ being higher in the glucose-containing media. The study shows that metals may render low-molecular-weight organic acids, such as citric acid, resistant to bacterial degradation. This stresses the importance of metals in influencing microbial decomposition of organic compounds, not only as a result of toxicity.     

Arabitol accumulation in Geotrichum candidum

Culture conditions which lead to the intracellular accumulation of arabitol and mannitol in Geotrichum candidum were investigated. The accumulation of arabitol was dependent on the concentrations of metabolizable hexoses, the non-metabolizable disaccharide sucrose, NaCl and KCl in the growth medium. In media containing 2% (w/v) glucose, fructose or l-sorbose cultures contained only mannitol after 48 h or 72 h growth. In media containing 10% (w/v) to 30% (w/v) glucose, or 25% (w/v) fructose or l-sorbose there was an increase in the total concentration of intracellular polyol due to the accumulation of arabitol. This pentitol was also found to accumulate intracellularly when the organism was grown in medium containing 34% (w/v) sucrose, 0.7 M NaCl or 0.7 M KCl in addition to 2% (w/v) glucose. Under the conditions tested no change in the accumulation of mannitol or ethanol-soluble carbohydrate, believed to be primarily composed of trehalose, was evident.Intracellular polyol was released during incubation of arthrospores obtained from media containing 25% or 10% glucose, in distilled water at 25° C, but no polyol was released under these conditions from arthrospores obtained from growth in 2% glucose medium.     

Effect of the pho85 mutation on the catabolite repression of the CIT1 gene in yeasts Saccharomyces cerevisiae

The Krebs cycle is one of the major metabolic pathways in a cell, which includes both catabolic and anabolic reactions. The first enzyme of the Krebs cycle, citrate synthase, catalyzes one of a few irreversible reactions of the cycle, citrate formation from acetyl-CoA and oxaloacetate. Expression of the CIT1 gene encoding the mitochondrial form of this enzyme in Saccharomyces cerevisiae is repressed on glucose- and glutamate-containing medium and activated on the raffinose-containing medium. In this work, the dependence of glucose repression of the CIT1 gene on the content of phosphate in the medium was studied. On the phosphate-deficient medium, the level of the CIT1 gene expression was increased twice. A low-molecular-weight (about 34 kDa) protein was identified and shown to interact with a region of the CIT1 gene promoter (from -367 to -346 bp), which controls the glucose repression. The results obtained suggest that the Pho4 protein is involved in regulation of the CIT1 gene expression on the glucose-containing and phosphate-deficient medium. Disruption of the PHO85 gene encoding phosphoprotein kinase (Pho4p is the substrate of this enzyme) leads to alleviation of glucose repression of the CIT1 gene. Thus, in yeast cells grown in the presence of glucose, the PHO85 gene mediates downregulation of the CIT1 expression.     

<Emphasis Type="Italic">Xylella fastidiosa</Emphasis> Cultivation on a Minimal Solid Defined Medium

A simple defined solid medium containing citrate and succinate, three amino acids (L-glutamine, L-asparagine, and L-cysteine), hemin chloride, potato starch, gellan gum (GelRite), and mineral salts supported the growth of grape strains of Xylella fastidiosa, the bacterial pathogen that causes Pierces disease of grape. Isolation efficiency from infected grape plant samples, determined by the number of colony forming units recovered, on the defined medium was slightly less (10-fold) or indistinguishable from two standard rich media used for culturing X. fastidiosa, PWG and PD3, respectively. The bacterium also grew on media with citrate and L-glutamine as the only carbon and nitrogen sources. Potato starch was not essential for bacterial growth, but no growth was observed on media without hemin chloride. Agar inhibited bacterial growth when used as the gelling agent.     

Comparative study of two culture media for the detection of phospholipase activity of Candida albicans and Cryptococcus neoformans strains

Phospholipase activity (PHA) is considered a virulence factor related to pathogenicity of Candida albicans and Cryptococcus neoformans. The aim of this work was to compare the ability of two culture media: malt egg-yolk agar (MEA) and Sabouraud-egg yolk agar (SEA), for the detection of phospholipase activity. Forty four strains of C. neoformans and 54 of C. albicans isolated from different clinical specimens of human origin were studied. The phospholipase production was determined as a ratio between the diameter of each colony and the corresponding lysis halo. The values ranged between 0 and 1, and the highest level of enzymatic activity was the nearest to 0. Enzymatic activity was observed in 34 C. neoformans strains, grown either in MEA or SEA media; 59% of enzyme producers were detected in SEA only, while five strains (15% of producers) were detected just in MEA medium. Phospholipase activity was observed in both media only in nine of 34 enzyme producer strains. Forty two out of 54 strains of C. albicans were detected as enzyme producers; 31 of them (73.8%) were detected in MEA medium only. On the other hand 10 strains (23.8% of the enzyme producers) showed phospholipase activity just in SEA medium. Detection of PHA could be done by both media in one case only. In order to evaluate the time needed to detect PHA, 41 C. albicans strains were incubated 72 h. They were read at 24 h intervals. No enzyme activity was detected at 24 h, 15 enzyme producer strains remain negative at 48 h and the halos of all strains with PHA were better distinguished after 72 h. It was possible to conclude that neither MEA nor SEA media were good enough as the unique medium to detect phospholipase activity. Nevertheless, MEA was better than SEA to detect PHA of C. albicans after 72 h incubation. The opposite situation was seen when we studied PHA in C. neoformans strains. In this case, greater sensibility was observed with SEA medium compared with MEA medium. Six days incubation, but not longer incubation times, were necessary to detect phospholipase activity in C. neoformans strains.     

Responses of Nicotiana tabacum to CO2 enrichment at low-photon flux density

Bean plants ( Phaseolus vulgaris ) grown in phosphate-deficient (–P) medium display deficiency symptoms after about 2 weeks of culture. A decrease in inorganic phosphate level in roots was observed after 10 days of culture, and after 17 days it was more than 30 times less than control. The dry weight of the shoots of phosphate-deficient plants was lower whereas the dry weight of the roots was higher as compared to the control roots. After 2 weeks of culture, the sucrose level in –P roots almost doubled compared to control roots. An increase in glucose and fructose was observed earlier than that of sucrose, and after 10 days of culture in phosphate-deficient medium the glucose level in –P roots was about 3 times higher than that in +P roots. At the same time, a decrease in hexose-phosphate level was observed. This decrease may reflect a higher drain from the hexose phosphate pool, or it may be due to the lower capacity for hexose phosphorylation of phosphate-deficient plants, as judged by the lower hexokinase and fructokinase activities. The ratio of non-phosphorylated to phosphorylated sugars in –P roots was about 5 times higher as compared to control roots. We propose that glucose and fructose accumulation in phosphate-deficient roots represents a non-metabolic, probably vacuolar pool which is not utilized for growth and metabolism of the roots.     

A comparison of a sucrose-based solution with other preservation media for cold storage of isolated hepatocytes

A sucrose-based solution has been compared with other preservation solutions (University of Wisconsin (UW) solution and Marshall's citrate solution, with Dulbecco's medium as control) during hypothermic preservation of isolated rat hepatocytes for up to 72 h. Studies on the stability of liver cells at low temperature by exclusion of trypan blue dye and morphological appearance were conducted. During storage beyond 24 h, there was a clear difference between cells stored in Dulbecco's medium and Marshall's citrate and those stored in sucrose-based solution and UW solution, with the former storage groups showing many cells developing large membrane "blebs" and the latter storage groups maintaining a more typical morphology and developing only small membrane protrusions. Dye exclusion was higher in sucrose-based solution (48 h, 75 +/- 7%; 72 h, 65 +/- 6%) and UW solution (48 h, 72 +/- 5%; 72 h, 63 +/- 4%) than in Marshall's citrate (48 h, 31 +/- 5%; 72 h, 10 +/- 1%) and Dulbecco's medium (48 h, 8 +/- 2%; 72 h, 5 +/- 1%). These data suggest that sucrose-based solution should be investigated further as a less complex alternative solution for storage of isolated hepatocytes.     

Aluminum Elicits Exocellular Phosphatidylethanolamine Production in Pseudomonas fluorescens

Pseudomonas fluorescens ATCC 13525 was found to grow in a minimal mineral medium supplemented with millimolar amounts of aluminum, a known environmental toxicant. During the stationary phase of growth, the trivalent metal was localized in a phosphatidylethanolamine (PE)-containing residue. The concentration of PE in pellets ranged from 1.7 to 13.9 mg ml of culture(sup-1) in media supplemented with 1 to 30 mM aluminum. Although the gelatinous residue was observed during the stationary phase of growth, ultracentrifugation and dialysis experiments revealed that PE was produced from earlier stages of incubation and was associated with aluminum. A sharp diminution in the levels of PE and aluminum in the spent fluid was concomitant with the formation of the insoluble deposit. The aluminum content of the soluble cellular fraction increased during growth and reached an optimum of 1.85 mM of test metal at 45 h in cultures with 15 mM aluminum. Further incubation, however, led to a marked decrease in the cellular aluminum content, and during the stationary phase of growth, only trace amounts of the trivalent metal were detected in this fraction. When 45-h cells were incubated in fresh citrate medium, most of the intracellular aluminum was secreted in the spent fluid and citrate was rapidly consumed. Aluminum efflux was also observed in cultures in which d-glucose was substituted for citrate. However, no efflux of this trivalent metal was evident in media devoid of either citrate or d-glucose. Scanning electron microscopic studies and X-ray energy-dispersive analyses of the dialyzed supernatant aided in the visualization of nodule-like aluminum- and phosphorus-rich bodies associated with thread-like carbon-, oxygen-, and phosphorus-containing structures. Transmission electron microscopic and electron energy loss spectroscopic analyses revealed the presence of aluminum within bacteria after 45 h of incubation. Cells harvested after aluminum insolubilization did not shown aluminum inclusions. This aluminum-tolerant microbe may have potential application in bioremediation processes.     

Frothy feedlot bloat in cattle: production of extracellular polysaccharides and development of viscosity in cultures of Streptococcus bovis.

Streptococcus bovis was cultured in a synthetic medium with three concentrations of sucrose. Initial viscosity of the media was 1.5 centipoise (cp). After incubation for 8 h, the viscosity of the medium with 0.5% sucrose was unchanged, that with 3% sucrose had increased to 8 cp, and that with 6% sucrose to 112 cp. Similar results were found with a rumen fluid medium. A slimy material, responsible for increased viscosity of these cultures, was digested by dextranase. The material appeared as a complex system of intercellular fibers when viewed under the electron microscope after freeze-etching. With proteins and other polymers released from lysed bacteria, this slimy material may contribute directly to increased viscosity and foam formation. In addition to these intercellular fibers, each cell was surrounded by a fibrous capsule that was not digested by dextranase. This capsule stained with lead citrate and uranyl acetate, but not with ruthenium red. The amount of capsular material produced was similar whether the media contained 0.5, 3.0, or 6% sucrose.     

Selection of mixed inoculants exhibiting growth-promoting activity in rice plants from undefined consortia obtained by continuous enrichment

Aims

The main objective of this study was to test the hypothesis that isolating and characterizing bacterial suspensions (undefined consortia) from the root/rhizosphere of rice will contribute to the selection of mixtures of rhizobacteria with better growth-promoting activity of rice plants.

Methods

Bacterial consortia were obtained from roots/rhizosphere soil samples of rice plants grown under upland and irrigated production systems. Those undefined consortia were subjected to five consecutive passes every 7 days in NFb (N-free broth) semisolid medium. Thereafter, strains of each growth-promoting consortia were isolated by plating on three different culture media. Then, undefined consortia, as well as mix and single bacterial strains, were characterized in terms of indoleacetic acid production, nitrogen fixation capacity, and growth promotion of rice plants.

Results

Of the 72 consortia analyzed, 41.7 % and 50.0 % increased nitrogenase activity and the production of indolic compounds, respectively, after 5 continuous passes in NFB medium. Three undefined consortia, 11 single strains and 5 strain mixtures, exhibited plant growth promotion in rice plants under greenhouse conditions.

Conclusions

Continuous enrichment in Nfb medium of undefined consortia from root/rhizosphere soil is a good strategy for the selection of plant growth-promoting bacteria for rice plants.     

Effect of the pho85 Mutation on Catabolite Repression of the CIT1 Gene in Yeasts Saccharomyces cerevisiae

The Krebs cycle is one of the major metabolic pathways in a cell, which includes both catabolic and anabolic reactions. The first enzyme of the Krebs cycle, citrate synthase, catalyzes one of a few irreversible reactions of the cycle, citrate formation from acetyl-CoA and oxaloacetate. Expression of the CIT1 gene encoding the mitochondrial form of this enzyme inSaccharomyces cerevisiae is repressed on glucose- and glutamate-containing medium and activated on the raffinose-containing medium. In this work, the dependence of glucose repression of the CIT1 gene on the content of phosphate in the medium was studied. On the phosphate-deficient medium, the level of the CIT1 gene expression was increased twice. A low-molecular-weight (about 34 kDa) protein was identified and shown to interact with a region of the CIT1gene promoter (from –367 to –348 bp), which controls the glucose repression. The results obtained suggest that the Pho4 protein is involved in regulation of the CIT1gene expression on the glucose-containing and phosphate-deficient medium. Disruption of the PHO85 gene encoding phosphoprotein kinase (Pho4p is the substrate of this enzyme) leads to alleviation of glucose repression of the CIT1 gene. Thus, in yeast cells grown in the presence of glucose, the PHO85gene mediates downregulation of theCIT1expression.     

Growth efficiencies of freshwater bacterioplankton

The growth efficiency of freshwater bacteria was examined in continuous cultures. One series of experiments was carried out using generation times from 50 to 200 hours and aged, normal, and enriched media, all of natural origin. Another series of experiments examined the bacterial growth efficiency during the growth season in eutrophic Frederiksborg Slotssø, in relation to changes in the planktonic communities and to factors controlling the bacterial incorporation of ³H-thymidine. Attachment of bacteria to the inner surfaces of the experimental flasks was examined using various types of bottles, adding glass tubes to the bottles, and measuring ³H-thymidine incorporation and direct cell counts of attached and free-living bacteria. Attachment of bacteria varied, and in one example up to 36% of the thymidine incorporation was by attached bacteria after 4 days. It was calculated that 36% of attached bacteria caused an underestimation of the growth efficiency of 11%. The mean growth efficiency tended to decrease with generation time using enriched medium (47 to 19%) and aged medium (35 to 12%), and tended to decrease with medium quality (enriched > normal > aged media) from 37% to 27%. The only significant difference in growth efficiency occurred in relation to generation time, in samples with enriched medium (unpaired t-test, P < 0.05). The overall mean value for all generation times and media was 30% (SEM = 3%, n = 24). From April to October, the growth efficiency was determined 5 times in samples from Frederiksborg Slotssø. The overall mean value was 31% (SEM = 3%, n = 30), and there was no significant change in the growth efficiency during the period measured. In June, three bioassay experiments revealed that carbon limitation controlled bacterial incorporation of ³H-thymidine, whereas additions of phosphate and nitrate did not change the incorporation rates. The narrow range of growth efficiencies obtained in this study (mean 31%, SEM = 2%, n = 54) suggests that changes in substratequality in the media applied and in the eutrophic samples examined causes only subtle changes in the growth efficiency.     

用于质粒DNA规模化生产的大肠杆菌发酵培养基的筛选

为降低质粒DNA的生产成本,在标准LB培养基的基础上,利用国产试剂配制成十种大肠杆菌液体培养基,以pEGFPC3、pcDNAlacZ和pcDNKLYZ质粒转化的JM109和DH5α大肠杆菌为指示菌进行小规模发酵培养,定时采样测量OD600值及质粒产量,获得一种高性价比培养基。用该培养基培养重组大肠肝菌,绘制生长曲线,并于其对数生长中期进行42℃诱导。结果表明经42℃诱导后,重组大肠肝菌JM109和DH5α的质粒产量均有提高,重组JM109的产量比重组DH5α约提高20％,为低成本、大规模生产重组质粒提供了良好的技术保障。     

In vitro modulation of proliferation and melanization of melanoma cells by citrate

B16/F10 murine melanoma cells were grown for 24 and 36 h in Dulbecco's modified Eagle medium in presence of 10-20 mM trisodium citrate. The intracellular melanin concentration and the melanin secreted in the extracellular medium was estimated. It is observed that 20 mM citrate stimulates extracellular melanin secretion in B16/F10 melanoma cells by 200% at 36 h treatment. The intracellular melanin content increased by 90%. This stimulatory effect of citrate was totally abolished when these cells were grown in presence of 1 mM phenyl thiourea, a specific inhibitor of tyrosinase activity. Citrate (0.1-5 mM) had no effect on dopa oxidase activity either at pH 5.0 or at pH 6.8. There was no increase in the tyrosinase specific activity in presence of citrate. The increased melanin synthesis was shown to be due to stimulation of cellular tyrosine hydroxylase activity by citrate. It has been suggested that enhanced melanin synthesis results in an increased production of metabolites that are toxic to the growth of melanoma cells. We have studied the effect of citrate on cellular proliferation. Following 24 and 36 h treatment with citrate, the cells exhibited a dose-dependent decrease in proliferation. In presence of 20 mM citrate the cell number was only up to 50% of the control cultures after 36 h of incubation. The growth retardation was not due to cytotoxicity. Citrate, a natural metabolite, is a unique molecule which may be involved in the regulation of melanin biosynthetic pathway, since it enhances melanogenesis by increasing the hydroxylase activity of tyrosinase which is the regulatory enzyme of this pathway. These observations add further support to the critical role of intramelanosomal pH in regulation of melanogenesis.     

Growth and Metabolic Characteristics of Mycobacterium paratuberculosis

The cultural characteristics of newly isolated strains of Mycobacterium paratuberculosis on a variety of media were studied. The mycobactin dependence ascribed to this species of Mycobacterium was found to be easily circumvented by incorporation of 1% ferric ammonium citrate in serum or egg yolk media, but the earliest and most abundant growth occurred on serum medium that contained mycobactin and sodium pyruvate. In the presence of ferric ammonium citrate, respiration did not decrease during 5 days; but in the presence of all other agents studied, respiration decreased after the first day.