High pH,ammonia toxicity,and the search for life on the Jovian planets

The Jovian planets have environments apparently suitable for the evolution of life, but, nevertheless, present severe challenges to organisms. One such challenge arises from the presence of ammonia. Ammonia is an efficient biocide, its effect being dependent on pH as well as on concentration. The effects of pH and ammonia concentration were studied separately, where possible, on a variety of organisms, including some isolated from natural environments of high pH and/or ammonia concentration. Escherichia coli andBacillus subtilis are both extremely sensitive to ammonia. An aerobic organism (growth up to pH 11.4) from an alkaline spring is more resistant, but exhibits a toxic response to ammonia at a pH much lower than its maximum for growth. The greatest ammonia resistance has been found in an unidentified organism growing at near neutral pH. Even in this case, however, survival at ammonia concentrations reasonably expected on the Jovian planets is measured in hours. This is, nevertheless, two to three orders of magnitude longer than forE. coli. Our data support the tentative conclusion that contamination of the Jovian planets with terrestrial organisms that can grow is unlikely. However, the range of toxic response noted, coupled with the observation that terrestrial life has not been exposed to high ammonia concentrations for millions of years, suggests that adaptation to greater ammonia tolerance may be possible.     

Influence of pH on Ammonia Accumulation and Toxicity in Halophilic, Methylotrophic Methanogens

We studied the effects of pH and ammonia concentration on the growth of three methanogens. These three halophilic, methylotrophic methanogens, Methanolobus bombayensis, Methanolobus taylorii, and Methanohalophilus zhilinaeae, grew at environmental pH ranges that overlapped with each other and spanned the pH range from 7.0 to 9.5. During growth they had reversed membrane pH gradients ((Delta)pH) at all pH values tested. The (Delta)pH was in the range of -0.4 to -0.9 pH units, with the cytosol being more acidic than the environmental pH. Methanohalophilus zhilinaeae had the most negative (Delta)pH (-0.9 pH units). These negative pH gradients resulted in the accumulation of ammonium (NH(inf4)(sup+)), and when grown at the highest external ammonia concentrations that allowed good growth, cells had cytosolic NH(inf4)(sup+) concentrations as high as 180 mM. The high concentrations of cytosolic NH(inf4)(sup+) were accompanied by greater (Delta)pH and lower concentrations of the major cytosolic cation K(sup+) (compared with cells grown in medium with only 5 mM ammonia). Methanolobus bombayensis and Methanolobus taylorii were more sensitive to total external ammonia at higher external pH values, but the inhibitory concentration of un-ionized ammonia that resulted in a 50% reduction of the growth rate was about 2 to 5 mM, regardless of the pH. This is consistent with growth inhibition by ammonia in other bacteria. However, Methanohalophilus zhilinaeae was more resistant to un-ionized ammonia than any other known organism. It had a 50% inhibitory concentration for un-ionized ammonia of 13 mM at pH 8.5 and 45 mM at pH 9.5. We examined the effects of pH on three ammonia-assimilating activities (glutamine synthetase, glutamate dehydrogenase, and alanine dehydrogenase) in cell lysates and found that the pH ranges were consistent with the observed ranges of intracellular pH.     

The prospect of life on Jupiter

Recent research into the possibility of there being life in the Solar System other than on the Earth has suggested that Jupiter may be a good candidate. The reactions within its atmosphere, both photolytic and electrical, lead to the formation of many nitrile and amino compounds. We have simulated electrical discharges in the Jovian atmosphere, using anhydrous methane-ammonia mixtures, and shown the formation of simple aliphatic nitriles, amino-nitriles, and their oligomers. Including hydrogen sulfide in the gas mixture, it appears that sulfur-containing amino-nitriles are not formed, since the hydrolysate of the products did not contain the corresponding amino-acids. There is a strong analogy between these reactions and the classical spark reactions simulating the primitive Earth's atmosphere. We are attempting a closer simulation of Jupiter's atmosphere by using appropriate temperature and pressure conditions. It seems that prebiotic synthesis on Jupiter may have reached an advanced state. As an alternative approach we have tested the survival ability of common terrestrial microorganisms in aqueous media at 102 atmospheres pressure and at 20°C. in a simulated Jovian atmosphere.E. coli, S. marcescens, A. aerogenes andB. subtilis will all tolerate 24 h. under these conditions with conditions with little death. The ability of terrestrial organisms to survive Jovian atmospheric conditions, coupled with the likelihood of advanced prebiotic synthesis, suggests that a parallel evolution of life may have occurred on Jupiter and Earth, and that the two forms need not be so different as has been supposed. Lunar Science Institute Contribution.     

On the coexistence of competing microbial species in a chemostat under cycling

If a microorganism has a growth coupled production or consumption of acid or alkali, it is possible to use the pH-auxostat as a means of control in continuous fermentation. In using the pH-auxostat, it is possible to separate the inlet substrate flow in two different streams. These will both be pH controlled, with one main flow, consisting of nutrients and a second minor but concentrated flow, of acid or alkali. Hereby, it is possible to vary the difference in pH between the fermentor and the inlet medium. This pH difference is proportional to the steady-state cell mass concentration.(1,2) It is shown that by separating the inlet flow in two different streams and cultivating without any substrate limitation, the maximum growth rate may be obtained while the cell mass concentration will be controlled. This will also give the possibility to reach high cell mass concentrations at mu(max) without the risk of wash-out. A modified expression, based on hydrogen, of the steady-state bio-mass concentration, X, is developed as \documentclass{article}\pagestyle{empty}\begin{document}$$ X = Y_{X/H} \cdot [F_{{\rm Hin}} /(F_{{\rm Hin}} + F_{{\rm Min}} )] \cdot (C_{{\rm Hin}} - C_{{\rm HFERM}} ) $$\end{document} where Y(X/H) is the yield coefficient of cell mass per acid produced. The indexes Hin and Min refer to the inflows of alkali and medium, respectively; C(Hin) is the inlet concentration of hydrogen ions. The boundary condition for the cell mass shows that S(in) > X/Y(X/S), where S(in) is the medium substrate concentration and Y(X/S) is the yield of biomass per consumed substrate. It is shown that when the cell mass concentration exceeds this value, the flow stops. The applicability of the pH-auxostat method is then verified from different experiments. It is hereby used to detect a deviation from the maximal growth rate showing effects on the microbial physiology. With Escherichia coli used as the model organism, the effect on the growth rate of temperature and high concentration of ammonia were investigated.     

Complementary functioning of the component proteins of nitrogenase from several bacteria

The nitrogenase proteins from eight organisms have been highly purified, and a survey of their cross-reactions shows that the nitrogenase proteins from a wide variety of organisms can interact with one another. An active cross-reaction is the complementary functioning of the MoFe protein and the Fe protein from different organisms. Of 64 possible combinations of component proteins, 8 yielded homologous nitrogenases (components from the same organism); 45 of the 56 possible heterologous crosses generated active hybrid nitrogenases; 4 heterologous crosses yielded no measurable nitrogenase activity but did form inactive tight-binding complexes; 6 crosses did not give measurable activity; and 1 cross was not made. All these crosses were assayed for acetylene reduction, and some also were assayed for ammonia formation, hydrogen evolution, and ATP hydrolysis activity. The activity generated by combining two complementary heterologous nitrogenase components depended on pH, component ratio, and protein concentration, the same factors that determine the activity of homologous nitrogenases. However, several crosses showed an unusual dependency on component ratio and protein concentration, and some cross-reactions showed interesting ATP hydrolysis activity.     

Fluorescence detection of organic molecules in the Jovian atmosphere.

A search for fluorescent emission due to the presence of possible organic molecules in the Jovian atmosphere is described. We first consider natural Jovian fluorescent emission excited by precipitating auroral particles. Due to our lack of knowledge of the Jovian precipitation particle energies and fluxes we next consider fluorescent emission excited by a laser system aboard a Jupiter spacecraft. Laser-induced fluorescence is routinely used to monitor trace constituents and pollutants in the terrestrial atmosphere. Several spacecraft laser systems are currently under development. Our calculations indicate that laser-induced fluorescent detection is approximately two orders of magnitude more sensitive than rocket ultraviolet measurements of possible Jovian absorption features at 2600 A that have been attributed to the presence of adenine or benzene.     

Fluorescence detection of organic molecules in the Jovian atmosphere

A search for fluorescent emission due to the presence of possible organic molecules in the Jovian atmosphere is described. We first consider natural Jovian fluorescent emission excited by precipitating auroral particles. Due to our lack of knowledge of the Jovian precipitating particle energies and fluxes we nest consider fluorescent emission excited by a laser system aboard a Jupiter spacecraft. Laser-induced fluorescence is routinely used to monitor trace constituents and pollutants in the terrestrial atmosphere. Several spacecraft laser systems are currently under development. Our calculations indicate that laser-induced fluorescent detection is approximately two orders of magnitude more sensitive than rocket ultraviolet measurements of possible Jovian absorption features at 2600 Å that have been attributed to the presence of adenine or benzene.     

A kinetic microplate method for quantifying the antibacterial properties of biological fluids

The microplate assay for measuring antibacterial activity was adapted by incorporating a known concentration range of gentamicin as an internal standard. Staphylococcus aureus subsp. aureus strain ATCC 6538P, Escherichia coli ATCC 25922, and Lactobacillus spp. were used as target organisms, although other indicator organisms and antibiotics can be examined. Assay range and sensitivity were dependent on the species and density of indicator organism, and conditions (e.g., type, concentration, and pH of growth medium). Plotting the area under the growth curve (AUGC) versus gentamicin concentration (log transformed) yielded a linear curve that was used to quantify in units of gentamicin the antibacterial activity of a secretory fluid (SCF; pancreatic juice) and for comparisons of samples collected at different times, analysed on different days, and from different studies. This adaptation of the microtiter broth method will be useful for investigating man-made compounds, and the antibacterial activity of secretory fluids and the influences of age, diet, and health status.     

Molecular cloning of a coding sequence of Bordetella pertussis filamentous hemagglutinin gene

Abstract The halophilic phototrophic bacterium Rhodospirillum salexigens was tested for growth on a variety of organic and inorganic nitrogenous compounds as sole nitrogen sources. In media containing acetate as carbon source, the amino acids glutamate, proline, and aspartate supported good growth of R. salexigens ; several other amino acids or ammonia did not support growth. Attempts to grow R. salexigens on ammonia led to the discovery that this organism excretes a highly basic substance under certain nitrogen nutritional conditions which raises the pH above that supporting growth. Cultures of R. salexigens transferred to media containing both pyruvate and acetate as carbon sources grew on ammonia as sole nitrogen source and the culture pH did not rise. Dual substrate experiments showed that R. salexigens utilized glutamate in preference to ammonia when both were present at equimolar concentrations.     

Urease-mediated destruction of bacteria is specific for Helicobacter urease and results in total cellular disruption

Abstract The survival of Helicobacter mustelae, Proteus mirabilis, Escherichia coli and Campylobacter jejuni in the presence of urea and citrate at pH 6.0 was examined. H. mustelae , which has urease activity similar to H. pylori , had a markedly reduced survival, median 2.5% (0–78%) ( P <0.001) when incubated nder these conditions. Only 7% of the ammonia produced by H. mutelae urease activity was recovered from the buffer, a similar percentage to that previously reported with H. pylori . None of the other organisms, all of which had lower urease activity, had impaired survival under these conditions. Electron microscopical studies demonstrated extensive structural damage to H. pylori following exposure to urea and citrate at pH 6.0. This structural damage to the organisms makes it unlikely that the low recovery of ammonia was due to retention of ammonia within the bacteria and suggests that the ammonia may have been incorporated into glutamate or other amino acids. Incorporation of ammonia into these compounds would deplete the cell of the key metabolic intermediate α-ketoglutarate and could thus explain the mechanism of the urease-dependent destruction of the organism.     

The effect of pH on the toxicity of ammonia to a murine hybridoma.

The growth inhibition of a murine hybridoma mediated by ammonium chloride was shown to vary with the pH of the culture medium. Values for the initial media concentration causing 50% growth inhibition (IC50) ranged from 4 mM to 7.6 mM as the pH was reduced from 7.8 to 6.8. A significant negative correlation was observed between the IC50 and the NH3 concentration of the medium, suggesting that ammonia and not ammonium may be the toxic species in the culture medium. The optimum initial pH for cell growth was 7.4. However, this optimum shifts to lower pH as ammonia accumulates in culture as a metabolic by-product. This suggests that in order to obtain high cell yields, it may be beneficial to adopt a culture strategy of lowering pH during cell growth to offset the inhibitory effects of accumulated ammonia.     

Effects of low pH and humus on the survivorship, growth and feeding of Gammarus pulex (L.) (Amphipoda)

SUMMARY. 1. The toxicity to the freshwater amphipod Gammarus pulex (L.) of soft water (pH6.0) with three concentrations of XAD-extracted aquatic humus was treated in the laboratory.
2. Exposure to pH6.0 water without humus added resulted in 92% mortality after 3 weeks. Humus, added at concentrations of 7 and 20 g Cm ³, decreased the mortality to 80% and 64% in the same period.
3. Surviving animals kept at pH6.0 had a lower growth rate, lower food conversion efficiency and higher body water content than animals kept at pH 7.3.
4. Humus had no significant effects on growth, food conversion or body composition of G. pulex kept at pH 6.0. However, there was a tendency for growth and food conversion to increase, and for body water content to decrease with increased humus concentration. The effects of humus on growth and food conversion of G. pulex observed in this study do not support the contention that humus acts as a free coupler to lower metabolic efficiency. On the contrary, humus tended to benefit food conversion.
5. It is suggested that low concentrations of humus can be directly beneficial to organisms in acidified water in ways other than by complexing toxic metals.     

Dialysis Culture of T-Strain Mycoplasmas

Using dialyzing cultures of T-strain mycoplasmas, it was possible to make some observations relevant to the growth and metabolism of these organisms which would not be possible in nondialyzing cultures due to growth inhibition of the organisms by elevated pH and increased ammonium ion concentration in media containing urea. The rate of ammonia accumulation was found to be related to the initial urea concentration in the medium and could not be accounted for by any change in the multiplication rate of the organisms. More ammonia was generated than could be accounted for by the added urea alone, suggesting that an ammonia-producing activity other than urease may be present in T-strain mycoplasmas. Titers above 10⁷ color change units per ml were achieved in dialysis cultures of a T-strain mycoplasma in the presence of urea, and such titers were maintained for approximately 60 h during dialysis culture in the absence of added urea.     

Evaluation of toxic potential of captan: Induction of hsp70 and tissue damage in transgenic Drosophila melanogaster (hsp70-lacZ) Bg9

The study investigated the working hypothesis that a widely used fungicide captan exerts toxic effects on nontarget organisms. Transgenic Drosophila melanogaster (hsp70-lacZ) was used as a model by assaying stress gene expression as an endpoint for cytotoxicity and also to evaluate whether stress gene expression is sufficient enough to protect and to prevent tissue damage against toxic insult of the chemical. The study was further extended to understand the effect of the pesticide on development, life cycle, and reproduction of the organism and finally to evaluate a concentration of the chemical to be nontoxic to the organism. The study showed that (i) captan causes cytotoxicity at and above 0.015 ppm; (ii) at 0.0015 ppm captan, absence of hsp70 expression in the exposed organism was evaluated as the concentration referred to as no observed adverse effect level (NOAEL) for Drosophila; (iii) emergence pattern of flies was affected only at the highest concentration of captan by 4 days, while hatching and survivorship were unaffected even at this concentration; (iv) reproductive performance was significantly affected only at 125.0 and 1250.0 ppm captan, while in the lower dietary concentrations no such deleterious effects were observed; (v) at 1250.0 ppm, hsp70 failed to protect the cells from toxicant assault after 48 h exposure, thus leading to tissue damage as revealed by Trypan Blue staining. The present study shows the cytotoxic potential of captan and further reveals the application of stress genes in determining NOAEL and its expression as bioindicator of exposure to environmental contaminants.     

Effect of pH of the Medium on the Availability of Chelated Iron for Chlamydomonas mundana

SYNOPSIS. Nutritional and cultural factors influenced growth of Chlamydomonas mundana at different H-ion concentrations. Growth at acid pH (5.5–6.0) primarily depended on the acetate and chelated iron concentration and on the method of preparation of the medium. The organism had increased Na, Ca, and Mn requirements at an acid pH. Sensitivity to increased levels of phosphate was greater at low than at high pH. The requirement for chelated as compared with non-chelated Fe was qualitatively easier to demonstrate at low pH. The procedure followed in preparing the growth media was more important with higher levels of chelator and Fe and with media of low pH. All 6 synthetic chelators studied were more effective, as measured by growth, if combined with iron before addition to the media. At high pH (7.0–7.5), the organism was much more adept at utilizing over-chelated Fe. This latter finding, and the greater sensitivity at low pH to trace-metal imbalance in ill-prepared media, indicate that the organism absorbs Fe much more efficiently at neutral or slightly alkaline pH.     

Population growth rate as a basis for ecological risk assessment of toxic chemicals

Assessing the ecological risks of toxic chemicals is most often based on individual-level responses such as survival, reproduction or growth. Such an approach raises the following questions with regard to translating these measured effects into likely impacts on natural populations. (i) To what extent do individual-level variables underestimate or overestimate population-level responses? (ii) How do toxicant-caused changes in individual-level variables translate into changes in population dynamics for species with different life cycles? (iii) To what extent are these relationships complicated by population-density effects? These issues go to the heart of the ecological relevance of ecotoxicology and we have addressed them using the population growth rate as an integrating concept. Our analysis indicates that although the most sensitive individual-level variables are likely to be equally or more sensitive to increasing concentrations of toxic chemicals than population growth rate, they are difficult to identify a priori and, even if they could be identified, integrating impacts on key life-cycle variables via population growth rate analysis is nevertheless a more robust approach for assessing the ecological risks of chemicals. Populations living under density-dependent control may respond differently to toxic chemicals than exponentially growing populations, and greater care needs to be given to incorporating realistic density conditions (either experimentally or by simulation) into ecotoxicological test designs. It is impractical to expect full life-table studies, which record changes in survival, fecundity and development at defined intervals through the life cycle of organisms under specified conditions, for all relevant species, so we argue that population growth rate analysis should be used to provide guidance for a more pragmatic and ecologically sound approach to ecological risk assessment.     

Toxicity of ammonia to algae in sewage oxidation ponds.

Ammonia, at concentrations over 2.0 mM and at pH values over 8.0, inhibits photosynthesis and growth of Scenedesmus obliquus, a dominant species in high-rate sewage oxidation ponds. Photosynthesis of Chlorella pyrenoidosa, Anacystis nidulans, and Plectonema boryanum is also susceptible to ammonia inhibition. Dark respiration and cell morphology were unaffected by any combination of pH and ammonia concentrations tested, thus limiting the apparent effect to inhibition of the normal function of the chloroplasts. Methylamine had the same effect as ammonia, and its penetration into the cells was found to be pH dependent. Therefore, the dependence of toxicity of amines to algae on pH apparently results from the inability to penetrate the cell membrane in the ionized form. When operated at 120-h detention time of raw wastewater, the high-rate oxidation pond maintained a steady state with respect to algal growth and oxygen concentration, and the concentration of ammonia did not exceed 1.0 mM. Shifting the pond to 48-h detention time caused an increase in ammonia concentration in the pond water to 2.5 mM, and the pond gradually turned anaerobic. Photosynthesis, which usually elevates the pH of the pond water to 9.0 to 10.0, could not proceed beyond pH 7.9 because of the high concentration of ammonia, and the algal population was washed out and reduced to a concentration that could maintain a doubling time of 48 h without photosynthesis bringing the pH to inhibitory levels. Under these conditions, the pH of the bond becomes a factor that limits the operational efficiency of the oxidation pond.     

Toxicity of ammonia to algae in sewage oxidation ponds.

Ammonia, at concentrations over 2.0 mM and at pH values over 8.0, inhibits photosynthesis and growth of Scenedesmus obliquus, a dominant species in high-rate sewage oxidation ponds. Photosynthesis of Chlorella pyrenoidosa, Anacystis nidulans, and Plectonema boryanum is also susceptible to ammonia inhibition. Dark respiration and cell morphology were unaffected by any combination of pH and ammonia concentrations tested, thus limiting the apparent effect to inhibition of the normal function of the chloroplasts. Methylamine had the same effect as ammonia, and its penetration into the cells was found to be pH dependent. Therefore, the dependence of toxicity of amines to algae on pH apparently results from the inability to penetrate the cell membrane in the ionized form. When operated at 120-h detention time of raw wastewater, the high-rate oxidation pond maintained a steady state with respect to algal growth and oxygen concentration, and the concentration of ammonia did not exceed 1.0 mM. Shifting the pond to 48-h detention time caused an increase in ammonia concentration in the pond water to 2.5 mM, and the pond gradually turned anaerobic. Photosynthesis, which usually elevates the pH of the pond water to 9.0 to 10.0, could not proceed beyond pH 7.9 because of the high concentration of ammonia, and the algal population was washed out and reduced to a concentration that could maintain a doubling time of 48 h without photosynthesis bringing the pH to inhibitory levels. Under these conditions, the pH of the bond becomes a factor that limits the operational efficiency of the oxidation pond.     

Effects of ammonia and lactate on hybridoma growth, metabolism, and antibody production

The influence of ammonia and lactate on cell growth, metabolic, and antibody production rates was investigated for murine hybridoma cell line 163.4G5.3 during batch culture. The specific growth rate was reduced by one-half in the presence of an initial ammonia concentration of 4 mM. Increasing ammonia levels accelerated glucose and glutamine consumption, decreased ammonia yield from glutamine, and increased alanine yield from glutamine. Although the amount of antibody produced decreased with increasing ammonia concentration, the specific antibody productivity remained relatively constant around a value of 0.22 pg/cell-h. The specific growth rate was reduced by one-half at an initial lactate concentration of 55 mM. Although specific glucose and glutamine uptake rates were increased at high lacatate concentration, they showed a decrease after making corrections for medium osmolarity. The yield coefficient of lactate from glucose decreased at high lactate concentrations. A similar decrease was observed for the ammonia yield coefficient from glutamine. At elevated lactate concentrations, specific antibody productivities increased, possibly due to the increase in medium osmolarity. The specific oxygen uptake rate was insensitive to ammonia and lactate concentrations. Addition of ammonia and lactate increased the calculated metabolic energy production of the cells. At high ammonia and lactate, the contribution of glycolysis to total energy production increased. Decreasing external pH and increasing ammonia concentrations caused cytoplasmic acidification. Effect of lactate on intracellular pH was insignificant, whereas increasing osmolarity caused cytoplasmic alkalinization.