Distribution and Metabolic Diversity of Microorganisms in Deep Igneous Rock Aquifers of Finland

Groundwater samples from 200- to 950-m depths in four igneous rock sites in Finland were investigated for different metabolic groups of microorganisms, and the data were compared with the available geochemical record. Samples were collected with a pressurized groundwater sampling system developed for gas and microbiological sampling. Two of the sites had groundwater that was fresh, with < 0.2 g/l dissolved solids, whereas that at the two other sites was much more saline, reaching a maximum of 24 g/l dissolved solids. The groundwater contained gases, 33 to 340 ml/l, with nitrogen or methane dominating. Total cell numbers were 10 5 to 10 6 cells/ml, which is typical for deep igneous rock aquifers. Growth media were designed to mimic the actual groundwater chemistry at each sampling point and used for most probable number enumeration of methanogens, acetogens, sulfate-reducing bacteria (SRB), and iron-reducing bacteria (IRB). SRB predominated in sites where iron sulfide fracture-filling minerals are common. IRB were the main population in one site where iron sulfide fracture minerals are not present, but iron hydroxide fracture minerals predominate. Fracture-filling minerals were a better indicator of microbial populations than was groundwater chemistry. Low numbers of autotrophic methanogens were cultured. One of several possible interpretations of stable isotope data suggested that most of the detected methane is thermogenic, which would correlate with few active methanogens. However, we concluded other interpretations were also possible.     

Quantitation of dissolved gas content in emulsions and in blood using mass spectrometric detection

Quantitation of dissolved gases in blood or in other biological media is essential for understanding the dynamics of metabolic processes. Current detection techniques, while enabling rapid and convenient assessment of dissolved gases, provide only direct information on the partial pressure of gases dissolved in the aqueous fraction of the fluid. The more relevant quantity known as gas content, which refers to the total amount of the gas in all fractions of the sample, can be inferred from those partial pressures, but only indirectly through mathematical modeling. Here we describe a simple mass spectrometric technique for rapid and direct quantitation of gas content for a wide range of gases. The technique is based on a mass spectrometer detector that continuously monitors gases that are rapidly extracted from samples injected into a purge vessel. The accuracy and sample processing speed of the system is demonstrated with experiments that reproduce within minutes literature values for the solubility of various gases in water. The capability of the technique is further demonstrated through accurate determination of O(2) content in a lipid emulsion and in whole blood, using as little as 20 μL of sample. The approach to gas content quantitation described here should greatly expand the range of animals and conditions that may be used in studies of metabolic gas exchange, and facilitate the development of artificial oxygen carriers and resuscitation fluids.     

Dissolved Oxygen Utilization as an Indicator of Total Microbial Activity on Non-metallic Materials in Contact with Potable Water

The technique for the assessment of growth of micro-organisms on non-metallic materials in contact with potable water employs standard microbiological counting techniques and a subjective visual assessment to measure the microbial activity that occurs in the test procedure. An alternative method of assessing microbial activity based on utilization of dissolved oxygen is described and compared with the existing method. The dissolved oxygen method was found to be a suitable alternative, the main advantages being reproducibility of results and a reduction in the cost of the test.     

Stopped flow mass spectrometry: applications to the carbonic anhydrase reaction

Membrane inlet mass spectrometry is combined with a stopped flow technique in order to allow the recording of moderately fast transients in concentrations of dissolved gases. The method was used for direct measurements of CO2 transients in the spontaneous and enzyme catalyzed hydrations of carbon dioxide. Rate constants and activation energies were determined.     

A numerical study of the nonsteady transport of gases in the pulmonary capillaries

A mathematical model is formulated for simulating the unsteady transport of gases in the blood flowing through the pulmonary capillaries. The formulation takes into account the transport mechanisms of molecular diffusion, convection and facilitated diffusion of the species due to haemoglobin. A time dependent situation is created by allowing to vary suddenly the partial pressures of the gases either in the venous blood or in the alveolar air. A numerical technique is described to solve the resulting time-dependent system of nonlinear coupled partial differential equations with the physiologically relevant boundary, entrance and initial conditions. The time required by the gases to achieve equilibrium is computed. It is shown that the dissolved oxygen takes longest in reaching equilibration whereas the carbon dioxide is the fastest. The various physiologically relevant unsteady situations have been examined.     

Dissolved atmospheric gas in xylem sap measured with membrane inlet mass spectrometry

A new method is described for measuring dissolved gas concentrations in small volumes of xylem sap using membrane inlet mass spectrometry. The technique can be used to determine concentrations of atmospheric gases, such as argon, as reported here, or for any dissolved gases and their isotopes for a variety of applications, such as rapid detection of trace gases from groundwater only hours after they were taken up by trees and rooting depth estimation. Atmospheric gas content in xylem sap directly affects the conditions and mechanisms that allow for gas removal from xylem embolisms, because gas can dissolve into saturated or supersaturated sap only under gas pressure that is above atmospheric pressure. The method was tested for red trumpet vine, Distictis buccinatoria (Bignoniaceae), by measuring atmospheric gas concentrations in sap collected at times of minimum and maximum daily temperature and during temperature increase and decline. Mean argon concentration in xylem sap did not differ significantly from saturation levels for the temperature and pressure conditions at any time of collection, but more than 40% of all samples were supersaturated, especially during the warm parts of day. There was no significant diurnal pattern, due to high variability between samples.     

Use of a portable quadrupole mass spectrometer for the measurement of dissolved gas concentrations in ovine rumen liquor in situ

The use of membrane-inlet mass spectrometry in the study of dissolved gas concentrations in the rumen was evaluated in order to assess the value of the technique as a tool for the study of microbial activity in ecosystems in situ. Four dissolved gases (CH₄, CO₂, H₂, and O₂) were measured simultaneously and continuously for short periods (up to 30 min) during the feeding period. These preliminary results have demonstrated the usefulness of the technique for monitoring microbial activity via gas production in a complex natural ecosystem.     

Large-scale plant cell culture: methods, applications and products.

Recent significant contributions to the design of large-scale plant cell cultures for secondary metabolite production include: the development of a strategy to control the concentration of dissolved gases at constant shear; a surface immobilization technique to retain cell mass at high mixing rates; and a modified stirred-tank reactor with a mesh cage to prevent damage of hairy root cultures.     

Observation of a chaotic multioscillatory metabolic attractor by real-time monitoring of a yeast continuous culture

We monitored a continuous culture of the yeast Saccharomyces cerevisiae by membrane-inlet mass spectrometry. This technique allows very rapid simultaneous measurements (one point every 12 s) of several dissolved gases. During our experiment, the culture exhibited a multioscillatory mode in which the dissolved oxygen and carbon dioxide records displayed periodicities of 13 h, 36 min and 4 min. The 36- and 4-min modes were not visible at all times, but returned at regular intervals during the 13-h cycle. The 4-min mode, which has not previously been described in continuous culture, can also be seen when the culture displays simpler oscillatory behavior. The data can be used to visualize a metabolic attractor of this system, i.e. the set of dissolved gas concentrations which are consistent with the multioscillatory state. Computation of the leading Lyapunov exponent reveals the dynamics on this attractor to be chaotic.     

The inhibition of mitochondrial cytochrome oxidase by the gases carbon monoxide, nitric oxide, hydrogen cyanide and hydrogen sulfide: chemical mechanism and physiological significance

The four gases, nitric oxide (NO), carbon monoxide (CO), hydrogen sulfide (H₂S) and hydrogen cyanide (HCN) all readily inhibit oxygen consumption by mitochondrial cytochrome oxidase. This inhibition is responsible for much of their toxicity when they are applied externally to the body. However, recently these gases have all been implicated, to greater or lesser extents, in normal cellular signalling events. In this review we analyse the chemistry of this inhibition, comparing and contrasting mechanism and discussing physiological consequences. The inhibition by NO and CO is dependent on oxygen concentration, but that of HCN and H₂S is not. NO and H₂S are readily metabolised by oxidative processes within cytochrome oxidase. In these cases the enzyme may act as a physiological detoxifier of these gases. CO oxidation is much slower and unlikely to be as physiologically important. The evidence for normal physiological levels of these gases interacting with cytochrome oxidase is equivocal, in part because there is little robust data about their steady state concentrations. A reasonable case can be made for NO, and perhaps CO and H₂S, inhibiting cytochrome oxidase in vivo, but endogenous levels of HCN seem unlikely to be high enough.     

Physical and Chemical Studies of Lago Tupé; a Central Amazonian Black Water “Ria Lake”

Lago Tupé, a Central Amazonian dystrophic black water “Ria lake” (blocked valley lake), lateral to the Rio Negro, was studied at all depths for a year. Physical and chemical results of that study are presented here as a prelude to the microbiological studies. The lake's origin and morphology are discussed. Results concerned with thermal properties, suspended matter and dissolved solids are presented, in addition to results on ionic composition, alkalinity, hardness, pH and dissolved oxygen. Nutrient studies include section of phosphate and nitrogen and conclude with silicate results.     

Molecular hydrogen from water radiolysis as an energy source for bacterial growth in a basin containing irradiating waste

Although being deionized, filtered and therefore normally deeply oligotrophic, the water from a basin containing irradiating waste presented relatively high bacterial concentrations (ca 10(5) cfu ml(-1)) and biofilm development at its surface and on the walls. This water was characterized by a high concentration of molecular H2 due to water radiolysis, while its electrochemical potential was around +400 mV due the presence of dissolved O2 and active oxygen compounds. This combination of H2 availability and of an oxidant environment is completely original and not described in nature. From surface and wall biofilms, we enumerated the autotrophic populations ( approximately 10(5) bacteria ml(-1)) able to grow in presence of H2 as energy source and CO2 as carbon source, and we isolated the most abundant ones among cultivable bacteria. They efficiently grew on a mineral medium, in the presence of H2, O2 and CO2, the presence of the three gases being indispensable. Two strains were selected and identified using their rrs gene sequence as Ralstonia sp. GGLH002 and Burkholderia sp. GGLH005. In pure culture and using isotope exchange between hydrogen and deuterium, we demonstrated that these strains are able to oxidize hydrogen as energy source, using oxygen as an electron acceptor, and to use carbon dioxide as carbon source. These chemoautotroph hydrogen-oxidizing bacteria probably represent the pioneer bacterial populations in this basin and could be primary producers in the bacterial community.     

Mass spectrometry as en ecological tool for in situ measurement of dissolved gases in sediment systems

Abstract The use of membrane-inlet quadrupole mass spectrometry, as a method for quantitative monitoring of dissolved gases in natural or semi-natural environments, is described. Its advantages over other methods lie in the fact that it provides an accurate, sensitive means for non-invasive, continuous analysis of several dissolved gases simultaneously. The potential of mass spectrometry as an ecological tool is illustrated by representative results from measurements made on undisturbed and experimentally amended estuarine and fresh-water sediments.
Dissolved gas profiles from the surface to a depth of 10 cm in the estuarine sediment showed that the dissolved oxygen decreased gradually until at 10 cm it was undetectable (< 0.25 μM); dinitrogen reached a maximum at 6 cm, where oxygen was 20 μM. In a fresh-water sediment, methane reached 1.5 mM at 10 cm depth. NO_x was also detected; quantitation of carbon dioxide necessitates a correction for the contribution of NO_x. Manipulation of conditions (gas phase, nitrogen and carbon sources) permitted ecological modelling.     

Direct interface of chemistry to microbiological systems: membrane inlet mass spectrometry.

Direct measurement of dissolved gases and low molecular weight volatiles through permeable membranes (e.g. 50-microm-thick silicone rubber), provides an invaluable tool for the investigation of the activities of microorganisms in the laboratory and in their natural environments. Multiple molecular species are monitored at a single point. Fast response times (t(90%)<1 min) and long-term stability, (<1% week(-1)); high specificity and high sensitivity (e.g. 0.2 microM for O(2), <0.5 mM for ethanol), provides a technique that can provide information on the kinetics of processes over many decades (10(0)-10(6)) of minutes. Spatial resolution of <1 mm enables 3D mapping of gases in complex ecosystems (sediments, peat, soils, biofilms, foodstuffs). Results with membrane inlet mass spectrometry (MIMS) when used in conjunction with confocal scanning laser microscopy, provides a powerful approach to the analysis of kinetic and spatial aspects of natural environments. Examples discussed are peat cores and cheese.     

Centroid search optimization of cultural conditions affecting the production of extracellular proteinase by Pseudomonas fragi ATCC 4973

The production of extracellular proteinase by Pseudomonas fragi ATCC 4973 grown in a defined citrate medium, containing glutamine as the sole nitrogen source, was determined under varying cultural conditions. Simultaneous evaluation of cultural conditions using a 'centroid search' optimization technique showed that the optimum cultural conditions for proteinase production by Ps. fragi were: incubation temperature, 12.5 degrees C; incubation time, 38 h; initial pH, 6.8; organic nitrogen concentration, 314 mmol nitrogen/l (glutamine); a gas mixture containing 16.4% oxygen flowing over the medium (7.42 ppm dissolved oxygen). Oxygen was the major factor influencing proteinase production by Ps. fragi. The results may have applications in the storage of fluid milk. Centroid search optimization was shown to be suitable for microbiological experiments.     

Effect of dissolved carbon dioxide on penicillin fermentations: Mycelial growth and penicillin production

The effect of dissolved carbon dioxide on the specific growth rate and the penicillin production rate of Penicillium chrysogenum was examined experimentally. The dissolved carbon dioxide was found to inhibit the specific growth rate and the penicillin production rate when the aerated submerged penicillin fermentation was exposed to influent gases of 12.6 and 20% carbon dioxide, respectively. Upon exposure to influent gases of 3 and 5% carbon dioxide, no pronounced metabolic inhibition was noted.     

NMR studies of water-gas interactions

Analysis of published data concerning the solubility of different gases in water as dependent on temperature was carried out. These dependences could be described by mono-or bi-exponential functions. Solubilities of nitrogen and oxygen are additive and depend on their percentage in the atmosphere over the liquid. The temperature dependence of oxygen and nitrogen dissolved in water corresponds to that in the atmospheric air. Measurements of water spin-lattice relaxation times, changing with the concentration of dissolved paramagnetic oxygen, showed that oxygen could be substantially but not completely eliminated by saturation with any gas. The best method is the contact with a water-immiscible liquid of gas capacity higher than water. However, all this leads to an unstable state of a gas-water system, converging to equilibrium.     

A Comparison of Methods to Measure Operator Protection Factors in Open-fronted Microbiological Safety Cabinets

Comparative tests to measure operator protection factors in microbiological safety cabinets in accordance with British Standard 5726 have demonstrated good agreement in the results obtained by a microbiological method using a Collison nebulizer and the technique producing an aerosol of potassium iodide. Either method is suitable for testing for operator protection factors in Class I and Class II safety cabinets.
The Collison nebulizer should be considered as the standard aerosol generator for the microbiological method; alternative nebulizers meeting the general requirements of BS 5726 should be compared in performance with this nebulizer if they are to be used for containment tests.
Any microbiological safety cabinet specified for a new installation should have been 'type' tested to ensure compliance with BS 5726. However, in order to ensure adequate performance, on-site commissioning tests (and routine maintenance checks thereafter) are necessary to verify that air velocity, filtration and operator protection factor requirements are met.     

Kinetics of nitrification and denitrification reactions

Nitrification and denitrification are important microbiological reactions of nitrogen. In this work, the kinetics of these reactions have been investigated based on a Monod-type expression involving two growth limiting substrates: ammonium nitrogen and dissolved oxygen for nitrification and nitrate nitrogen and dissolved organic carbon for denitrification. The kinetic constants and yield coefficients were evaluated for both these reactions. Past experimental work was used to determine the constants for the nitrification reaction. For the denitrification reaction, experiments were performed in a stirred tank reactor under conditions such that only one substrate was growth limiting. Steady-state values of the substrate concentrations in the reactor were determined at various dilution rates. These data were analyzed to obtain the kinetic and stoichiometric constants. From these constants it was concluded that in the range of nitrate nitrogen concentrations encountered in waste water, the denitrification reaction can be considered a first-order reaction. It was also found that three times as much organic carbon is required as nitrate nitrogen for complete nitrogen removal.     

The secretion of inert gas into the swim-bladder of fish

The composition of the gas mixture secreted into the swim-bladders of several species of fish has been determined in the mass spectrometer. The secreted gas differed greatly from the gas mixture breathed by the fish in the relative proportions of the chemically inert gases, argon, neon, helium, and nitrogen. Relative to nitrogen the proportion of the very soluble argon was increased and the proportions of the much less soluble neon and helium decreased. The composition of the secreted gas approaches the composition of the gas mixture dissolved in the tissue fluid. A theory of inert gas secretion is proposed. It is suggested that oxygen gas is actively secreted and evolved in the form of minute bubbles, that inert gases diffuse into these bubbles, and that the bubbles are passed into the swim-bladder carrying with them inert gases. Coupled to a preferential reabsorption of oxygen from the swim-bladder this mechanism can achieve high tensions of inert gas in the swim-bladder. The accumulation of nearly pure nitrogen in the swim-bladder of goldfish (Carassius auratus) is accomplished by the secretion of an oxygen-rich gas mixture followed by the reabsorption of oxygen.