Solid Medium for Culturing Black Smoker Bacteria at Temperatures to 120°C

A solid, highly thermostable medium, based on the new gelling agent GELRITE, was devised to facilitate the culturing of extremely thermophilic microorganisms from submarine hydrothermal vents. The medium remained solid at temperatures to 120°C at vapor pressures and hydrostatic pressures to 265 atm. It proved useful to its maximum tested limits in isolating colonies of black smoker bacteria from hydrothermal fluids recently collected at the Juan de Fuca Ridge in the Pacific Ocean.     

Formation and function of Vibrio parahaemolyticus lateral flagella.

Formation of the lateral flagella (L-flagella) of Vibrio parahaemolyticus was studied immunologically, using specific antiserum against L-flagella. On solid medium, L-flagella were formed at both high (37 degrees C) and low (25 degrees C) temperatures, although at high temperatures they became dissociated from the cells and decomposed in the medium. L-flagella were not formed in liquid or soft-agar medium. Formation of L-flagella was decreased by lowering the pH of the medium and repressed by transferring the cells from solid medium to liquid medium. Mutants possessing L-flagella but not a polar monotrichous flagellum (M-flagellum) swarmed on solid medium, whereas mutants were grown on solid medium and then transfered to liquid medium, the cells oscillated until they lost L-flagella. It is postulated that L-flagella are locomotive organelles on solid medium and in some cases also in liquid medium, whereas M-flagella are locomotive organelles only in liquid medium.     

Occurrence, recognition, and reversion of spontaneous, sporulation-deficient Bacillus anthracis mutants that arise during laboratory culture

Bacillus anthracis is a spore-forming, soil-dwelling bacterium. This review describes the occurrence of spontaneous mutations leading to loss of sporulation and the selective pressures that can lead to their enrichment. We also discuss recognition of the associated phenotypes on solid medium, thereby allowing researchers to employ measures that either prevent or favor selection of sporulation-deficient mutants.     

The self-stimulation reaction of rabbits in a helium-oxygen environment under increased pressure]

Change of intensity of hypothalamic self-stimulation was determined in rabbits during their stay in normoxic helium-oxygen medium under the pressures of 10, 15 and 40 kgf/cm2 at various speeds of compression. The experiments conducted testify to depressive influence on the hypothalamus self-stimulation of helium-oxygen medium under increased pressure; the influence was more expressed at higher pressures and great speed of compression. It is supposed that the decrease in frequency of pedal pressures was connected with the appearance of nervous syndrome of high pressures.     

Survival and growth of two heterotrophic hydrothermal vent archaea, <Emphasis Type="Italic">Pyrococcus</Emphasis> strain GB-D and <Emphasis Type="Italic">Thermococcus fumicolans</Emphasis>, under low pH and high sulfide concentrations in combination with high temperature and pressure regimes

Growth and survival of hyperthermophilic archaea in their extreme hydrothermal vent and subsurface environments are controlled by chemical and physical key parameters. This study examined the effects of elevated sulfide concentrations, temperature, and acidic pH on growth and survival of two hydrothermal vent archaea (Pyrococcus strain GB-D and Thermococcus fumicolans) under high temperature and pressure regimes. These two strains are members of the Thermococcales, a family of hyperthermophilic, heterotrophic, sulfur-reducing archaea that occur in high densities at vent sites. As actively growing cells, these two strains tolerated regimes of pH, pressure, and temperature that were in most cases not tolerated under severe substrate limitation. A moderate pH of 5.5–7 extends their survival and growth range over a wider range of sulfide concentrations, temperature and pressure, relative to lower pH conditions. T. fumicolans and Pyrococcus strain GB-D grew under very high pressures that exceeded in-situ pressures typical of hydrothermal vent depths, and included deep subsurface pressures. However, under the same conditions, but in the absence of carbon substrates and electron acceptors, survival was generally lower, and decreased rapidly when low pH stress was combined with high pressure and high temperature. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Primordial Ocean Chemistry and its Compatibility with the RNA World

We examine the stability of three key components needed to establish an RNA World under a range of potential conditions present on the early earth. The stability of ribose, cytosine, and the phosphodiester bond are estimated at different pH values and temperatures by extrapolating available experimental data. The conditions we have chosen range from highly acidic or alkaline hydrothermal vents, to the milder conditions in a primordial ocean at a range of atmospheric CO₂ partial pressures.     

A high-pressure thermal gradient block for investigating microbial activity in multiple deep-sea samples

Details about the construction and use of a high-pressure thermal gradient block for the simultaneous incubation of multiple samples are presented. Most parts used are moderately priced off-the-shelf components that easily obtainable. In order to keep the pressure independent of thermal expansion of the sample vessels, a back-pressure system with a constant leak rate was installed. Pressure is applied through high-pressure liquid chromatography (HPLC) pumps that run in constant pressure mode with variable flow rate, thereby regulating any pressure fluctuations. The device allows incubations along a wide range of temperatures and pressures and can easily be modified to accommodate different experiments, either biological or chemical. As an application, we present measurements of bacterial sulfate reduction rates in hydrothermal sediments from Guyamas Basin over a wide range of temperatures and pressures. Sulfate reduction rates increase with increasing pressure and show maximum values at pressures higher than in situ.     

High-pressure-temperature gradient instrument: use for determining the temperature and pressure limits of bacterial growth.

A pressurized temperature gradient instrument allowed a synoptic determination of the effects of temperature and pressure on the reproduction of bacteria. The instrument consisted of eight pressure vessels housed parallel to each other in an insulated aluminum block in which a linear temperature gradient was supported. For a given experiment, eight pressures between 1 and 1,100 bars were chosen; the linear temperature gradient was established over an interval within -20 to 100 degrees C. Pure cultures and natural populations were studied in liquid or solid medium either in short (ca. 2-cm) culture tubes or in long (76.2-cm) glass capillaries. In the case of a pure culture, experiments with the pressurized temperature gradient instrument determined values of temperature and pressure that bounded its growth. Feasibility experiments with mixed populations of bacteria from water samples from a shallow depth of the sea showed that the instrument may be useful in identifying the extent to which a natural population is adapted to the temperatures and pressures at the locale of origin of the sample. Additional conceived uses of the instrument included synoptic determinations of cell functions other than reproduction and of biochemical activities.     

Hydrothermal treatment and ethanol pulping of sunflower stalks

The influence of temperature in the hydrothermal treatment of sunflower stalks on the composition of the liquid fraction obtained was examined. The remaining solid fraction was subjected to ethanol pulping in order to obtain pulp that was used to produce paper sheets. The pulp was characterized in terms of yield, kappa index, viscosity, and cellulose, hemicellulose and lignin contents; and the paper sheets in terms of breaking length, stretch, burst index and tear index. Hydrothermal treatment of the raw material at 190 degrees C provided a liquid phase with maximal hemicellulose-derived oligomers and monosaccharide (glucose, xylose and arabinose) contents (26.9 and 4.2 g/L, respectively). Pulping the solid fraction obtained by hydrothermal treatment at 180 degrees C, with 70% ethanol at a liquid/solid ratio of 8:1 at 170 degrees C for 120 min provided pulp with properties on a par with those of soda pulp from the sunflower stalks, namely: 36.3% yield, 69.1% cellulose, 12.6% hemicellulose, 18.2% lignin and 551 ml/g viscosity. Also, paper sheets obtained from the ethanol pulp were similar in breaking length (3.8 km), stretch (1.23%), burst index (1.15 kN/g) and tear index (2.04 m Nm(2)/g) to those provided by soda pulp.     

Numerical Data Concerning the Sensitivity of Anaerobic Bacteria to Oxygen

Upon comparison of results obtained from growing eight different species of anaerobic bacteria in deep agar under two and three atm of pure oxygen with results of growing them in air under atomospheric pressure, it appears possible that the old terms of "microaerophobic," "very strict anaerobe," and "microaerophilic" can be replaced by precise values corresponding to the size of the inhibition zone produced by oxygen under various pressures when the bacteria are grown in a solid medium. The simultaneous effect of the dilution of the inoculum and the pressure of oxygen used was determined with the help of an electronic computer according to the standard multiple regression method. Two indices have been worked out which express the sensitivity of the species at different oxygen pressures when varying numbers of anaerobic bacteria are used: X sp, which is the size in millimeters of the inhibition zone when 1:10 dilutions of the culture are grown under pure oxygen at atmospheric pressure (lp=0), and B sp, which is the result of multiplying by 1.43 the difference in size of inhibition zone when 1:10 dilutions of the culture are grown under atmospheric air (lp = -0.7) and under pure oxygen at 1 atm absolute.     

Prebiotic Synthesis of Glycine from Ethanolamine in Simulated Archean Alkaline Hydrothermal Vents

Submarine hydrothermal vents are generally considered as the likely habitats for the origin and evolution of early life on Earth. In recent years, a novel hydrothermal system in Archean subseafloor has been proposed. In this model, highly alkaline and high temperature hydrothermal fluids were generated in basalt-hosted hydrothermal vents, where H₂ and CO₂ could be abundantly provided. These extreme conditions could have played an irreplaceable role in the early evolution of life. Nevertheless, sufficient information has not yet been obtained for the abiotic synthesis of amino acids, which are indispensable components of life, at high temperature and alkaline condition. This study aims to propose a new method for the synthesis of glycine in simulated Archean submarine alkaline vent systems. We investigated the formation of glycine from ethanolamine under conditions of high temperature (80–160 °C) and highly alkaline solutions (pH = 9.70). Experiments were performed in an anaerobic environment under mild pressure (0.1–8.0 MPa) at the same time. The results suggested that the formation of glycine from ethanolamine occurred rapidly and efficiently in the presence of metal powders, and was favored by high temperatures and high pressures. The experiment provides a new pathway for prebiotic glycine formation and points out the phenomenal influence of high-temperature alkaline hydrothermal vents in origin of life in the early ocean.     

Numerical simulation of blood and interstitial flow through a solid tumor

A theoretical framework is presented for describing blood flow through the irregular vasculature of a solid tumor. The tumor capillary bed is modeled as a capillary tree of bifurcating segments whose geometrical construction involves deterministic and random parameters. Blood flow along the individual capillaries accounts for plasma leakage through the capillary walls due to the transmural pressure according to Sterling’s law. The extravasation flow into the interstitium is described by Darcy’s law for a biological porous medium. The pressure field developing in the interstitium is computed by solving Laplace’s equation subject to derived boundary conditions at the capillary vessel walls. Given the arterial, venous, and tumor surface pressures, the problem is formulated as a coupled system of integral and differential equations arising from the interstitium and capillary flow transport equations. Numerical discretization yields a system of linear algebraic equations for the interstitial and capillary segment pressures whose solution is found by iterative methods. Results of numerical computations document the effect of the interstitial hydraulic and vascular permeability on the fractional plasma leakage. Given the material properties, the fractional leakage reaches a maximum at a particular grade of the bifurcating vascular tree.     

Polysaccharide hydrolysis accelerated by adding carbon dioxide under hydrothermal conditions

Polysaccharides such as agar, guar gum, starch, and xylan were hydrolyzed to produce mono- and oligosaccharides under hydrothermal conditions with and without carbon dioxide in a small batch reactor. The molecular weight distributions of the polysaccharide hydrolyzates shifted to lower molecular weights by increasing the carbon dioxide load, corresponding to higher pressures of carbon dioxide. For example, the yield of glucose produced from the hydrolysis of starch at 200 degrees C was increased significantly from 3.7% to 53.0% (on a carbon weight basis) of the initial polysaccharide by increasing carbon dioxide load in a reaction time of 15 min. Carbonic acid generated from water and carbon dioxide appeared to lower the pH of high-temperature and high-pressure water. Polysaccharide hydrolysis under hydrothermal conditions in the presence of carbon dioxide is an environmentally benign method to produce mono- and oligosaccharides because the process does not require the use of conventional acids and bases followed by neutralization and separation.     

MORPHOGENESIS OF POLLEN CALLUS CULTURES OF HYOSCYAMUS NIGER

Morphogenesis of calluses of single pollen grain origin shed from anthers of Hyoscyamus niger cultured in a liquid medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) was followed upon their transfer to a solid medium with or without 2,4-D. In a solid medium lacking 2,4-D, small calluses consisting of one to five nodular groups of cells at the time of inoculation differentiated root and shoot systems and formed miniature seedlings. In the same medium large calluses with several nodules initially formed a crop of bipolar somatic embryoids with well-defined root and shoot axes which subsequently differentiated into seedlings. Irrespective of their size at the time of transfer, calluses grown in a solid medium containing 2,4-D continued to proliferate without showing signs of organogenesis or embryogenesis.     

Effects of temperature and pressure on sulfate reduction and anaerobic oxidation of methane in hydrothermal sediments of Guaymas Basin

Rates of sulfate reduction (SR) and anaerobic oxidation of methane (AOM) in hydrothermal deep-sea sediments from Guaymas Basin were measured at temperatures of 5 to 200 degrees C and pressures of 1 x 10(5), 2.2 x 10(7), and 4.5 x 10(7) Pa. A maximum SR of several micromoles per cubic centimeter per day was found at between 60 and 95 degrees C and 2.2 x 10(7) and 4.5 x 10(7) Pa. Maximal AOM was observed at 35 to 90 degrees C but generally accounted for less than 5% of SR.     

Optimization of Ethanol Production From Microfluidized Wheat Straw by Response Surface Methodology

In this study, wheat straw was pretreated with a microfluidizer to improve its enzymatic hydrolysis and ethanol yields. The pretreatment was performed at various pressures (500, 1000, and 1500 bar) and solid loadings (1, 2, and 3%). The microfluidized biomass was then subjected to hydrolysis and simultaneous saccharification and co-fermentation (SSCF) experiments at different enzyme loadings (5, 10, and 15 FPU/g dry wheat straw) using a mutant yeast. The results indicated that the microfluidization method alters the structure of biomass and leads to a reduction in lignin content. The samples pretreated at 1% solid loading contained the minimum lignin concentration and provided the maximum sugar and ethanol yields. These results signified that the microfluidization method is more effective on biomass at low solid loadings. The process conditions were optimized for higher ethanol and sugar yields using response surface methodology (RSM). The optimum pressure and solid and enzyme loadings were found as 1500 bar, 1%, and 15 FPU/g dry wheat straw, respectively. The yields obtained at this condition were 82%, 94%, and 65% for glucose, xylose, and ethanol, respectively. High sugar yields implied that microfluidization is an effective pretreatment method for cellulosic ethanol production. On the other hand, low ethanol yield may indicate that the microorganism was sensitive to inhibitory compounds present in the fermentation medium.     

Effects of suspension density on microbial metabolic processes

Respiration of yeasts Saccharomyces cerevisiae, Rhodotorula glutinis, Endomyces magnusii, and Candida utilis, and of bacteria Escherichia coli and Bacillus megaterium, anaerobic production of CO2 by S. cerevisiae, active transport of quinovose by R. glutinis and of L-proline and L-leucine by S. cerevisiae were highly dependent on cell suspension density. Respiration of S. cerevisiae in the presence of glucose decreased in a biphasic fashion from 140 to 40 nmol O2 per mg dry solid per min as suspension density increased from 0.01 to 2 mg/mL. Higher partial pressures of oxygen further enhanced the trend. The active transports were affected monophasically in the maximum rate of uptake which was as much as ten times greater at low than at high suspension densities. A component of the external medium is suspected to cause the decrease of metabolic functions at higher cell densities, acting as a noncompetitive inhibitor. The component was present and mutually active in suspensions of the various yeasts as well as of bacteria. Its properties and results of model experiments suggest it to be dissolved carbon dioxide.     

Avian Salmonella-Stained Microtest Antigens Produced on Solid Media

Procedures are described for the preparation of Salmonella pullorum and Salmonella typhimurium tetrazolium-stained microagglutination and microantiglobulin antigens with solid media for culture propagation. Cell suspensions were found to be more easily harvested from solid medium than from previously used liquid medium, and greater yields of antigen were obtained. Liquid medium microagglutination and microantiglobulin test antigens were found to be more sensitive than those prepared on solid medium. The microtest antigens produced on solid medium were easier to prepare and in serological tests gave titers more comparable to those demonstrated with the standard macroscopic tube agglutination test.     

Anaerobic Spirochete from a Deep-Sea Hydrothermal Vent

An obligately anaerobic spirochete, designated strain GS-2, was selectively isolated from samples collected at a deep-sea (2,550 m) hydrothermal vent of the Galapagos Rift ocean floor spreading center. The morphological and physiological characteristics of strain GS-2 resembled those of Spirochaeta strains. However, strain GS-2 failed to grow consistently in any liquid medium tested. In addition, strain GS-2 grew more slowly and to lower yields than other Spirochaeta species. The occurrence of obligately anaerobic bacteria in hydrothermal vents indicates that the water in at least some of the vent areas is anoxic. The presence of strain GS-2 shows that these areas are favorable for anaerobic marine spirochetes.     

On the infusion of a therapeutic agent into a solid tumor modeled as a poroelastic medium

The direct infusion of an agent into a solid tumor, modeled as a spherical poroelastic material with anisotropic dependence of the tumor hydraulic conductivity upon the tissue deformation, is treated both by solving the coupled fluid/elastic equations, and by expressing the solution as an asymptotic expansion in terms of a small parameter, ratio between the driving pressure force in the fluid system, and the elastic properties of the solid. Results at order one match almost perfectly the solutions of the full system over a large range of infusion pressures. Comparison with experimental results is acceptable after the hydraulic conductivity of the medium is properly calibrated. Given the uncertain estimates of some model constants, the order zero solution of the expansion, for which fluid and porous matrix are decoupled, yields acceptable values and trends for all the physical fields of interest, rendering the coupled analysis (in the limit of small displacements) of little use. When the deformation of the tissue becomes large nonlinear elasticity theory must be resorted to.