Solid Medium for Culturing Black Smoker Bacteria at Temperatures to 120 degrees 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 degrees 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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Micropropagation of adult avocado

A procedure has been developed to successfully micropropagate the IV-8 selection, an adult avocado rootstock. Cultures were initiated from basal shoots obtained after pruning a tree back to ground level. Buds sprouted in Murashige and Skoog solid medium with macroelements at half strength and a 1.3 μM benzyladenine supplement. To induce proliferation, shoots were cultured for 2 weeks in liquid medium in a rollordrum with the Gamborg salt formulation and 1.3 μM benzyladenine, followed by 6 weeks in double phase conditions (solid medium with a layer of liquid medium on the top) using the same salt formulation and two different benzyladenine supplements, 2.8 μM in the solid phase and 0.4 μM in the liquid phase. Ninety percent of the shoots rooted after a 3-day culture in liquid medium in a rollordrum with MS macroelements at 0.3× and 4.9 μM indolebutyric acid, followed by transfer to solid medium in the absence of auxin but with 1 g l⁻¹ activated charcoal. Survival rate during the acclimatization in the greenhouse was 70%. This revised version was published online in June 2006 with corrections to the Cover Date.     

Development of a new medium for the culture of agallian leafhopper cells

Summary The optimal composition of a medium for tissue culture of cells from the leafhopperAgallia constricta was estimated from experiments in which the rate of growth of the cells was measured at different concentrations of each component. Wound tumor virus and theconstricta variety of potato yellow dwarf virus multiply, inA. constricta when these viruses are transmitted from one plant to another. Differences weredetected in the suitablility of different batches of fetal bovine serum (after heating at 56°C for 30 min), and histidine as components in the tissue culture medium. Without heat treatment even thebest fetal bovine serum was not suitable. Estimated optimal concentrations of fetal bovine serum, histidine, salts, dextrose, lactalbumin hydrolysate and yeast autolysate were determined. Fungizone (amphotericin B) at 50 or 100 mg per 1 caused harmful effects; effective concentrations of penicillin, neomycin and steptomycin did not. Combinations of histidine-HCl and histidine (free-base) made it possible to prepare buffered medium at my pH between 6.0 and 7.0. Optimal growth ofA. constricta cells occurred at pH 6.43, and ofAceratagallia sanguinolenta at pH 6.30. Osmotic pressures of the new medium between 360 and 405 mOSM were better than lower osmotic pressures. The new medium was still suitable for growth of the cells after, storage for 6 months at 4°C. Portion of a thesis submitted for the Ph.D. degree by the senior author to the Graduate College of the University of Illinois. This research was supported in part by Grant GB 20915 from the National Science Foundation and Grant AI 6392 from the National Institutes of Health.     

Production of lipases by solid state fermentation using vegetable oil-refining wastes

Lipases were produced by a microbial consortium derived from a mixture of wastewater sludges in a medium containing solid industrial wastes rich in fats, under thermophilic conditions (temperature higher than 45 °C for 20 days) in 4.5-L reactors. The lipases were extracted from the solid medium using 100 mM Tris–HCl, pH 8.0 and a cationic surfactant agent (cetyltrimethylammonium chloride). Different doses of surfactant and buffer were tested according to a full factorial experimental design. The extracted lipases were most active at 61–65 °C and at pH 7.7–9. For the solid samples, the lipolytic activity reached up to 120,000 UA/g of dry matter. These values are considerably higher than those previously reported in literature for solid-state fermentation and highlight the possibility to work with the solid wastes as effective biocatalysts.     

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.     

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.     

Influence of salinity and cadmium on capsule strength in Pacific herring eggs

Eggs of Pacific herring (Clupea pallasi) were incubated at 5°C in salinities of 5, 20, and 35‰, and in cadmium concentrations of 0.05, 0.1, 1, 5, and 10 ppm (20‰ S). Bursting pressures of eggs in the eight groups were measured throughout incubation. In general, bursting pressures rose to a primary maximum after fertilization, declined, rose to a secondary maximum, then declined again toward hatching. Rate of attainment of the primary maximum was related to salinity of the incubation medium. Bursting pressures at the primary and secondary maxima reached final values of about 1300 and 700 g, respectively, in incubation salinities at and above 20‰. Corresponding egg volumes were greatest in low salinities (5‰) and near minimum values in salinities of 20‰ or greater (35‰). Cadmium in the incubation medium delayed attainment of primary maximum bursting pressures and primary and secondary maxima were reduced to 200–350 g at Cd concentrations near 1 ppm. Egg volumes also decreased with increased Cd concentration. It appears that Ca⁺⁺/Cd⁺⁺ ratios, depending on salinity and Cd levels in the incubation medium, influence the properties of both the jelly coat and the capsule of herring eggs. Changes in the properties of these layers could make the eggs more susceptible to mechanical damage, particularly at combinations of higher (?1 ppm) Cd concentration and lower (?20 ‰) salinities.     

Conjugal Transfer but Not Quorum-Dependent tra Gene Induction of pTiC58 Requires a Solid Surface

Donors of Agrobacterium tumefaciens harboring a transfer-constitutive derivative of the nopaline-type Ti plasmid pTiC58 transferred this element at frequencies 3 to 4 orders of magnitude higher in matings conducted on solid surfaces than in those conducted in liquid medium. However, as measured with a lacZ reporter fusion, the tra genes of the wild-type Ti plasmid were inducible by opines to indistinguishable levels on solid and in liquid medium. Donors induced in liquid transferred the Ti plasmid at high frequency when mated with recipients on solid medium. We conclude that while formation of stable mating pairs and subsequent transfer of the Ti plasmid is dependent on a solid stratum, the regulatory system can activate tra gene expression to equivalent levels in liquid and on solid surfaces.     

Sorption of bisphenol A, 17α-ethinyl estradiol and phenanthrene on thermally and hydrothermally produced biochars

Thermal and hydrothermal biochars were characterized, and adsorption of bisphenol A (BPA), 17α-ethinyl estradiol (EE2) and phenanthrene (Phen) was determined to investigate the sorption characteristic difference between the two types of biochars. Thermal biochars were composed mostly of aromatic moieties, with low H/C and O/C ratios as compared to hydrothermal ones having diverse functional groups. Single-point organic carbon-normalized distribution coefficients (logK_OC) of EE2 and BPA of hydrothermal biochars were higher than thermal biochars, while Phen logK_OC values were comparable among them. X-ray diffraction and solid state nuclear magnetic resonance results suggested that hydrothermal biochars consisted of more amorphous aliphatic-C, possibly being responsible for their high sorption capacity of Phen. This study demonstrated that hydrothermal biochars could adsorb a wider spectrum of both polar and nonpolar organic contaminants than thermally produced biochars, suggesting that hydrothermal biochar derived from poultry and animal waste is a potential sorbent for agricultural and environmental applications.