W. Heinen

Organic sulfur compounds resulting from the interaction of iron sulfide, hydrogen sulfide and carbon dioxide in an anaerobic aqueous environment.

The reaction of iron sulfide (FeS) with H2S in water, in presence of CO2 under anaerobic conditions was found to yield H2 and a variety of organic sulfur compounds, mainly thiols and small amounts of CS2 and dimethyldisulfide. The same compounds were produced when H2S was replaced by HCl, in the H2S-generating system FeS/HCl/CO2. The identification of the products was confirmed by GC-MS analyses and the incorporation of H2 in the organic sulfur compounds was demonstrated by experiments in which all hydrogen compounds were replaced by deuterium compounds. Generation of H2 and the synthesis of thiols were both dependent upon the relative abundance of FeS and HCl or H2S, i.e. the FeS/HCl- or FeS/H2S-proportions. Whether thiols or CS2 were formed as the main products depended also on the FeS/HCl-ratio: All conditions which create a H2 deficiency were found to initiate a proportional increase in the amount of CS2. The quantities of H2 and thiols generated depended on temperature: the production of H2 was significantly accelerated from 50°C onward and thiol synthesis above 75°C. The yield of thiols increased with the amount of FeS and HCl (H2S), given a certain FeS/HCl-ratio and a surplus of CO2. A deficiency of CO2 results in lower thiol systhesis. The end product, pyrite (FeS2), was found to appear as a silvery granular layer floating on the aqueous surface. The identity of the thiols was confirmed by mass spectrometry, and the reduction of CO2 demonstrated by the determination of deuterium incorporation with DCl and D2O. The described reactions can principally proceed under the conditions comparable to those obtaining around submarine hydrothermal vents, or the global situation about 4 billion years ago, before the dawn of life, and could replace the need for a reducing atmosphere on the primitive earth.

Characteristics and properties of a caldo-active bacterium producing extracellular enzymes and two related strains

SummaryThe caldo-active strain YT-P was found to produce a variety of extracellular enzymes, including an amylase and a protease, which were further examined. With azo-casein as a substrate, optimum conditions with respect to enzyme and substrate concentration were determined for the protease. The optimum temperature was found to be 70°C, with a sharp decline to both lower and higher temperatures. The enzyme was found to be extremely heat-stabile, with unaltered activity after 8 hours at 80°C.Optimum conditions for the amylase were also examined. This enzyme was shown to be less heat-stabile, though the temperature optimum was again at 70°C. The activity or stability was not influenced by absence or presence of Ca-ions. The main activity of the amylase was found in the 20–40% ammonium sulfate fraction, which also contained the bulk of the proteolytic enzyme.This strain growth optimally on a variety of carbon sources at 72°C. Typical submicroscopical features are the double-layered cell wall, and a cytoplasmic membrane with a varying number of small dots and dot-free patches.Furthermore the nutritional requirements and submicroscopical features of two other strains, YT-G and YT-F, are described and compared to strain YT-P.Based on the fatty acid composition of the three spore forming caldo-active strains we suggest that they belong to the genus Bacillus, and propose the names B. caldolyticus for strain YT-P, B. caldovelox for strain YT-F, and B. caldotenax for strain YT-G.

Bacillus flavothermus, a newly isolated facultative thermophile

A sample from a hot spring on the northern island of New Zealand contained five different thermophilic bacterial strains. One strain with peculiar properties, i.e. the formation of dark yellow colonies at 30°C as well as at 70°C, was further characterized. It was found to be a gram-positive, facultatively aerobic, motile Bacillus species, with terminal endospores. According to the physiological properties the strain closely resembled B. coagulans. However, two typical characteristics were contradictory to this conclusion, namely the intense yellow pigmentation of the colonies and the range of growth temperature. The latter was found to reach from 30 to 70°C, with an optimum at 60°C under aerobic and at 65°C under anaerobic conditions. Growth at moderate temperatures was slower than at 60°C, but the final cell yields were almost equal. The strain can therefore be considered as facultatively thermophilic. The pigment, which was found to be located in the cytoplasmic membrane, was spectroscopically identified as a carotenoid.Because the characteristics of this strain did not correspond with any of the Bacillus species described thus far, we concluded, that we had isolated a novel strain, for which the name Bacillus flavothermus is proposed.

Bio-degradation and utilization of silica and quartz.

SummaryA mesophilic bacterium,Proteus mirabilis, which was known to be able to accumulate monomer silicate ions, a thermophilic bacterium,Bacillus caldolyticus, originating from a habitat with high silica concentrations, and a silicautilizing plant,Equisetum arvense, were all found to produce monomer silica from its polymer. The monomer silica, resulting from the mineralysis of either experimentally polymerized silica, or from quartz, is taken up byP. mirabilis cells, and also byEquisetum, which then deposits the silica again as a polymer in its stem and leaves. WithB. caldolyticus, which does not utilize the depolymerized product under the given conditions, we found that the intensity of the mineralysis depends on the growth rate of the organism.

Zur Kenntnis des Fettsäureabbaus durch Schimmelpilze

Zusammenfassung1.Ein Fettsäureabbau zu Methylketonen, der bisher nur für Aspergillaceen bekannt war, wurde allgemeiner, jedoch nicht ubiquitär bei Phycomyceten, Ascomyceten und Fungi imperfecti verbreitet gefunden. Die aktivsten Methylketonbildner stellten die Gattungen Rhizopus, Aspergillus, Penicillium, Neurospora, Sordaria, Botrytis und Cladosporium.2.Das Maximum der Methylketonbildung aus Laurinsäure tritt nach 4–7 tägiger Inkubation bei 28° auf. Das pH-Optimum liegt im schwach sauren Gebiete (pH 5–7). Von den geradzahligen Fettsäuren liefert Buttersäure (C4) am meisten und am raschesten Methylketon (Aceton), von den höheren Fettsäuren (langsamer) die Laurinsäure (C12). Relativ schwach ist die Methylketonbildung bei den Säuren C8 und C10; sie fehlt bei Säuren oberhalb C14.

Separation and partial purification of extracellular amylase and protease from Bacillus caldolyticus

SummaryThe extracellular amylase and protease from Bacillus caldolyticus can be concentrated by ammonium sulfate precipitation after growth on either solid or in liquid media containing starch, glucose, and brain-heart infusion. Using the Diaflo ultrafiltration system with membranes of various permeability, the enzymes could be separated from each other by extensive flushing with buffer. Best results were obtained with the 50–70% ammonium sulfate fraction as starting material, yielding 72% of the total amylase activity in the low molecular weight fraction (UM-10 fraction: 10000–30000), while 54 and 25% respectively of the protease were retained in the two high molecular weight fractions (50000–100000, and more than 100000). Similar results were obtained with the 20–50% ammonium sulfate fraction, while the fraction of 0–20% saturation contained a low molecular weight protease. The native amylase seems to consist of a number of sub-units, which after extensive flushing accumulate in the fraction with an approximate molecular weight between 10000 and 30000. The enzyme could also be precipitated from cell-free liquid media with ammonium sulfate, followed by separation and purification on ultra-filtration cells. According to the specific activity of the UM-10 fractions a 400-fold purification was obtained compared to the amylase activity of the cell-free medium.Direct concentration and separation from liquid media, omitting ammonium sulfate treatment, was also found to be possible, although prolonged flushing with buffer was necessary to obtain satisfactory separation.During purification from the ammonium sulfate fractions, amylase activity was found to decrease but could be restored by Ca-ions. At 70°C, a final concentration of 0.5 mM CaCl2, was sufficient for full restoration, while three times that amount was necessary at 80°C. Determination of the Km-values for Ca at different temperatures resulted in an asymptotically increasing curve at temperatures beyond 75°C. Addition of Ca had a pronounced effect on the stability of the amylase at 80°C but not at 90°C. Protease activity and stability was not affected by Ca-ions.

Hypophosphite oxidase fromBacillus caldolyticus

Bacillus caldolyticus can utilize phosphorus either as phosphate, phosphite, or hypophosphite. When cultures are supplied with PO2 as the sole source of phosphorus, the hypophosphite is oxidized to phosphate, which accumulates in the medium prior to the beginning of the log phase, and is then metabolised during growth. Resting cell suspensions also have the ability to oxidise PO2 to PO4. The reaction is specific for hypophosphite: PO3 is not oxidised to PO4, regardless of whether the cells are grown in PO3- or PO2-medium. The hypophosphite oxidase works optimally between pH 7.0 to 7.5, with a temperature optimum at 75°C; theKm for NaH2PO2 is 320 μM. Sonication of cells, followed by high-speed centrifugation and ammonium sulfate fractionation of the cell-free extract showed that the PO2 oxidation, which is accompanied by the formation of NADH, requires at least three components: An ammonium sulfate fraction of the cell-free extract, the residue fraction containing the respiratory chain, and NAD as cofactor. Most probably a second cofactor, so far not characterized, is required to accomplish full activity.

Growth of bacteria at 100°C and beyond

Bacillus caldolyticus, a caldoactive bacterium originating from a hot spring at Yellowstone Park, was grown in a defined medium, whose composition resembled that of the pool water. Using a chemostat culture, which simulated the natural conditions, the organism could be adapted to grow at 100°C at a reasonable rate. Under increased pressure growth occurred also at 105° C.

Silicium-Stoffwechsel bei Mikroorganismen

ZusammenfassungUntersuchungen zur Mobilität der aufgenommenen Kieselsäure in Bakterien zeigen, daß die Kieselsäure in Abwesenheit von Glucose rasch wieder gegen Phosphat ausgetauscht werden kann. Wenn jedoch die Aufnahme der Kieselsäure aus glucosehaltigen Si-Lösungen erfolgte, so ergibt sich, daß nur ein geringer Teil dieser Kieselsäure durch Inkubation in Phosphatlösungen wieder ausgetauscht werden kann. Dieser Si-Anteil wird jedoch bei Inkubation der Zellen in Carbonatlösung vollständig ausgeschieden. Die Möglichkeit, daß diese Kieselsäure in Form von C−Si-Bindungen in der Zelle vorliegt, wird diskutiert. Weitere Untersuchungen zu diesen Fragen sind im Gange.SummaryStudies on the mobility of silicon in bacterial cells show, that Si is readily excanged against phosphate if the cells are incubated in absence of glucose. However if the bacteria are preincubated in silicate solutions in the presence of glucose, the silicon taken up is not mobile any more during incubation in phosphate solutions. The element is extruded completely by incubating the cells in carbonate solutions. Evidence has been made that silicon is deposited within the cells as organic compound in which C−Si-bonds are formed.

A combined micro-and semi-micro colorimetric determination of long-chain fatty acids from plant cutin

SummaryRe-examination of the colorimetric fatty acid determination with copper nitrate, followed by complex formation with DIECA has shown that the method is not reliable if applied as described by Duncombe (1962, 1963): The Cu concentration is too high, the DIECA concentration much too low and the wavelength chosen (440 mμ) is suitable only for very low fatty acid concentrations.According to the results reported here the following alterations have to be adopted: The concentration of the copper nitrate solution should be 3%, a 0.5% solution of DIECA in butanol has to be used and measurements should be done at 492 mμ. The method described here offers the opportunity to determine fatty acid concentrations in the semi-micro range by measuring the filtered chloroform phase directly at 691 mμ, covering a range between 175 μg/ml to 1.2 mg/ml. If the concentration turns out to be lower than 200 μg F. A./ml, the same sample can be used for a micro-determination (up to 200 μg/ml) at 492 mμ, after formation of the yellowish-brown complex by addition of 0.1 ml 0.5% butanolic DIECA solution to 1.0 ml of the chloroform phase.The method has been applied to determine the amount of free F. A. in cutin layers and cutin powder, revealing that the latter contains 5.6 times more free F. A. than the intact material. The free F. A. within the polymer seem to serve as interconnections for the main units of the cutin polylipid.