Stress-induced morphological and physiological changes in γ-linolenic acid production by Mucor fragilis in batch and continuous cultures

Batch and continuous cultures conditions were studied in order to increase γ-linolenic acid production by Mucor fragilis CCMI 142, in response to the presence of ethanol. Continuous cultures were used to add ethanol pulses to steady state pellet cultures. It was demonstrated that pellet size, which allowed homogeneous fungal cultures, can be obtained by means of pH adjustment, thus enabling steady state continuous growth at 2.90±0.05.

The 5 and 2% (v/v) ethanol pulses induced hyphal morphological changes with arthrospore formation. A 1% (v/v) pulse of ethanol did not immediately affect growth, but induced morphological changes, which led to autolysis at the pellet core. A 0.5% (v/v) pulse of ethanol did not affect neither the morphology nor the physiology of the microorganism to any significant extent. The 0.5 and 1% (v/v) ethanol pulses resulted in an increase in the proportion of γ-linolenic acid production up to 11%. Data from batch cultures showed a higher enhancement of ethanol, attaining 30% of γ-linolenic acid.

The increase of γ-linolenic acid content observed in batch and continuous conditions appears to be a response associated with stress induced by the ethanol which seems to be of value as an industrial medium component.     

Sterols of the fungus Mucor hiemalis sporangiospores

We earlier reported that the viability and lipid composition of Mucor hiemalis F-1156 sporangiospores, as well as their capacity to develop yeast-like cells, depend on the age of the spore-forming culture [1]. With the increase in the time of the culture growth, sporangiospores exhibit a decrease in the levels of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, cardiolipin, and reserve lipids, as well as in the degree of fatty acid unsaturation, whereas the level of glycolipids increases. However, sterol composition was not been studied. Sterols are involved in morphogenetic processes [2, 3], and therefore we suggested that sporangiospores from senescent cultures, which, upon germination, develop mycelium and yeast-like cells, may exhibit a distinctive sterol pattern, along with the changed composition of fatty acids and polar and neutral lipids.     

Analysis of factors influencing the rate of germination of spores of Bacillus subtilis by very high pressure

AIMS: To elucidate the factors that determine the rate of germination of Bacillus subtilis spores with very high pressure (VHP) and the mechanism of VHP germination. METHODS AND RESULTS: Spores of B. subtilis were germinated rapidly with a VHP of 500 MPa at 50 degrees C. This VHP germination did not require the spore's nutrient-germinant receptors, as found previously, and did not require diacylglycerylation of membrane proteins. However, the spore's pool of dipicolinic acid (DPA) was essential. Either of the two redundant enzymes that degrade the spore's peptidoglycan cortex, and thus allow completion of spore germination, was essential for completion of VHP germination. However, neither of these enzymes was needed for DPA release triggered by VHP treatment. Completion of spore germination as well as DPA release with VHP had an optimum temperature of approx. 60 degrees C, in contrast to an optimum temperature of 40 degrees C for germination with the moderately high pressure of 150 MPa. The rate of spore germination by VHP decreased approx. fourfold when the sporulation temperature increased from 23 degrees C to 44 degrees C, and decreased twofold when 1 mol l(-1) salt was present in sporulation. However, large variations in levels of unsaturated fatty acids in the spore's inner membranes did not affect rates of VHP germination. Complete germination of spores by VHP was not inhibited significantly by killing of spores with several oxidizing agents, and was not inhibited by ethanol, octanol or o-chlorophenol at concentrations that abolish nutrient germination. Completion of spore germination by VHP was also inhibited by Hg(2+), but this ion did not inhibit DPA release caused by VHP. In contrast, dodecylamine, a surfactant that can trigger spore germination, strongly inhibited DPA release caused by VHP treatment. CONCLUSIONS: VHP does not cause spore germination by acting upon the spore's nutrient-germinant receptors, but by directly causing DPA release. This DPA release then leads to subsequent completion of germination. VHP likely acts on the spore's inner membrane to cause DPA release, targeting either a membrane protein or the membrane itself. However, the precise identity of this target is not yet clear. SIGNIFICANCE AND IMPACT OF THE STUDY: There is significant interest in the use of VHP to eliminate or reduce levels of bacterial spores in foods. As at least partial spore germination by pressure is almost certainly essential for subsequent spore killing, knowledge of factors involved and the mechanism of VHP germination are crucial to the understanding of spore killing by VHP. This work provides new insight into factors that can affect the rate of B. subtilis spore germination by VHP, and into the mechanism of VHP germination itself.     

Mechanisms of killing spores of Bacillus subtilis by acid, alkali and ethanol

AIMS: To determine the mechanisms of killing of Bacillus subtilis spores by ethanol or strong acid or alkali. METHODS AND RESULTS: Killing of B. subtilis spores by ethanol or strong acid or alkali was not through DNA damage and the spore coats did not protect spores against these agents. Spores treated with ethanol or acid released their dipicolinic acid (DPA) in parallel with spore killing and the core wet density of ethanol- or acid-killed spores fell to a value close to that for untreated spores lacking DPA. The core regions of spores killed by these two agents were stained by nucleic acid stains that do not penetrate into the core of untreated spores and acid-killed spores appeared to have ruptured. Spores killed by these two agents also did not germinate in nutrient and non-nutrient germinants and were not recovered by lysozyme treatment. Spores killed by alkali did not lose their DPA, did not exhibit a decrease in their core wet density and their cores were not stained by nucleic acid stains. Alkali-killed spores released their DPA upon initiation of spore germination, but did not initiate metabolism and degraded their cortex very poorly. However, spores apparently killed by alkali were recovered by lysozyme treatment. CONCLUSIONS: The data suggest that spore killing by ethanol and strong acid involves the disruption of a spore permeability barrier, while spore killing by strong alkali is due to the inactivation of spore cortex lytic enzymes.SIGNIFICANCE AND IMPACT OF THE STUDY: The results provide further information on the mechanisms of spore killing by various chemicals.     

Isolation of an ethanol-tolerant endospore-forming Gram-negative Brevibacillus sp. as a covert contaminant in grape tissue cultures

AIMS: To characterize the alcohol-surviving bacterial isolate ARBG1 from in vitro grapes (Vitis vinifera). METHODS AND RESULTS: Two bacterial strains that survived in covert form in grape cultures were isolated from the spent alcohol used for disinfecting the tools of which one (ARBG2) was characterized earlier. The present study describes characterization of the second isolate, ARBG1. Nutrient agar (NA)-derived colonies of ARBG1 displayed consistently Gram-negative staining rods (2-4x0.5-0.6 micro) substantiated by KOH mucoid thread test. Older cultures (3-7 days) showed emergence of Gram-negative staining, oblong, phase-refractile cells with ellipsoidal spores. The growth and sporulation were modified by growth medium and incubation temperature with the optimum around 37 degrees C. Identification attempts involving microscopic, biochemical, Biolog or fatty acid profiling approaches brought in mixed and inconclusive results. PCR amplification of 16S rDNA was not successful with the standard primers 27F and 1492R but with 27F and a modified primer ARBG1-RP1. The identity of the isolate was established as Brevibacillus sp. based on partial 16S rDNA sequence data from eight single colonies with Gram-positive Brevibacillus choshinensis as the closest match (99.5%). Spotting tests on NA employing spore suspension in aqueous ethanol (0%, 25%, 50%, 60%, 70%, 80% or 90%, v/v) indicated unhindered bacterial-survival in alcohol for 1 month, and that at 2 or 4 months revealed 90% ethanol as more sporicidal than lower levels, corroborated by plating results. Grape microcuttings inoculated with ARBG1 showed substantial general colonization of shoots, roots and medium but low endophytic colonization. CONCLUSIONS: The rare type of spore-producing consistently Gram-negative bacterial isolate ARBG1 was identified as Brevibacillus sp. based on 16S rDNA sequence similarity. The alcohol-defying organism was nonpathogenic and survived in covert form in grape cultures. Aqueous 90% ethanol appeared more sporicidal than lower levels. SIGNIFICANCE AND IMPACT OF THE STUDY: Characterization of an unusual endospore-forming Gram-negative bacterium, observation that some bacteria may fall outside the purview of standard 16S rDNA primers, elucidation of the threats of covert bacteria in plant tissue cultures and alcohol-mediated lateral transmission of spore formers, and the revelation that 70-80% ethanol may not be the most effective bactericidal concentration for all bacteria.     

Analysis of the action of compounds that inhibit the germination of spores of Bacillus species

AIMS: To determine the mechanism of action of inhibitors of the germination of spores of Bacillus species, and where these inhibitors act in the germination process. METHODS AND RESULTS: Spores of various Bacillus species are significant agents of food spoilage and food-borne disease, and inhibition of spore germination is a potential means of reducing such problems. Germination of the following spores was studied: (i) wild-type B. subtilis spores; (ii) B. subtilis spores with a nutrient receptor variant allowing recognition of a novel germinant; (iii) B. subtilis spores with elevated levels of either the variant nutrient receptor or its wild-type allele; (iv) B. subtilis spores lacking all nutrient receptors and (v) wild-type B. megaterium spores. Spores were germinated with a variety of nutrient germinants, Ca2+-dipicolinic acid (DPA) and dodecylamine for B. subtilis spores, and KBr for B. megaterium spores. Compounds tested as inhibitors of germination included alkyl alcohols, a phenol derivative, a fatty acid, ion channel blockers, enzyme inhibitors and several other compounds. Assays used to assess rates of spore germination monitored: (i) the fall in optical density at 600 nm of spore suspensions; (ii) the release of the dormant spore's large depot of DPA; (iii) hydrolysis of the dormant spore's peptidoglycan cortex and (iv) generation of CFU from spores that lacked all nutrient receptors. The results with B. subtilis spores allowed the assignment of inhibitory compounds into two general groups: (i) those that inhibited the action of, or response to, one nutrient receptor and (ii) those that blocked the action of, or response to, several or all of the nutrient receptors. Some of the compounds in groups 1 and 2 also blocked action of at least one cortex lytic enzyme, however, this does not appear to be the primary site of their action in inhibiting spore germination. The inhibitors had rather different effects on germination of B. subtilis spores with nutrients or non-nutrients, consistent with previous work indicating that germination of B. subtilis spores by non-nutrients does not involve the spore's nutrient receptors. In particular, none of the compounds tested inhibited spore germination with dodecylamine, and only three compounds inhibited Ca2+-DPA germination. In contrast, all compounds had very similar effects on the germination of B. megaterium spores with either glucose or KBr. The effects of the inhibitors tested on spores of both Bacillus species were largely reversible. CONCLUSIONS: This work indicates that inhibitors of B. subtilis spore germination fall into two classes: (i) compounds (most alkyl alcohols, N-ethylmaleimide, nifedipine, phenols, potassium sorbate) that inhibit the action of, or response to, primarily one nutrient receptor and (ii) compounds [amiloride, HgCl2, octanoic acid, octanol, phenylmethylsulphonylfluoride (PMSF), quinine, tetracaine, tosyl-l-arginine methyl ester, trifluoperazine] that inhibit the action of, or response to, several nutrient receptors. Action of these inhibitors, is reversible. The similar effects of inhibitors on B. megaterium spore germination by glucose or KBr indicate that inorganic salts likely trigger germination by activating one or more nutrient receptors. The lack of effect of all inhibitors on dodecylamine germination suggests that this compound stimulates germination by creating channels in the spore's inner membrane allowing DPA release. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides new insight into the steps in spore germination that are inhibited by various chemicals, and the mechanism of action of these inhibitors. The work also provides new insights into the process of spore germination itself.     

The effect of acid shock on sporulating Bacillus subtilis cells

AIMS: To study the effect of acid shock in sporulation on the production of acid-shock proteins, and on the heat resistance and germination characteristics of the spores formed subsequently. METHODS AND RESULTS: Bacillus subtilis wild-type (SASP-alpha+beta+) and mutant (SASP-alpha-beta-) cells in 2 x SG medium at 30 degrees C were acid-shocked with HCl (pH 4, 4.3, 5 and 6 against a control pH of 6.2) for 30 min, 1 h into sporulation. The D85-value of B. subtilis wild-type (but not mutant) spores formed from sporulating cells acid-shocked at pH 5 increased from 46.5 min to 78.8 min, and there was also an increase in the resistance of wild-type acid-shocked spores at both 90 degrees C and 95 degrees C. ALA- or AGFK-initiated germination of pH 5-shocked spores was the same as that of non-acid-shocked spores. Two-dimensional gel electrophoresis showed only one novel acid-shock protein, identified as a vegetative catalase 1 (KatA), which appeared 30 min after acid shock but was lost later in sporulation. CONCLUSIONS: Acid shock at pH 5 increased the heat resistance of spores subsequently formed in B. subtilis wild type. The catalase, KatA, was induced by acid shock early in sporulation, but since it was degraded later in sporulation, it appears to act to increase heat resistance by altering spore structure. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first proteomic study of acid shock in sporulating B. subtilis cells. The increasing spore heat resistance produced by acid shock may have significance for the heat resistance of spores formed in the food industry.     

PHOTOMORPHOGENESIS IN PTERIS VITTATA III. PROTECTIVE ACTION OF ETHANOL ON BLUE-LIGHT-INDUCED INHIBITION OF SPORE GERMINATION

In the fern Pteris vittata, the low-energy blue-light-induced inhibition of phytochrome dependent spore germination was protected by 0.1 mol ethanol. This protective action was observed only with ethanol in a sampling of 9 alcohols. Ethanol does not induce dark germination of spores and may act directly on the inhibition induced by blue light. It takes about a 15 times more intense dose of blue light for the induction of 50% inhibition of spore germination in 0.1 mol ethanol containing media compared with control media. The protective action of ethanol was apparent within 2 days after treatment and reached a maximum level at the 4th day. The effect of ethanol continues for about 32 hours after withdrawal of the ethanol. This action of ethanol was also observed in the case of far-red light irradiation but the mode of action of ethanol may be different from the case of blue light irradiation.     

Mechanisms of Bacillus subtilis spore killing by and resistance to an acidic Fe-EDTA-iodide-ethanol formulation

AIMS: To determine the mechanisms of Bacillus subtilis spore killing by and resistance to an acidic solution containing Fe(3+), EDTA, KI and ethanol termed the KMT reagent. METHODS AND RESULTS: Wild-type B. subtilis spores were not mutagenized by the KMT reagent but the wild-type and recA spores were killed at the same rate. Spores (alpha(-)beta(-)) lacking most DNA-protective alpha/beta-type small, acid-soluble spore proteins were less resistant to the KMT reagent than wild-type spores but were also not mutagenized, and alpha(-)beta(-) and alpha(-)beta(-)recA spores exhibited nearly identical resistance. Spore resistance to the KMT reagent was greatly decreased if spores had defective coats. However, the level of unsaturated fatty acids in the inner membrane did not determine spore sensitivity to the KMT reagent. Survivors in spore populations killed by the KMT reagent were sensitized to killing by wet heat or nitrous acid and to high salt in plating medium. KMT reagent-killed spores had not released their dipicolinic acid (DPA), although these killed spores released their DPA more readily when germinated with dodecylamine than did untreated spores. However, KMT reagent-killed spores did not germinate with nutrients or Ca(2+)-DPA and were recovered only poorly by lysozyme treatment in a hypertonic medium. CONCLUSIONS: The KMT reagent does not kill spores by DNA damage and a major factor in spore resistance to this reagent is the spore coat. KMT reagent treatment damages the spore's ability to germinate, perhaps by damaging the spore's inner membrane. However, this damage is not oxidation of unsaturated fatty acids. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide information on the mechanism of spore resistance to and killing by the KMT reagent developed for killing Bacillus spores.     

Effect of red light and gibberellic acid on lipid metabolism in germinating spores of the fern Anemia phyllitidis

The spores of the fern Anemia phyllitidis contain abundant quantities of lipid as reserve material. Germination of these spores can be induced either by red light or, even in the dark, by gibberellic acid. The effects of both these factors on lipid degradation, lipase and isocitrate lyase activities, and on the fatty acid composition have been studied in the course of the germination process. During germination in darkness with gibberellic acid, the fatty acid composition remained similar to that in the ungerminated spore. In contrast, when spores were germinated in red light, α-linolenic acid was synthesized. Little activity of lipase and isocitrate lyase could be detected in the dry spore. Red light or gibberellic acid affected a dramatic increase of the activities of these enzymes. Lipid breakdown and lipase activity were more active in red light, however. Permanent stimuli were necessary for growth and complete lipid degradation. Induction of germination simultaneously with both factors revealed an additivity of the effects of red light and gibberellic acid.     

Inhibition of Clostridium perfringens sporulation by Bacteroides fragilis and short-chain fatty acids

The effect a common fecal organism, Bacteroides fragilis, has on the sporulation of Clostridium perfringens, an organism linked to some cases of antibiotic associated diarrhea, was examined. Established B. fragilis cultures significantly decreased the number of heat resistant spores formed by C. perfringens ATCC 12915 and increased the number of vegetative cells. To determine if short-chain fatty acids (SCFA), fermentation products of B. fragilis, inhibited sporulation, the SCFA were added to sporulation broth. Sporulation decreased in the presence of acetate, isobutyrate, isovalerate, and succinate. Vegetative cell number for C. perfringens decreased in the cultures with isobutyrate. Propionate did not affect sporulation or vegetative cell number. The data support the hypothesis that the decrease in short-chain fatty acid concentration following antibiotic therapy predisposes patients to diarrheas caused by C. perfringens.     

Mycelial lipids as an aid in identifying rust fungi in culture-Cronartium fusiforme.

The sterol and fatty acid content of mycelium from germinating basidiospores of Cronartium fusiforme was determined. The mycelium contained stigmast-7-enol, fungisterol, and possibly stigmasta-5,7-dienol. No ergosterol was detected. The mycelium contained the expected fatty acids and low relative proportions of 9,10-epoxyoctadecanoic acid. The absence of ergosterol, and presence of the epoxy C18 acid and sterols typical of certain rust spores may be used for a relatively rapid confirmation of rust fungi in culture. Based on these chemical criteria, yeast-like cells isolated from the cultures of germinating basidiospores appear not to be C. fusiforme.     

Survival of spores of Rhizopus stolonifer, Aspergillus niger, Botrytis cinerea and Alternaria alternata after exposure to ethanol solutions at various temperatures

AIMS: To quantify and model the toxicity of brief exposures of spores of Rhizopus stolonifer, Aspergillus niger, Botrytis cinerea and Alternaria alternata to heated, aqueous ethanol solutions. These fungi are common postharvest decay pathogens of fresh grapes and other produce. Sanitation of produce reduces postharvest losses caused by these and other pathogens. METHODS AND RESULTS: Spores of the fungi were exposed to solutions containing up to 30% (v/v) ethanol at 25-50 degrees C for 30 s, then their survival was determined by germination on semisolid media. Logistical, second-order surface-response models were prepared for each fungus. Subinhibitory ethanol concentrations at ambient temperatures became inhibitory when heated at temperatures much lower than those that cause thermal destruction of the spores by water alone. At 40 degrees C, the estimated ethanol concentrations that inhibited the germination of 50% (LD(50)) of the spores of B. cinerea, A. alternata, A. niger and R. stolonifer were 9.7, 13.5, 19.6 and 20.6%, respectively. CONCLUSIONS: Ethanol and heat combinations were synergistic. Control of spores of these fungi could be accomplished with much lower temperatures and ethanol concentrations when combined compared with either used alone. Botrytis cinerea and A. alternata were less resistant to the combination than A. niger or R. stolonifer.     

Simultaneous ethanol and bacterial ice nuclei production from sugar beet molasses by a Zymomonas mobilis CP4 mutant expressing the inaZ gene of Pseudomonas syringae in continuous culture

AIM: The aim of this work was to construct a Zymomonas mobilis mutant capable of simultaneous ethanol and ice nuclei production from agricultural by-product such as sugar beet molasses, in steady-state continuous culture. METHODS AND RESULTS: A sucrose-hypertolerant mutant of Z. mobilis strain CP4, named suc40, capable of growing on 40% (w/v) sucrose medium was isolated following N-methyl-N'-nitro-N-nitrosoguanidine treatment. Plasmid pDS3154 carrying the inaZ gene of Pseudomonas syringae was conjugally transferred and expressed in suc40. The potential for simultaneous ethanol and bacterial ice nuclei production was assessed in steady-state continuous cultures over a range of dilution rates from 0.04 to 0.13 h(-1). In addition, the fatty acid and phospholipid profile of the three strains was also investigated. Ethanol production up to 43 g l(-1) was achieved at dilution rates below 0.10 h(-1) in sugar beet molasses. Ice nucleation activity gradually increased with increasing dilution rate and the greatest activity, -3.4 log (ice nuclei per cell), was observed at the highest dilution rate (0.13 h(-1)). Both mutant strains displayed a different fatty acid and phospholipid profile compared with the wild-type strain. CONCLUSIONS: The ability of the mutant and recombinant plasmid-containing strains to grow on high sugar concentrations and in high osmotic pressure environments (molasses) can be attributed to their phospholipid and fatty acid contents. SIGNIFICANCE AND IMPACT OF THE STUDY: Taking into account that sugar beet molasses is a low cost agricultural by-product, the simultaneous ethanol and bacterial ice nucleation production achieved under the studied conditions is considered very promising for industrial applications.     

Germination of spores of Bacillus subtilis with dodecylamine

AIMS: To determine the properties of Bacillus subtilis spores germinated with the alkylamine dodecylamine, and the mechanism of dodecylamine-induced spore germination. METHODS AND RESULTS: Spores of B. subtilis prepared in liquid medium were germinated efficiently by dodecylamine, while spores prepared on solid medium germinated more poorly with this agent. Dodecylamine germination of spores was accompanied by release of almost all spore dipicolinic acid (DPA), degradation of the spore's peptidoglycan cortex, release of the spore's pool of free adenine nucleotides and the killing of the spores. The dodecylamine-germinated spores did not initiate metabolism, did not degrade their pool of small, acid-soluble spore proteins efficiently and had a significantly lower level of core water than did spores germinated by nutrients. As measured by DPA release, dodecylamine readily induced germination of B. subtilis spores that: (a) were decoated, (b) lacked all the receptors for nutrient germinants, (c) lacked both the lytic enzymes either of which is essential for cortex degradation, or (d) had a cortex that could not be attacked by the spore's cortex-lytic enzymes. The DNA in dodecylamine-germinated wild-type spores was readily stained, while the DNA in dodecylamine-germinated spores of strains that were incapable of spore cortex degradation was not. These latter germinated spores also did not release their pool of free adenine nucleotides. CONCLUSIONS: These results indicate that: (a) the spore preparation method is very important in determining the rate of spore germination with dodecylamine, (b) wild-type spores germinated by dodecylamine progress only part way through the germination process, (c) dodecylamine may trigger spore germination by a novel mechanism involving the activation of neither the spore's nutrient germinant receptors nor the cortex-lytic enzymes, and (d) dodecylamine may trigger spore germination by directly or indirectly activating release of DPA from the spore core, through the opening of channels for DPA in the spore's inner membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide new insight into the mechanism of spore germination with the cationic surfactant dodecylamine, and also into the mechanism of spore germination in general. New knowledge of mechanisms to stimulate spore germination may have applied utility, as germinated spores are much more sensitive to processing treatments than are dormant spores.     

Studies of the release of small molecules during pressure germination of spores of Bacillus subtilis

AIMS: To measure rates of release of small molecules during pressure germination of Bacillus subtilis spores, and the role of SpoVA proteins in dipicolinic acid (DPA) release. METHODS AND RESULTS: Rates of DPA release during B. subtilis spore germination with pressures of 150 or 500 megaPascals were much higher in spores with elevated levels of SpoVA proteins, and spores with a temperature-sensitive mutation in the spoVA operon were temperature-sensitive in DPA release during pressure germination. Spores also released arginine and glutamic acid, but not AMP, during pressure germination. CONCLUSIONS: Pressure germination of B. subtilis spores causes release of many small molecules including DPA. SpoVA proteins are involved in the release of DPA, perhaps because SpoVA proteins are a component of a DPA channel in the spore's inner membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides new insight into the mechanism of pressure germination of spores of Bacillus species, a process that has significant potential for usage in the food industry.     

The lipid content of cell walls obtained from juvenile,yeast-like and filamentous cells ofCandida albicans

Purified cell walls ofCandida albicans obtained from juvenile cells, mature yeast-like cells and filamentous cells were analyzed for their lipid components. Chloroform: methanol (2:1 v v) extraction of the acetone-treated dried cell walls indicated the total lipid content to be 2.1% of the dry weight of the juvenile cell walls, 1.8% of the mature yeast-like cell walls and 4.5% of the filamentous cell walls. Separation of the chloroform: methanol extractable fraction through a silicie acid column and quantitative determination of the fractions showed significant amounts of sterol esters, triglycerides, sterols, free fatty acids, and phospholipids in these extracts. Following acetone extraction sterols were shown to constitute a greater percentage of the cell wall of juvenile cells than mature cells. Thin-layer chromatography separated the acetone-extractable lipids into at least four components. Diethyl ether extracts of the cell walls indicated the presence of small amounts of glycerol phospholipids in the cell walls of juvenile and mature yeast cells. Boiling 95% ethanol also removed a small lipid fraction from the cell walls of both juvenile and mature yeast which could include sphingosine phosphatides or glycosides.     

Bacillus megaterium spore germination is influenced by inoculum size

AIMS: The effect of spore density on the germination (time-to-germination, percent germination) of Bacillus megaterium spores on tryptic soy agar was determined using direct microscopic observation. METHODS AND RESULTS: Inoculum size varied from approximately 10(3) to 10(8) cfu ml(-1) in a medium where pH=7 and the sodium chloride concentration was 0.5% w/v. Inoculum size was measured by global inoculum size (the concentration of spores on a microscope slide) and local inoculum size (the number of spores observed in a given microscope field of observation). Both global and local inoculum sizes had a significant effect on time-to-germination (TTG), but only the global inoculum size influenced the percentage germination of the observed spores. CONCLUSIONS: These results show that higher concentrations of Bacillus megaterium spores encourage more rapid germination and more spores to germinate, indicating that low spore populations do not behave similarly to high spore populations. SIGNIFICANCE AND IMPACT OF THE STUDY: A likely explanation for the inoculum size-dependency of germination would be chemical signalling or quorum sensing between Bacillus spores.     

Spore lipids and germination in Agaricus bisporus

Summary The effects of a number of organic compounds on the germination of spores of Agaricus bisporus (J. Lange) Pilat has been investigated and a preliminary analysis of spore lipids carried out. Germination was stimulated by isocaproic acid but not by straight-chain C₅ to C₁₁ fatty acids or by the amino acids leucine and iso-leucine. Cholesterol at a concentration of 1 ppm was inhibitory. The lipid reserve of the spore comprised mono-, di- and tri-glycerides, free fatty acids and sterols. The phospholipid fraction was unusually small and contained a lecithin and cephalin fraction, phosphatidylinositol and cardiolipin phosphatidic acid; phosphatidylcholine being the most prominent component. The role of lipids and various germination stimulants in the physiology of A. bisporus spores is discussed.     

Kinetics of hydrogen production with continuous anaerobic cultures utilizing sucrose as the limiting substrate

In this study, local sewage sludge was acclimated to establish H2-producing enrichment cultures, which were used to convert sucrose to H2 with continuously stirred anaerobic bioreactors. The steady-state behaviors of cell growth, substrate utilization, and product formation were closely monitored. Kinetic models were developed to describe and predict the experimental results from the H2-producing cultures. Operation at dilution rates (D) of 0.075-0.167 h(-1) was preferable for H2 production, resulting in a H2 concentration of nearly 0.02 mol/l. The optimal hydrogen production rate was 0.105 mol/h occurring at D=0.125 h(-1). The major volatile fatty acid produced was butyric acid (HBu), while acetic acid and propionic acid were also produced in lesser quantities. The major solvent product was ethanol, whose concentration was only 15% of that of HBu, indicating that the metabolic flow favors H2 production. The proposed model was able to interpret the trends of the experimental data. The maximum specific growth rate (mu(max)), Monod constant (Ks), and yield coefficient for cell growth (Y(x/s)) were estimated as 0.172 h(-1), 68 mg COD/l, and 0.1 g/g, respectively. The model study also suggests that product formation in the continuous hydrogen-producing cultures was essentially a linear function of biomass concentration.