Sporulation of Clostridium putrefaciens and the Resistance of the Spores to Heat, γ-Radiation and Curing Salts

Clostridium putrefaciens grew well in most media used routinely for culturing anaerobes, but produced spores only on lactose-egg yolk agar. The D_80° was 8–14 min, z value was 4°–6° and D _γ, 0.16 Mrad. The inhibitory interactions of pH, NaCl, NaNO₂ and incubation temperature are described.     

Nitrite-induced germination of putefactive anaerobe 3679h spores

Sodium nitrite alone has been shown to stimulate germination of PA 3679h spores. The process was accelerated by using increased concentrations of sodium nitrite, a low pH, and a high temperature of incubation. At low concentrations of nitrite (0.01 to 0.2%), the delay of 36 to 48 hr occurred before germination commenced at 37 C. However, with 3.45% nitrite at 45 C and pH 6.0, most of the spores germinated within 1 hr. At pH 7.0, the germination rate decreased markedly, and at pH 8.0 it was nil. The greatest acceleration in germination rate occurred near 60 C. Hydroxylamine was completely inhibitory to nitrite-induced germination. Sodium nitrite, in turn, inhibited germination by l-alanine, the degree of inhibition being influenced by nitrite concentration and pH.     

A precise potentiometric method for determination of algal activity in an open CO2 system

Abstract. The activity of the green alga Scenedesmus obliquus was studied in simplified nutrient solutions (20 mol m₋₃ NaNO₃, 20 mol m₋₃ NH₄C1, 20 mol m₋₃ NH₄NO₃, and 20 mol m₋₃ NaCl, respectively) at 25 °C. The experiments were performed under welldefined incident photon density fluxes ranging from 10 to 200 μmol m₂ s₋₁, Light-dependent changes in pH and alkalinity (A) were followed by means of a potentiometric method using a glass electrode. In the experiments, carbon dioxide with known partial pressure was bubbled through the algal suspension, and during dark periods ul intervals of 1 h, the solution was allowed to equilibrate with the gas phase. This technique was applied to calculate equilibrium values of pH and alkalinity at regular intervals during a 12-h period. Results obtained in NaNO₃, solution show a linear increase in A with time, at each level of illumination studied. After an initial drop, A also increases in NH₄NO₃, solution in a similar way to that in NaNO₃ solution. The change in A with time was also found to increase linearly with the photon density flux studied and no saturation level could be defined. In experiments in NaCl solution, no changes in A were registered while measurements in NH₄Cl solution showed a decrease in A with time.     

Nitrite in the root zone and its effects on ion uptake and growth of wheat seedlings

The immediate and posteffects of various concentrations of NaNO₂ on ion uptake of wheat ( Triticum aestivum L. cv. GK Öthalom) seedlings were studied at different pH values. Without pretreatment, the higher the concentration of NaNO₂ the greater was the decrease in uptake of K⁺ into the roots, both at pH 4 and pH 6. At pH 6 but not at pH 4 the reverse was true when the seedlings were pretreated with NaNO₂. Due to the high Na⁺ content of the roots, an effect of Na⁺ in this process cannot be excluded. Nitrite was taken up by the roots more rapidly than nitrate. Nitrite at 0.1 m M in the medium induced the development of an uptake system for both NO⁻₂ and NO⁻₃ in wheat roots. At higher concentrations pretreatment with NO⁻₂ decreased NO⁻₃ uptake by the roots, but NO₃ did not inhibit the uptake of NO₂. The toxic effect of NO⁻₂ was strongly pH dependent. Lower pH of the external solution led to an increased inhibition by NO⁻₂ of both ion uptake and growth of seedlings. The inhibitory effect of NO⁻₂ differed considerably for roots and shoots. The roots and especially the root hairs were particularly sensitive to NO⁻₂ treatment.     

Mechanism of chemical manipulation of the heat resistance of Clostridium perfringens spores

The mechanism(s) of chemical manipulation of the heat resistance of Clostridium perfringens type A spores was studied. Spores were converted to various ionic forms by base-exchange technique and these spores were heated at 95°C. Of the four ionic forms, i.e. Ca²⁺, Na⁺, H⁺ and native, only hydrogen spores appeared to have been rapidly inactivated at this temperature, when survivors were enumerated on the ordinary plating medium. However, the recovery of the survivors was improved when the plating medium was supplemented with lysozyme, and more dramatically when the heated spores were pretreated with alkali followed by plating in the medium containing lysozyme. In contrast to crucial damage to germination, in particular to spore lytic enzyme, no appreciable amount of DPA was released from the heat-damaged H-spores. These results suggest that a germination system is involved in the thermal inactivation of the ionic forms of spores, and that exchangeable cation load plays a role in protection from thermal damage of the germination system within the spore. An enhancement of thermal stability of spore lytic enzyme in the presence of a high concentration of NaCl was consistent with the hypothesis.     

Hydrogen peroxide release from bacterial spores during germination

The release of free H₂O₂ from spores of Clostridium perfringens and Bacillus megaterium during germination has been demonstrated using the scopoletin fluorescence assay. Scopoletin oxidation was markedly inhibited when exogenous catalase was added, and was also influenced by the concentration of spores. H₂O₂ release into the germination medium was observed to parallel the O₂ consumption during germination, suggesting that the H₂O₂ may arise from certain O₂-dependent metabolism associated with initiation of spore germination.     

The Effect of Oxygen on the Germination and Outgrowth of Clostridium butyricum Spores and Changes in the Oxidation-Reduction Potential of Cultures

S ummary . The E_h fall observed during incubation of Clostridium butyricum spores occurred during germination and emergence, not during the log phase; it is attributed to the H₂ tension resulting from metabolism. When the O₂ tension in the medium was increased, the E_h fell only after a few spores outgrew and replicated; germination of remaining spores then followed. It is suggested that the few cells able initially to metabolize can (a) elaborate NADH etc. which reduce the O₂ tension to a level non-inhibitory for the remaining spores, and (b) produce the H₂ tension recorded by the Pt electrode.     

Effects of sulfate and nitrate on K+ uptake and growth of wheat and cucumber

The uptake of K⁺ ion was studied in the roots of wheat ( Triuicum aestivum L. cv. GK Szeged) and cucumber ( Cucumis sativus L. cv. Budai csemege) seedlings grown in nutrient solution under nitrogen and sulfate stress conditions. Seedlings pretreated with 1 or 10 m M NaNO₃, absorbed more K⁺ than those treated with 0.1 m M NaNO₃. However, the posteffect of NaNO₃ was considerably influenced by the Na₂SO₄, treatment. The results suggest that, at least partly, a feed-back regulation of K⁺ uptake may occur. However, due to the high Na⁺ contents of the roots, a Na⁺ effect in this process cannot be excluded. The growth and dry matter yields of the roots and shoots were strongly influenced by the SO²⁻/₄ and NO⁻/₃ supply of the plants. Appreciable differences were experienced between wheat and cucumber seedlings. The optimum SO²⁻/₄ concentration of the growth solution for maximal growth varied considerably between the species, and was also different for the roots and the shoots in a given species.     

A rapid method for the determination of bacterial fatty acid composition

Heat treatment of spores of non-proteolytic strains of Clostridium botulinum at 75–90°C, and enumeration of survivors on a nutrient medium containing lysozyme gave biphasic survival curves. A majority of spores were inactivated rapidly by heating, and the apparent heat-resistance of these spores was similar to that observed by enumeration on medium without lysozyme. A minority of spores showed much greater heat-resistance, due to the fact that the spore coat was permeable to lysozyme, which diffused into the spore from the medium and replaced the heat-inactivated germination system. The proportion of heated spores permeable to lysozyme was between 0.2 and 1.4% for spores of strains 17B (type B) and Beluga (type E), but was about 20% for spores of strain Foster B96 (type E). After treatment of heated spores with alkaline thioglycolate, all were permeable to lysozyme. D-values for heated spores that were permeable to lysozyme (naturally and after treatment with thioglycolate) were: for strain 17B, D₈₅°C, 100 min; D₉₀°C, 18.7 min; D₉₅°C, 4.4 min; for strain Beluga, D₈₅°C, 46 min; D₉₀°C, 11.8 min; D₉₅°C, 2.8 min. The z-values for these spores of strains 17B and Beluga were 7.6°C and 8.3°C.     

Recovery of heat-injured spores of Clostridium perfringens types B, C and D by lysozyme and an initiation protein

R.G. LABBÉ AND C.-A. CHANG. 1995. Heat-injured spores of several strains of Clostridium perfringens types B, C and D could be partially recovered if lysozyme was included in the recovery medium. As little as 25 ng ml^-1was effective. D _90°C values of 1.3–2.6 were obtained with an approximate 2–3-fold increase in the presence of 1 μg ml^-1of lysozyme. In the absence of lysozyme, prolonged heating of spores resulted in the appearance of satellite colonies surrounding colonies of surviving spores. An initiation protein, previously reported in the case of type A strains, was also produced by type B, C and D strains. When added to the recovery medium it too promoted the recovery of spores from thermal injury though not as effectively as lysozyme.     

Culture conditions affect the chemical composition of the exopolysaccharide synthesized by the fungus Aureobasidium pullulans

Aims:  To identify if culture conditions affect the chemical composition of exopolysaccharide (EPS) produced by Aureobasidium pullulans .
Methods and Results:  In batch airlift and continuously stirred tank (CSTR) reactors the EPS produced with low (0·13 g l⁻¹ N) initial NaNO₃ or (NH₄)₂SO₄ levels contained pullulan, with maltotriose as its major component, similar to that synthesized in the airlift reactor with high (0·78 g l⁻¹ N) initial NaNO₃ levels. EPS produced by CSTR grown cultures with high (NH₄)₂SO₄ levels contained little pullulan, possibly because of a population shift from unicells to mycelium. This chemical difference may explain why total EPS yields did not fall as they did with cultures grown under identical conditions with high NaNO₃ levels, where the pullulan component of the EPS disappeared. EPS synthesized in N-limiting chemostat cultures of A. pullulans changed little with growth rate or N source, being predominantly pullulan consisting of maltotriose units.
Conclusions:  While the EPS chemical composition changed little under N-limiting conditions, high initial medium N levels determined maltotriose content and/or pullulan content possibly by dictating culture morphology.
Significance and Impact of the Study:  These results emphasize the requirement of all studies to determine EPS chemical composition when examining the influence of culture conditions on EPS yields.     

Nitrite-induced Germination of Putrefactive Anaerobe 3679h Spores

Sodium nitrite alone has been shown to stimulate germination of PA 3679h spores. The process was accelerated by using increased concentrations of sodium nitrite, a low pH, and a high temperature of incubation. At low concentrations of nitrite (0.01 to 0.2%), the delay of 36 to 48 hr occurred before germination commenced at 37 C. However, with 3.45% nitrite at 45 C and pH 6.0, most of the spores germinated within 1 hr. At pH 7.0, the germination rate decreased markedly, and at pH 8.0 it was nil. The greatest acceleration in germination rate occurred near 60 C. Hydroxylamine was completely inhibitory to nitrite-induced germination. Sodium nitrite, in turn, inhibited germination by l-alanine, the degree of inhibition being influenced by nitrite concentration and pH.     

Sensitivity of Bacillus coagulans spores to combinations of high hydrostatic pressure, heat, acidity and nisin

We investigated the combined effects of pressure, temperature, pH, initial spore concentration and the presence of nisin on the survival of spores of Bacillus coagulans. Spores were more sensitive to pressure both at lower pH and at higher treatment temperatures. An additional 1.5-log₁₀ reduction in cfu ml^-1 was observed when pH was lowered from 7.0 to 4.0 during pressurization at 400 Mpa and 45°C. A 4-log₁₀ cfu ml^-1 reduction was observed when the temperature was increased from 25°C to 70°C during pressurization at 400 Mpa. The spores were sensitive to nisin at concentrations as low as 0.2 IU ml^-1. At least a 6-log₁₀ reduction was generally achieved with pressurization at 400 Mpa in pH 4.0 buffer at 70°C for 30 min when plated in nutrient agar containing 0.8 IU ml^-1 nisin.     

Production of clover broomrape seed germination stimulants by red clover root requires nitrate but is inhibited by phosphate and ammonium

The effect of nutrients (nitrate, ammonium, urea, phosphate and potassium) on the production and/or exudation of germination stimulants for clover broomrape ( Orobanche minor Sm.), a root holoparasite, by its host red clover ( Trifolium pratense L.) was examined using hydroponically grown material. Potassium (K₂SO₄) concentrations up to 100 mg l^–1 (based on K) did not affect the production of germination stimulants by red clover while, in contrast, phosphate (NaH₂PO₄) was highly inhibitory even at concentrations as low as 1 mg l^–1 (based on P). Nitrate (NaNO₃) markedly promoted stimulant production in a dose-dependent manner from 2 to 50 mg l^–1 (based on N). Ammonium [(NH₄)₂SO₄] had no effect at 2 mg l^–1 (based on N) but was inhibitory at higher concentrations. Ammonium is known to be a seed germination inhibitor of root parasites, indicating that ammonium has a dual inhibitory action. Urea had no effect at 2 mg l^–1 (based on N) but was promotive at higher concentrations. These results provide a basis for the inhibitory effects of nitrogen fertilizer on infection by root parasitic weeds, broomrapes and witchweeds, and explain why these parasites prevail in areas where there is lower phosphorus availability in soils.     

Factors Affecting Germination and the Mode of Germination of Zygospores of Choanephora cucurbitarum

Treatment of zygospores of Choanephora cucurbitarum with KMnO₄, NaClO or H₂O₂ effectively activated the spores and induced their germination. The optimum concentration of KMnO₄ for activation of zygospores was 0.25 to 0.5%. Zygospores were not able to germinate in darkness even after activation by KMnO₄. When zygospores from 40 to 50-day-old cultures were treated with 0.5% KMnO₄ solution for 60 min before incubation on water agar at 24% under light, about 50% germinated in 10 days. KMnO₄ treatment killed more than 99% of residual mycelial fragments, sporangiospores and sporangiola in the zygospore suspension. During germination disappearance of oil droplets in zygospores occurred prior to the cracking on zygospore wall. Both sporangial germination and mycelial germination were found. Moreover, sporangiole germination was observed for the first time.     

Effects on acid-base balance, methaemoglobinemia and nitrogen excretion of European eel after exposure to elevated ambient nitrite

Haemoglobin, methaemoglobin, blood nitrite concentration and acid-base balance were measured in European eel Anguilla anguilla following exposure to 0 (control), 0·142, 0·356, 0·751 and l·549 mM nitrite in fresh water for 24 h. Blood GOT (glutamate oxaloacetate transaminase) and GPT (glutamate pyruvate transaminase) activities and whole animal ammonia-N and urea-N excretions were also measured. Blood nitrite, methaemoglobin, PO ₂ (oxygen partial pressure), GOT, and whole animal ammonia-N excretion and urea-N excretion increased directly with increasing ambient nitrite concentrations, whereas blood pH, PCO ₂, and HCO⁻₃ were inversely related to ambient nitrite concentration. An accumulation of nitrite in the blood of A. anguilla following 24 h exposure to elevated ambient nitrite as low as 0·751 mM increased its blood methaemoglobin, PO ₂, GOT and nitrogen excretion, but decreased its PCO ₂ (carbon dioxide partial pressure), HCO⁻₃ and functional haemoglobin.     

Effect of some factors on determination of viable microbial cells by peroxyoxalate chemiluminescence method

The effects of physical and chemical factors on the production of H₂O₂ from Escherichia coli cells were studied. When 20 mmol 1^-1 Tris-HCl buffer was used for this purpose the electron transport system (ETS) showed the highest activity at pH 7.6-8.2. KCN promoted the production of H₂O₂ from E. coli cells, and the optimum concentration was changed in different reaction times and pH values. Glucose, 5 mg ml^-1, increased the ETS activity about twofold. The other substrates and surfactants did not increase the chemiluminescence intensity. NaNO₂ and Na₂SO₄ in inorganic salts significantly reduced the ETS activity above 70%. In addition, the optimum temperature for the production of H₂O₂ was 30°C in this study. When glucose (5 mg ml^-1) and KCN (0.2 mmol 1^-1) were added to the reaction buffer containing 0.5 mmol 1^-1 menadione, the detectable minimum cell densities (averages of triplicate assay) of E. coli, Enterobacter cloacae and Serratia marcescens were 5 times 10³ cells ml^-1, 10⁴ cells ml^-1 and 10⁴ cells ml^-1 respectively.     

Response surface methodology to optimize the nutritional parameters for enhanced production of jasmonic acid by Lasiodiplodia theobromae

Aims:  To find out the cumulative effect of the nutritional parameters and to enhance the production of jasmonic acid (JA) in static fermentation by Lasiodiplodia theobromae using response surface methodology (RSM).
Method and Results:  Malt extract, sucrose, NaNO₃ and MgSO₄.7H₂O were analysed by a 30-trial central composite design using RSM for optimizing their concentrations in the medium and the effect of their mutual interaction on JA production. Sucrose and NaNO₃ were found highly significant in influencing the JA production. Malt extract and MgSO₄.7H₂O showed an effect on the JA production in interaction with other variables. When the optimum values of the parameters obtained through RSM (19·95 g l⁻¹ malt extract, 50 g l⁻¹ sucrose, 7·5 g l⁻¹ NaNO₃ and 3·51 g l⁻¹ MgSO₄.7H₂O) were applied, 32% increase in JA production (299 mg l⁻¹) was observed in comparison with 225 mg l⁻¹ of JA produced with same media components not analysed by RSM and subsequently validated the statistical model.
Conclusions:  Increase in JA production was achieved by optimizing the nutritional parameters.
Significance and Impact of the Study:  This is the first report of using RSM for optimizing a medium for JA production. It resulted in an increase in JA production without augmentation of costly additives.     

An Investigation of the Oxidation-reduction Potential and of the Effect of Oxygen on the Germination and Outgrowth of Clostridium butyricum Spores, using Platinum Electrodes

S ummary . The changes in electrode potential recorded by a platinum foil electrode, and in pH, during the incubation of Clostridium butyricum spores in a medium continuously sparged with a sterile gas mixture have been correlated with the biological events occurring simultaneously. The marked fall in E _h which occurs during incubation is recorded by this type of electrode as occurring during the period of rapid cell replication; this observation may be an artefact associated with foil electrodes and the authors feel that results obtained with such electrodes should be interpreted with caution. The stimulating effect of CO₂ and the increasingly inhibitory effect on germination of O₂, as the concentration of the latter in the gas mixture is increased, have been noted. The inhibitory effect is thought to be due to the O₂ itself and not to the high E _h produced in the medium by the O₂.     

A parametric study of the destruction efficiency of Bacillus spores in low pressure oxygen-based plasmas

The destruction of Bacillus spores in oxygen-based plasmas sustained in the millitorr pressure range has been studied as functions of various biological and plasma parameters, namely Bacillus species, surface concentration of spores, treatment temperature, and gas composition. In an oxygen plasma, Bacillus stearothermophilus appears less plasma-resistant than the other spores tested. Oxygen, H₂O₂ and chiefly CO₂ plasmas are clearly shown to be much more efficient in destroying Bacillus subtilis spores than pure argon plasma. The bacterial surface concentration on the spore carriers and the treatment temperature also lead to significant variations in the destruction efficiency of spores when using CO₂ plasma.